CN106480003B - The combination of helicobacter pylori dominant antigen and screening technique based on CD4+T cellular immunity - Google Patents

Abstract

The invention relates to a combination of dominant antigens of Helicobacter pylori based on CD4+T cell immunization and a preparation method. The combination of dominant antigens includes the following three and their homologous proteins: hypoxanthine dehydrogenase, type II citric acid Synthase and urease B subunits; their amino acid sequences are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, respectively. The CD4+T cell immunodominant antigen combination screened by the invention has obvious immune protection effect, and its protection effect is better than that of H. pylori whole protein antigen, has stronger Helicobacter pylori removal ability, and causes lighter pathological damage at the same time. The three immunodominant antigens provided by the present invention can all induce the body to produce a strong immune response against the antigens. Therefore, by inducing the body to respond to the immunoprotective dominant antigen, or directly immunizing the body with the protective antigen, it will play an effective immune protection role against Helicobacter pylori infection, which can be used for further Helicobacter pylori prophylactic and therapeutic multivalent vaccines. Research.

Description

Helicobacter pylori dominant antigen combination and screening method based on CD4+T cell immunization

technical field

The invention belongs to the technical field of medicine and biology, and relates to a combination of dominant antigens of Helicobacter pylori, in particular to a combination of dominant antigens of Helicobacter pylori based on CD4+T cell immunization and a screening method.

Background technique

Helicobacter pylori (H. pylori) colonizes the gastric mucosa and is a class I pathogenic factor for gastric cancer. main causative factor. With a global infection rate of over 50%, there is an urgent need for an effective Helicobacter pylori vaccine.

The screening of candidate antigens is the core of vaccine development. Unlike viruses with a limited number of antigens, bacteria are difficult to systematically screen to identify protective antigens for their immunodominance due to the large number of antigens they contain. This has been hindering the development of antibacterial vaccines.

Studies have shown that local th1 and th17 cells in gastric mucosa play an important role in resisting H.pylori infection. Using the CD4+ T cell-dominant epitope H.pyloriaA(88-100), it was demonstrated that the H.pyloriaA(88-100) epitope-specific CD4+T response was associated with the severity of gastric disease in the HLA-DRB1*1501-restricted population closely related. Hitzler, I. Analysis of B-cell and CD4+ T-cell responses in H. pylori-immunized mice shows that th1 and th17 cells play a major protective role, rather than B-cell-mediated humoral immunity. Similarly, the study by DeLyria, E.S. also supports the protective role of th17 in H. pylori immunity.

However, the immunodominant antigens of th1 and th17 have never been systematically screened, and a new Helicobacter pylori vaccine based on them has not been developed.

SUMMARY OF THE INVENTION

In view of this, the purpose of the present invention is to provide a combination of Helicobacter pylori dominant antigens based on CD4+ T cell immunization.

The present invention also provides a screening method for the above-mentioned advantageous antigen combination.

To achieve the above object, the present invention provides the following technical solutions:

Helicobacter pylori dominant antigen combination based on CD4+ T cell immunity, including the following three and their homologous proteins: inosine 5'-monophosphate dehydrogenase (IMPDH), type II citrate synthase (type II citrate synthase, CS II) and urease B subunit (urease subunit beta, UreB); their amino acid sequences are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively.

Preferably, the mass ratio of the three antigens is 1:1:1, and the total antigen content of the vaccine is 100 μg.

The application of the above advantageous antigen combination in the preparation of a vaccine for preventing or treating Helicobacter pylori infection.

Preferably, the vaccine is a protein or nucleic acid vaccine.

Preferably, the vaccine further comprises a medically acceptable immune adjuvant.

Further preferably, the immune adjuvant is any one or more of Freund's adjuvant, aluminum adjuvant, CPG ODN 1826 or AddaVax.

The screening method for the above-mentioned advantageous antigen combination comprises the following steps:

1) lysing the Helicobacter pylori cell to obtain the Helicobacter pylori whole antigen lysate, and then dividing it into 30 components by molecular sieve according to the molecular weight of each protein;

2) Use the 30 components of step 1) to stimulate spleen CD4+ T cells from Helicobacter pylori-infected and immunized mice respectively, detect the proliferation of CD4+ T cells by ³ H-TdR incorporation method, and use the proliferation index as Figure, the dominant component PC05 and the subdominant component PC17 with the strongest ability to stimulate the proliferation of CD4+ T cells were obtained;

3) Using the dominant component PC05 and sub-dominant component PC17 screened in step 2) to immunize mice respectively for challenge protection experiment, with H. pylori whole bacteria immunization and PBS as control, it is determined that the dominant component PC05 has stronger immune clearance while causing less inflammatory damage;

4) Determine the protein components contained in the dominant component PC05 obtained in step 3) by using high performance liquid chromatography-mass spectrometry coupled with LC-MS/MS, and confirm that PC05 contains 11 kinds of proteins;

5) Utilize the method of genetic engineering to synthesize 11 kinds of proteins determined in step 4);

6) PC05-specific CD4+ T cells were stimulated with 11 proteins synthesized in step 5), and three H. pylori antigens with the strongest Th1 and Th17 responses were screened and identified.

Preferably, in step 1), 8M urea is used to lyse Helicobacter pylori cells.

The beneficial effects of the present invention are:

After H.pylori infects the gastric mucosa, H.pylori antigens are recognized, processed and presented by antigen-presenting cells (APCs) such as dendritic cells (DCs) and macrophages. APCs in turn activate naive CD4+ T cells Induces IFN-γ-secreting Th1 cell responses and IL-17A-secreting Th17 cell responses. IL-17 promotes the secretion of cytokines such as IL-1, IL-6, IL-8 and TNF-α from gastric mucosal epithelial cells, mesenchymal cells, endothelial cells, and lamina propria mononuclear cells that express IL-17 receptors. , and recruit neutrophils to clear H. pylori.

Therefore, the applicant attempted to systematically screen out the immunodominant antigens of Th1 and Th17 from the whole H. pylori bacteria, and based on this, a novel Helicobacter pylori vaccine was developed. During this process, each antigen of the whole H. pylori bacteria was included in the simultaneous evaluation until the immunodominant antigen was screened out. At the same time, indicators such as the immune protection rate and inflammation score of each antigen were verified in the mouse model. As a result, three dominant antigens of Th1 and Th17 were successfully screened: IMPDH, CS II and UreB. The immunoprotection experiment proved that it has stronger H.pylori clearance and caused less immunopathological damage. This provides new candidate antigens for the design and development of future novel Helicobacter pylori vaccines.

The results showed that the CD4+T cell immunodominant antigen combination screened had obvious immune protection, and its protective effect was better than that of H. The three immunodominant antigens provided by the present invention can all induce the body to produce a strong immune response against the antigens. Therefore, by inducing the body to respond to the immune protective dominant antigen, or directly immunizing the body with the protective antigen, it will have an effective immune protection effect on Helicobacter pylori infection, which can be used for further Helicobacter pylori prophylactic and therapeutic multivalent vaccines. Research.

Description of drawings

In order to make the purpose, technical solutions and beneficial effects of the present invention clearer, the present invention provides the following drawings for description:

Figures 1A to 1D show that H. pylori pan-antigen has an immunoprotective effect and stimulates IFN-γ and IL-17A responses in the gastric mucosa, wherein Figure 1A is the bacterial colonization amount, and Figure 1B-1 and Figure 1B-2 are gastric monocytogenes The cell suspension was detected by flow cytometry. Figure 1C shows the number of CD3+T and CD4+T cells, and Figure 1D shows the expression levels of IFN-γ and IL-17A mRNA in the gastric mucosa of mice.

Figure 2 shows that the H. pylori whole antigen was divided into 30 components according to molecular weight by molecular sieve chromatography.

Figures 3A and 3B are the screening of dominant components, in which Figure 3A is the proliferation of CD4+ T lymphocytes in the immune/infected group stimulated by each component, and Figure 3B is the proliferation of CD4+ T lymphocytes in the immune/uninfected group stimulated by each component .

Figures 4A to 4C are the evaluation of H. pylori whole antigen, PC05, and PC17 immune protection, wherein Figure 4A is the amount of bacterial colonization in the gastric mucosa, Figure 4B is the gastric histopathological score, and Figure 4C is the gastric tissue pathological section.

Figures 5A to 5C show that the dominant component PC05 stimulated stronger Th1 and Th17 responses, and Figure 5A shows the expression levels of IFN-γ and IL-17A mRNA in gastric mucosa of mice after H. pylori pan-antigen, PC05, and PC17 immunization challenge , Figure 5B shows the flow cytometry detection of antigen-specific CD4+ T cells, and Figure 5C shows that PC05 stimulates specific Th1 and Th17 response levels stronger than PC17 and H. pylori whole bacteria antigens.

Figures 6A-6D show that P5 (IMPDH), P10 (CS II) and P11 (UreB) elicit stronger specific Th1 and Th17 responses.

Figure 7 shows that the immunodominant antigens IMPDH, CS II and UreB have better immune protection functions.

Detailed ways

The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

The Helicobacter pylori strain B0 involved in the present invention is domesticated in BALB/c mice.

Materials and Methods

(1) Bacteria and mice

Helicobacter pylori strain B0: This strain is a domesticated strain of Helicobacter pylori acclimated in BALB/c mice, which is characterized by easier colonization in the stomach of BALB/c mice and leads to pathological reactions. Helicobacter pylori strains were isolated and cultured from gastric tissue of Helicobacter pylori carriers, and were inoculated into brain heart infusion agar medium containing 10% (volume ratio) rabbit blood by "three-line method". A single colony was picked for culture, identified as Helicobacter pylori by gene sequencing, and named as Helicobacter pylori strain M. BALB/c mice were then infected with H. pylori strain M. Then repeat the above separation and purification scheme to isolate Helicobacter pylori from gastric tissue of BALB/c mice. The thus obtained Helicobacter pylori strain, which is easier to colonize BALB/c mice, was named as Helicobacter pylori strain B0.

Helicobacter pylori strain B0 was cultured in brain heart infusion agar medium containing 10% (v/v) rabbit blood at 37°C under microaerophilic conditions. Two days later, the H. pylori strains were transferred from the plates to 10% (v/v) fetal bovine serum (FBS) Brucella broth for expansion. Helicobacter pylori in exponential growth phase was used for infection and in vitro experiments. Specific pathogen-free (SPF) female BALB/c mice aged six to eight weeks were purchased from the Experimental Animal Center of the Third Military Medical University.

(2) Main reagents and sources

See Table 1 for details.

Table 1. Main reagents and sources of the present invention

The screening method of the advantageous antigen combination of the present invention comprises the following steps:

1) use 8M urea to split Helicobacter pylori microbial cells to obtain Helicobacter pylori full antigen urea lysate, then divide into 30 components by molecular sieve according to the molecular weight of each protein;

2) Use the 30 components of step 1) to stimulate spleen CD4+ T cells from Helicobacter pylori-infected and immunized mice respectively, detect the proliferation of CD4+ T cells by ³ H-TdR incorporation method, and use the proliferation index as Figure, the dominant component PC05 and the subdominant component PC17 with the strongest ability to stimulate the proliferation of CD4+ T cells were obtained;

3) Using the dominant component PC05 and sub-dominant component PC17 screened in step 2) to immunize mice respectively for challenge protection experiment, with H. pylori whole bacteria immunization and PBS as control, it is determined that the dominant component PC05 has stronger immune clearance while causing less inflammatory damage;

4) Determine the protein components contained in the dominant component PC05 obtained in step 3) by using high performance liquid chromatography-mass spectrometry coupled with LC-MS/MS, and confirm that PC05 contains 11 kinds of proteins;

5) Utilize the method of genetic engineering to synthesize 11 kinds of proteins determined in step 4);

6) Stimulate PC05-specific CD4+ T cells with 11 kinds of proteins synthesized in step 5) respectively, and screen and identify the H. pylori antigens with the strongest Th1 and Th17 responses, three in total;

7) Using the immunodominant antigens of Th1 and Th17 cells screened in step 6) to immunize mice respectively to carry out a challenge protection experiment, with PC05 and PBS immunization as controls. It was determined that all three immunodominant antigens have obvious immune clearance ability while causing less inflammatory damage.

Example 1:

Evaluation of CD4+ T cell responses in H.pylori whole-bacteria immunized mice

1. Animal immunization and challenge experimental protocol

Experimental animals: BALB/c mice were female 6-8 weeks old.

antigen:

(1) Immunization/challenge group (I/C): H. pylori inactivated whole bacteria. 100μg/only.

(2) Unimmunized/challenged group (U/C): phosphate buffered saline (PBS).

Adjuvant: Freund's adjuvant. 100μl / only.

Immunization method: subcutaneous injection.

Immune volume: 200μl/only.

Immunization scheme: subcutaneous immunization 3 times (week 0, 2, 4). Complete Freund's adjuvant was used for the first time, incomplete Freund's adjuvant was used for the second time, and no adjuvant was added for the third time.

One week after the last immunization, 1.0×10 ⁹ CFU of Helicobacter pylori was administered by gavage, once a day for 4 consecutive days. The mice were sacrificed at the 4th week after the challenge, and the quantitative amount of Helicobacter pylori and CD4+T cell response in the gastric tissue of the mice were detected, and the immune protection effect was analyzed.

2. Quantitative detection of Helicobacter pylori in gastric mucosa of mice.

Real-time quantitative PCR was used to detect the colonization of Helicobacter pylori in the stomach. Bacterial DNA was extracted with a bacterial genome extraction kit, and its colonization was detected based on Helicobacter pylori 16SrDNA. The results are shown in Figure 1A.

The sequence of Helicobacter pylori 16S rDNA is as follows:

Upstream primer: 5'-TTTGTTAGAGAAGATAATGACGGTATCTAAC-3', as shown in SEQ ID NO.4;

Downstream primer: 5'-CATAGGATTTCACACCTGACTGACTATC-3', as shown in SEQ ID NO.5.

The sequence of the fluorescent probe is as follows:

FAM-CGTGCCAGCAGCCGCGGT-TAMRA is shown in SEQ ID NO.6.

3. Detection of CD4+ T cell responses in mouse gastric mucosa

Mouse gastric tissue was dissected along the greater and lesser curvatures of the stomach and gently washed twice with sterile PBS to remove food debris. Then put into 10ml Hank's Balanced Salt Solution (HBSS, without Ca, My), containing 1mM dithiothreitol (DTT), 1mM ethylenediaminetetraacetic acid (EDTA), and 2% fetal calf serum (FCS), Incubate at 37°C for 45min. The resulting mixture was then passed through a sterile steel mesh to remove undigested tissue to obtain a single cell suspension. The digested single cell suspension was washed twice with sterile PBS. It was then stained with immunofluorescently labeled antibodies (FITC-CD3, APC-CD4) and detected by flow cytometry. Reference is made to Figures 1B-1, 1B-2 and 1C.

Real-time quantitative PCR was used to detect the local mRNA level of mouse gastric mucosa CD4+ T cell immune response. Total RNA from gastric mucosa was extracted by Trizol method, reverse transcribed into cDNA, and the expressions of IFN-γ and IL-17A were detected by real-time quantitative PCR using SYBR Green incorporation method. The results are shown in Figure 1D.

The sequence of IFN-γ is as follows:

Upstream primer: 5'-GATCCTTTGGACCCTCTGACTT-3', as shown in SEQ ID NO.7;

Downstream primer: 5'-TGACTGTGCCGTGGCAGTAA-3' as shown in SEQ ID NO.8.

The sequence of IL-17A is as follows:

Upstream primer: 5'-CTCCAGAAGGCCCTCAGACTAC-3', as shown in SEQ ID NO.9;

Downstream primer: 5'-GGGTCTTCATTGCGGTGG-3', as shown in SEQ ID NO.10.

Example 2:

Molecular sieve chromatography divides H. pylori whole bacterial antigens into different components according to molecular weight

First, the Helicobacter pylori strain B0 was dissolved in 8 mol/L urea, containing 10 mmol/L dithiothreitol (DTT) 1 g/6 ml, and stirred gently for 18 hours at 4°C. Next, centrifuge at 12000g, collect the supernatant, and filter with a 0.2 μM filter. Thirdly, by molecular sieve chromatography, proteins with different molecular weights were divided into 30 groups, and the mixed protein fractions were named PC01-PC30 (Fig. 2). A 10/300GL Superdex 200 chromatography column was used for molecular screening, the loading volume was 1ml, the flow rate was set to 0.5mL/min, and the collection was 1ml/tube. The obtained protein fractions were analyzed by sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) electrophoresis, and the concentration of each protein fraction was determined with a bicinchoninic acid (BCA) detection kit. The results are shown in Figure 2.

Example 3:

³ H-TdR incorporation assay to detect the proliferation of CD4+ T cells stimulated by different components

The peritoneal macrophages of BALB/c mice were taken as antigen presenting cells (APC). 1×10 ⁵ APC and 200 μL of complete RPMI1640 medium were added per well in a 96-well round bottom plate. It was co-cultured with the whole antigen of H. pylori strain B0 or PC01-PC30 of each protein component in a 37°C 5% CO ₂ incubator, the final concentration of each antigen was 50 μg/mL, and each group had 3 duplicate wells. Ten hours later, splenic CD4+ T lymphocytes were sorted by magnetic beads from H. pylori immunized/infected and H. pylori immunized/uninfected mice 4 weeks later. 1×10 ⁵ spleen CD4+ T lymphocytes were co-cultured with the above APCs for 96 hours. 1 μCi/well ^of3H -TdR was added during the last 18 hours. After that, the CPM value of each well was detected by a liquid scintillation counter and expressed as stimulation index (SI), and SI was defined as CPM of experimental group/CPM of negative control. The results are shown in Figures 3A and 3B.

Example 4:

Evaluation of the immune protection function of H.pylori whole bacteria, PC05 and PC17

1. Animal immunization and challenge experimental protocol

Experimental animals: BALB/c mice were female 6-8 weeks old.

Antigen: H.pylori inactivated whole bacteria, PC05, PC17, 100μg/only. Equal volume of PBS control.

Adjuvant: Freund's adjuvant. 100μl / only.

Immunization method: subcutaneous injection.

Immune volume: 200μl/only.

Immunization scheme: subcutaneous immunization 3 times (week 0, 2, 4). Complete Freund's adjuvant was used for the first time, incomplete Freund's adjuvant was used for the second time, and no adjuvant was added for the third time.

One week after the last immunization, 1.0×10 ⁹ CFU of Helicobacter pylori was administered by gavage, once a day for 4 consecutive days. The mice were sacrificed at the 4th week after the challenge, and the quantitative amount of Helicobacter pylori, pathological damage and CD4+ T cell response in the gastric tissue of the mice were detected, and the immune protection effect was analyzed.

2. Quantitative detection of Helicobacter pylori in gastric mucosa of mice.

Real-time quantitative PCR was used to detect the colonization of Helicobacter pylori in the stomach. Bacterial DNA was extracted with a bacterial genome extraction kit, and its colonization was detected based on Helicobacter pylori 16SrDNA. The results are shown in Figure 4A.

The sequence of Helicobacter pylori 16S rDNA is as follows:

Upstream primer: 5'-TTTGTTAGAGAAGATAATGACGGTATCTAAC-3', as shown in SEQ ID NO.4;

Downstream primer: 5'-CATAGGATTTCACACCTGACTGACTATC-3', as shown in SEQ ID NO.5.

The sequence of the fluorescent probe is as follows:

FAM-CGTGCCAGCAGCCGCGGT-TAMRA is shown in SEQ ID NO.6.

3. Mouse gastric mucosal inflammation score

A small amount of gastric tissue was taken longitudinally along the greater curvature of the stomach, fixed with formalin, embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin-eosin. Histopathological scoring was then performed under the microscope. The results are shown in Figure 4B and Figure 4C.

The scoring criteria were: 0, no significant lesions; 0.5, mild abnormalities, such as small inflammatory infiltrates or extensive mucosal metaplasia without inflammation; 1.0, a mild inflammatory cell infiltration, usually involving Gland base; 1.5, mild infiltration, plus mild epithelial hyperplasia or extensive mucinous cell metaplasia; 2.0, inflammatory cell invasion of the gland and/or submucosa; 2.5, inflammatory cell invasion of the gland, At the same time, there is mucinous cell metaplasia and/or mild epithelial hyperplasia in the submucosa; 3.0, extensive inflammation invades the glands and submucosa, often accompanied by moderate mucinous cell metaplasia and mild to moderate epithelial hyperplasia; 3.5, 3.0 The above inflammations are accompanied by obvious epithelial cell hyperplasia; 4.0. Intense inflammatory infiltration invading the mucous layer, the normal structure of the gland is destroyed, and usually accompanied by obvious epithelial hyperplasia and extensive mucous cell metaplasia; 4.5. Severe inflammation accompanied by Focal mucosal ulceration; 5.0. Inflammation of extensive mucosal and submucosal involvement, with destruction of glandular structures and ulceration.

4. Detection of CD4+ T cell response in mouse gastric mucosa

Mouse gastric tissue was dissected along the greater and lesser curvatures of the stomach and gently washed twice with sterile PBS to remove food debris. Then put into 10ml Hank's Balanced Salt Solution (HBSS, without Ca, My), containing 1mM dithiothreitol (DTT), 1mM ethylenediaminetetraacetic acid (EDTA), and 2% fetal calf serum (FCS), Incubate at 37°C for 45min. The resulting mixture was then passed through a sterile steel mesh to remove undigested tissue to obtain a single cell suspension. The digested single cell suspension was washed twice with sterile PBS. Then the total RNA was extracted by Trizol method, reverse transcribed into cDNA, and the expression of IFN-γ and IL-17A was detected by real-time quantitative PCR using SYBR Green incorporation method. The results are shown in Figure 5A.

The sequence of IFN-γ is as follows:

Upstream primer: 5'-GATCCTTTGGACCCTCTGACTT-3', as shown in SEQ ID NO.7;

Downstream primer: 5'-TGACTGTGCCGTGGCAGTAA-3' as shown in SEQ ID NO.8.

The sequence of IL-17A is as follows:

Upstream primer: 5'-CTCCAGAAGGCCCTCAGACTAC-3', as shown in SEQ ID NO.9;

Downstream primer: 5'-GGGTCTTCATTGCGGTGG-3', as shown in SEQ ID NO.10.

5. Detection of mouse splenocyte antigen-specific CD4+ T cell response.

The spleen lymphocytes of H.pylori-infected mice were isolated by grinding method. Then 1×10 ⁷ lymphocytes were co-cultured with 100 μg H.pylori strain B0 whole antigen, 5U/ml recombinant mouse interleukin-2 (rmIL-2), 3ml/well complete RPMI1640 medium, 12-well plate. After 5 days in a 37°C, 5% CO ₂ incubator, dead cells were removed by Ficoll gradient centrifugation. The living cells were passaged, added with 20U/ml rmIL-2, and cultured in complete RPMI1640, until the twelfth day to obtain H. pylori whole bacteria-specific lymphocytes. During this period, half of the medium was changed when necessary. The prepared specific lymphocytes 1×10 ⁵ and APC cells presenting the corresponding antigen 1×10 ⁵ were co-cultured for 5 hours in a 96-well round bottom plate, and 200 μL of complete RPMI1640 medium containing BD golgistop was added to each well. It was then stained with fluorescently labeled antibodies FITC-CD3, APC-CD4, PE-IFN-γ, PerCP-Cy5.5-IL-17A and analyzed by flow cytometry. The results are shown in Figure 5B and Figure 5C.

Example 5:

Analysis of protein components by high performance liquid chromatography-mass spectrometry

The PC05 protein fractions were resolved by high performance liquid chromatography-mass spectrometry (LC-MS/MS Analysis). The PC05 protein band was transferred from the SDS-PAGE gel to EP tubes and cleaved with protease, and the cleavage products were detected by a Maxis 4G UHR Q-TOF mass spectrometer. All recorded data were analyzed with Proteome Discoverer ^™ 1.4 Software (Thermo Fisher Scientific, USA) and compared with the ncbi.fasta database (ftp://ftp.ncbi.nih.gov/) to identify proteins Specific protein components of group PC05.

PC05 protein composition and amino acid sequence

PC05 includes 11 protein components (Table 2).

Table 2. Eleven protein components of PC05

P5inosine 5'-monophosphate dehydrogenase (IMPDH) inosine nucleotide dehydrogenase

MRILQRALTFEDVLMVPRKSSVLPKDVSLKSRLTKNISLNIPFISAAMDTVTEHKTAIAMARLGGIGIVHKNMDIQTQVKEITKVKKSESGVINDPIFIHAHRTLADAKVITDNYKISGVPVVDDKGLLIGILTNRDVRFETDLSKKVGDVMTKMPLVTARVGISLEEARDLMHKHKIEKLPIVDKDNVLKGLITIKDIQKRIEYPEANKDDFGRLRVGAAIGVGQLDRAEMLVKAGVDALVLDSAHGHSANILHTLEEIKKSLVVDVIVGNVVTKEATSDLISAGADAIKVGIGPGSICTTRIVAGVGMPQVSAIDNCVEVASKFDIPVIADGGIRYSGDVAKALALGASSVMIGSLLAGTEESPGDFMIYQGRQYKSYRGMGSIGAMTKGSSDRYFQEGVASEKLVPEGIEGRVPYRGKVSDMIFQLVGGVRSSMGYQGAKNILELYQNAEFVEITSAGLKESHVHGVDITKEAPNYYG

P10type II citrate synthase (CS II) type II citrate synthase

MSVTLVNNENNERYEFETIESTRGPKAVDFSKLFETTGFFSYDPGYSSTAGCQSKISYVNGKKGELYYRGHRIEDLVAKYKYVDVCKLLLTGELPKNQDESLEFELELRHRSFVHESLLNMFSAFPSNAHPMAKLSSGVSILSTLYSTHQNMHTEEDYQTMARRIVAKIPTLAAICYRNEVGAPIIYPDIARSYVENILFMLRGYPYSRLKHTTQGEVEITPLEVEAFDKILTLHADHSQNASSTTVRNVASTGVHPYAAISAGISALWGHLHGGANEKVLLQLEEIGDVKNVDKYIARVKDKNDNFKLMGFGHRVYKSYDPRAKILKGLKDELHQKGVKMDERLSEIAAKVEEIALKDEYFIERNLYPNVDFYSGTILRALKIPVRFFTPVFVIGRTVGWCAQLLEHVKSPQARITRPRQVYVGD

P11urease subunit beta(UreB) urease B subunit

MKKISRKEYASMYGPTTGDKVRLGDTDLIAEVEHDYTIYGEELKFGGGKTLREGMSQSNNPSKEELDLIITNALIVDYTGIYKADIGIKDGKIAGIGKGGNKDMQDGVKNNLSVGPATEALAGEGLIVTAGGIDTHIHFISPQQIPTAFASGVTTMIGGGTGPADGTNATTITPGRRNLKFMLRAAEEYSMNIGFLAKGNASNDASLADQIEAGAIGLKIHEDWGTTPSAINHALDVADKYDVQVAIHTDTLNEAGCVEDTMAAIAGRTMHTYHTEGAGGGHAPDIIKVAGEHNILPASTNPTIPFTVNTEAEHMDMLMVCHHLDKSIKEDVQFADSRIRPQTIAAEDTLHDMGIFSITSSDSQAMGRVGEVITRTWQTADKNKKEFGRLKEEKGDNDNFRIKRYLSKYTINPAIAHGISEYVGSVEVGKVADLVLWSPAFFGVKPNMIIKGGFIALSQMGDANASIPTPQPVYYREMFAHHGKAKYDANITFVSQAAYDKGIKEELGLERQVLPVKNCRNITKKDMQFNDTTAHIEVNSETYHVFVDGKEVTSKPANKVSLAQLFSIF

Example 6:

Cloning and expression of 11 proteins in PC05 fractions

H. pylori DNA was extracted with a bacterial genome extraction kit. The upstream and downstream primers were designed according to the gene sequences of 11 proteins, and the target genes were obtained by PCR amplification. Select pGEX-6P-1 as the vector plasmid to induce expression in engineering bacteria E.Coli. The GST-tagged protein of interest was isolated with GST-tagged protein purification media (Glutathione Sepharose 4Fast Flow). Then, the GST tag was excised with Precision Protease to obtain the target protein. The concentration was determined by BCA method.

Example 7:

Identification of CD4+ T lymphocyte predominant antigens in PC05 fractions

The spleen lymphocytes of the PC05 fraction immunized mice were isolated by grinding method. Then 1×10 ⁷ lymphocytes were co-cultured with 100 μg PC05 component antigen, 5 U/ml recombinant mouse interleukin-2 (rmIL-2), 3 ml/well complete RPMI1640 medium, 12-well plate was added. After 5 days in a 37°C, 5% CO ₂ incubator, dead cells were removed by Ficoll gradient centrifugation. The viable cells were passaged, added with 20U/ml rmIL-2, and cultured in complete RPMI1640, until the twelfth day to obtain H. pylori whole bacteria-specific or single-protein antigen-specific lymphocytes. During this period, half of the medium was changed when necessary. The prepared specific lymphocytes 1×10 ⁵ were co-cultured with 1×10 ⁵ APC cells that presented 11 antigens in PC05 in a 96-well round bottom plate for 5 hours, and 200 μL of complete RPMI1640 medium containing BDgolgistop was added to each well. It was then stained with fluorescently labeled antibodies FITC-CD3, APC-CD4, PE-IFN-γ, PerCP-Cy5.5-IL-17A and analyzed by flow cytometry. The results are shown in Figures 6A and 6B.

Example 8:

Evaluation of immunoprotective function of dominant antigens IMPDH, CS Ⅱ and UreB

1. Animal immunization and challenge experimental protocol

Experimental animals: BALB/c mice were female 6-8 weeks old.

Antigens: IMPDH, CS II and UreB, 100 μg/only. Equal volume of PBS control.

Adjuvant: Freund's adjuvant. 100μl / only.

Immunization method: subcutaneous injection.

Immune volume: 200μl/only.

Immunization scheme: subcutaneous immunization 3 times (week 0, 2, 4). Complete Freund's adjuvant was used for the first time, incomplete Freund's adjuvant was used for the second time, and no adjuvant was added for the third time.

One week after the last immunization, 1.0×10 ⁹ CFU of Helicobacter pylori was administered by gavage, once a day for 4 consecutive days. The mice were sacrificed at the 4th week after the challenge, and the quantitative amount of Helicobacter pylori, pathological damage and CD4+ T cell response in the gastric tissue of the mice were detected, and the immune protection effect was analyzed.

2. Quantitative detection of Helicobacter pylori in gastric mucosa of mice.

Real-time quantitative PCR was used to detect the colonization of Helicobacter pylori in the stomach. Bacterial DNA was extracted with a bacterial genome extraction kit, and its colonization was detected based on Helicobacter pylori 16SrDNA. The results are shown in Figure 6C.

The sequence of Helicobacter pylori 16S rDNA is as follows:

Upstream primer: 5'-TTTGTTAGAGAAGATAATGACGGTATCTAAC-3', as shown in SEQ ID NO.4;

Downstream primer: 5'-CATAGGATTTCACACCTGACTGACTATC-3', as shown in SEQ ID NO.5.

The sequence of the fluorescent probe is as follows:

FAM-CGTGCCAGCAGCCGCGGT-TAMRA is shown in SEQ ID NO.6.

3. Mouse gastric mucosal inflammation score

A small amount of gastric tissue was taken longitudinally along the greater curvature of the stomach, fixed with formalin, embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin-eosin. Histopathological scoring was then performed under the microscope. The results are shown in Figure 6D.

The scoring criteria were: 0, no significant lesions; 0.5, mild abnormalities, such as small inflammatory infiltrates or extensive mucosal metaplasia without inflammation; 1.0, a mild inflammatory cell infiltration, usually involving Gland base; 1.5, mild infiltration, plus mild epithelial hyperplasia or extensive mucinous cell metaplasia; 2.0, inflammatory cell invasion of the gland and/or submucosa; 2.5, inflammatory cell invasion of the gland, At the same time, there is mucinous cell metaplasia and/or mild epithelial hyperplasia in the submucosa; 3.0, extensive inflammation invades the glands and submucosa, often accompanied by moderate mucinous cell metaplasia and mild to moderate epithelial hyperplasia; 3.5, 3.0 The above inflammations are accompanied by obvious epithelial cell hyperplasia; 4.0. Intense inflammatory infiltration invading the mucosal layer, the normal structure of the gland is destroyed, and usually accompanied by obvious epithelial hyperplasia and extensive mucous cell metaplasia; 4.5. Severe inflammation accompanied by Focal mucosal ulceration; 5.0. Inflammation of extensive mucosal and submucosal involvement, with destruction of glandular structures and ulceration.

4. Detection of CD4+ T cell response in mouse gastric mucosa

Mouse gastric tissue was dissected along the greater and lesser curvatures of the stomach and gently washed twice with sterile PBS to remove food debris. Then put into 10ml of Hank's Balanced Salt Solution (HBSS, without Ca, My), containing 1mM dithiothreitol (DTT), 1mM ethylenediaminetetraacetic acid (EDTA), and 2% fetal calf serum (FCS), Incubate at 37°C for 45min. The resulting mixture was then passed through a sterile steel mesh to remove undigested tissue to obtain a single cell suspension. The digested single cell suspension was washed twice with sterile PBS. Then the total RNA was extracted by Trizol method, reverse transcribed into cDNA, and the expression of IFN-γ and IL-17A was detected by real-time quantitative PCR using SYBR Green incorporation method. The results are shown in Figure 7.

The sequence of IFN-γ is as follows:

Upstream primer: 5'-GATCCTTTGGACCCTCTGACTT-3', as shown in SEQ ID NO.7;

Downstream primer: 5'-TGACTGTGCCGTGGCAGTAA-3' as shown in SEQ ID NO.8.

The sequence of IL-17A is as follows:

Upstream primer: 5'-CTCCAGAAGGCCCTCAGACTAC-3', as shown in SEQ ID NO.9;

Downstream primer: 5'-GGGTCTTCATTGCGGTGG-3', as shown in SEQ ID NO.10.

Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should Various changes may be made in details without departing from the scope of the invention as defined by the claims.

SEQUENCE LISTING

<110> The Third Military Medical University of the Chinese People's Liberation Army

<120> Helicobacter pylori dominant antigen combination and screening method based on CD4+ T cell immunization

<130>

<160> 10

<170> PatentIn version 3.3

<210> 1

<211> 481

<212> PRT

<213> Artificial sequences

<220>

<223> inosine nucleotide dehydrogenase

<400> 1

Met Arg Ile Leu Gln Arg Ala Leu Thr Phe Glu Asp Val Leu Met Val

1 5 10 15

Pro Arg Lys Ser Ser Val Leu Pro Lys Asp Val Ser Leu Lys Ser Arg

20 25 30

Leu Thr Lys Asn Ile Ser Leu Asn Ile Pro Phe Ile Ser Ala Ala Met

35 40 45

Asp Thr Val Thr Glu His Lys Thr Ala Ile Ala Met Ala Arg Leu Gly

50 55 60

Gly Ile Gly Ile Val His Lys Asn Met Asp Ile Gln Thr Gln Val Lys

65 70 75 80

Glu Ile Thr Lys Val Lys Lys Ser Glu Ser Gly Val Ile Asn Asp Pro

85 90 95

Ile Phe Ile His Ala His Arg Thr Leu Ala Asp Ala Lys Val Ile Thr

100 105 110

Asp Asn Tyr Lys Ile Ser Gly Val Pro Val Val Asp Asp Lys Gly Leu

115 120 125

Leu Ile Gly Ile Leu Thr Asn Arg Asp Val Arg Phe Glu Thr Asp Leu

130 135 140

Ser Lys Lys Val Gly Asp Val Met Thr Lys Met Pro Leu Val Thr Ala

145 150 155 160

Arg Val Gly Ile Ser Leu Glu Glu Ala Arg Asp Leu Met His Lys His

165 170 175

Lys Ile Glu Lys Leu Pro Ile Val Asp Lys Asp Asn Val Leu Lys Gly

180 185 190

Leu Ile Thr Ile Lys Asp Ile Gln Lys Arg Ile Glu Tyr Pro Glu Ala

195 200 205

Asn Lys Asp Asp Phe Gly Arg Leu Arg Val Gly Ala Ala Ile Gly Val

210 215 220

Gly Gln Leu Asp Arg Ala Glu Met Leu Val Lys Ala Gly Val Asp Ala

225 230 235 240

Leu Val Leu Asp Ser Ala His Gly His Ser Ala Asn Ile Leu His Thr

245 250 255

Leu Glu Glu Ile Lys Lys Ser Leu Val Val Asp Val Ile Val Gly Asn

260 265 270

Val Val Thr Lys Glu Ala Thr Ser Asp Leu Ile Ser Ala Gly Ala Asp

275 280 285

Ala Ile Lys Val Gly Ile Gly Pro Gly Ser Ile Cys Thr Thr Arg Ile

290 295 300

Val Ala Gly Val Gly Met Pro Gln Val Ser Ala Ile Asp Asn Cys Val

305 310 315 320

Glu Val Ala Ser Lys Phe Asp Ile Pro Val Ile Ala Asp Gly Gly Ile

325 330 335

Arg Tyr Ser Gly Asp Val Ala Lys Ala Leu Ala Leu Gly Ala Ser Ser

340 345 350

Val Met Ile Gly Ser Leu Leu Ala Gly Thr Glu Glu Ser Pro Gly Asp

355 360 365

Phe Met Ile Tyr Gln Gly Arg Gln Tyr Lys Ser Tyr Arg Gly Met Gly

370 375 380

Ser Ile Gly Ala Met Thr Lys Gly Ser Ser Asp Arg Tyr Phe Gln Glu

385 390 395 400

Gly Val Ala Ser Glu Lys Leu Val Pro Glu Gly Ile Glu Gly Arg Val

405 410 415

Pro Tyr Arg Gly Lys Val Ser Asp Met Ile Phe Gln Leu Val Gly Gly

420 425 430

Val Arg Ser Ser Met Gly Tyr Gln Gly Ala Lys Asn Ile Leu Glu Leu

435 440 445

Tyr Gln Asn Ala Glu Phe Val Glu Ile Thr Ser Ala Gly Leu Lys Glu

450 455 460

Ser His Val His Gly Val Asp Ile Thr Lys Glu Ala Pro Asn Tyr Tyr

465 470 475 480

Gly

<210> 2

<211> 426

<212> PRT

<213> Artificial sequences

<220>

<223> Type II citrate synthase

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Met Ser Val Thr Leu Val Asn Asn Glu Asn Asn Glu Arg Tyr Glu Phe

1 5 10 15

Glu Thr Ile Glu Ser Thr Arg Gly Pro Lys Ala Val Asp Phe Ser Lys

20 25 30

Leu Phe Glu Thr Thr Gly Phe Phe Ser Tyr Asp Pro Gly Tyr Ser Ser

35 40 45

Thr Ala Gly Cys Gln Ser Lys Ile Ser Tyr Val Asn Gly Lys Lys Gly

50 55 60

Glu Leu Tyr Tyr Arg Gly His Arg Ile Glu Asp Leu Val Ala Lys Tyr

65 70 75 80

Lys Tyr Val Asp Val Cys Lys Leu Leu Leu Thr Gly Glu Leu Pro Lys

85 90 95

Asn Gln Asp Glu Ser Leu Glu Phe Glu Leu Glu Leu Arg His Arg Ser

100 105 110

Phe Val His Glu Ser Leu Leu Asn Met Phe Ser Ala Phe Pro Ser Asn

115 120 125

Ala His Pro Met Ala Lys Leu Ser Ser Gly Val Ser Ile Leu Ser Thr

130 135 140

Leu Tyr Ser Thr His Gln Asn Met His Thr Glu Glu Asp Tyr Gln Thr

145 150 155 160

Met Ala Arg Arg Ile Val Ala Lys Ile Pro Thr Leu Ala Ala Ile Cys

165 170 175

Tyr Arg Asn Glu Val Gly Ala Pro Ile Ile Tyr Pro Asp Ile Ala Arg

180 185 190

Ser Tyr Val Glu Asn Ile Leu Phe Met Leu Arg Gly Tyr Pro Tyr Ser

195 200 205

Arg Leu Lys His Thr Thr Gln Gly Glu Val Glu Ile Thr Pro Leu Glu

210 215 220

Val Glu Ala Phe Asp Lys Ile Leu Thr Leu His Ala Asp His Ser Gln

225 230 235 240

Asn Ala Ser Ser Thr Thr Val Arg Asn Val Ala Ser Thr Gly Val His

245 250 255

Pro Tyr Ala Ala Ile Ser Ala Gly Ile Ser Ala Leu Trp Gly His Leu

260 265 270

His Gly Gly Ala Asn Glu Lys Val Leu Leu Gln Leu Glu Glu Ile Gly

275 280 285

Asp Val Lys Asn Val Asp Lys Tyr Ile Ala Arg Val Lys Asp Lys Asn

290 295 300

Asp Asn Phe Lys Leu Met Gly Phe Gly His Arg Val Tyr Lys Ser Tyr

305 310 315 320

Asp Pro Arg Ala Lys Ile Leu Lys Gly Leu Lys Asp Glu Leu His Gln

325 330 335

Lys Gly Val Lys Met Asp Glu Arg Leu Ser Glu Ile Ala Ala Lys Val

340 345 350

Glu Glu Ile Ala Leu Lys Asp Glu Tyr Phe Ile Glu Arg Asn Leu Tyr

355 360 365

Pro Asn Val Asp Phe Tyr Ser Gly Thr Ile Leu Arg Ala Leu Lys Ile

370 375 380

Pro Val Arg Phe Phe Thr Pro Val Phe Val Ile Gly Arg Thr Val Gly

385 390 395 400

Trp Cys Ala Gln Leu Leu Glu His Val Lys Ser Pro Gln Ala Arg Ile

405 410 415

Thr Arg Pro Arg Gln Val Tyr Val Gly Asp

420 425

<210> 3

<211> 569

<212> PRT

<213> Artificial sequences

<220>

<223> Urease B subunit

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Met Lys Lys Ile Ser Arg Lys Glu Tyr Ala Ser Met Tyr Gly Pro Thr

1 5 10 15

Thr Gly Asp Lys Val Arg Leu Gly Asp Thr Asp Leu Ile Ala Glu Val

20 25 30

Glu His Asp Tyr Thr Ile Tyr Gly Glu Glu Leu Lys Phe Gly Gly Gly

35 40 45

Lys Thr Leu Arg Glu Gly Met Ser Gln Ser Asn Asn Pro Ser Lys Glu

50 55 60

Glu Leu Asp Leu Ile Ile Thr Asn Ala Leu Ile Val Asp Tyr Thr Gly

65 70 75 80

Ile Tyr Lys Ala Asp Ile Gly Ile Lys Asp Gly Lys Ile Ala Gly Ile

85 90 95

Gly Lys Gly Gly Asn Lys Asp Met Gln Asp Gly Val Lys Asn Asn Leu

100 105 110

Ser Val Gly Pro Ala Thr Glu Ala Leu Ala Gly Glu Gly Leu Ile Val

115 120 125

Thr Ala Gly Gly Ile Asp Thr His Ile His Phe Ile Ser Pro Gln Gln

130 135 140

Ile Pro Thr Ala Phe Ala Ser Gly Val Thr Thr Met Ile Gly Gly Gly

145 150 155 160

Thr Gly Pro Ala Asp Gly Thr Asn Ala Thr Thr Ile Thr Pro Gly Arg

165 170 175

Arg Asn Leu Lys Phe Met Leu Arg Ala Ala Glu Glu Tyr Ser Met Asn

180 185 190

Ile Gly Phe Leu Ala Lys Gly Asn Ala Ser Asn Asp Ala Ser Leu Ala

195 200 205

Asp Gln Ile Glu Ala Gly Ala Ile Gly Leu Lys Ile His Glu Asp Trp

210 215 220

Gly Thr Thr Pro Ser Ala Ile Asn His Ala Leu Asp Val Ala Asp Lys

225 230 235 240

Tyr Asp Val Gln Val Ala Ile His Thr Asp Thr Leu Asn Glu Ala Gly

245 250 255

Cys Val Glu Asp Thr Met Ala Ala Ile Ala Gly Arg Thr Met His Thr

260 265 270

Tyr His Thr Glu Gly Ala Gly Gly Gly His Ala Pro Asp Ile Ile Lys

275 280 285

Val Ala Gly Glu His Asn Ile Leu Pro Ala Ser Thr Asn Pro Thr Ile

290 295 300

Pro Phe Thr Val Asn Thr Glu Ala Glu His Met Asp Met Leu Met Val

305 310 315 320

Cys His His Leu Asp Lys Ser Ile Lys Glu Asp Val Gln Phe Ala Asp

325 330 335

Ser Arg Ile Arg Pro Gln Thr Ile Ala Ala Glu Asp Thr Leu His Asp

340 345 350

Met Gly Ile Phe Ser Ile Thr Ser Ser Asp Ser Gln Ala Met Gly Arg

355 360 365

Val Gly Glu Val Ile Thr Arg Thr Trp Gln Thr Ala Asp Lys Asn Lys

370 375 380

Lys Glu Phe Gly Arg Leu Lys Glu Glu Lys Gly Asp Asn Asp Asn Phe

385 390 395 400

Arg Ile Lys Arg Tyr Leu Ser Lys Tyr Thr Ile Asn Pro Ala Ile Ala

405 410 415

His Gly Ile Ser Glu Tyr Val Gly Ser Val Glu Val Gly Lys Val Ala

420 425 430

Asp Leu Val Leu Trp Ser Pro Ala Phe Phe Gly Val Lys Pro Asn Met

435 440 445

Ile Ile Lys Gly Gly Phe Ile Ala Leu Ser Gln Met Gly Asp Ala Asn

450 455 460

Ala Ser Ile Pro Thr Pro Gln Pro Val Tyr Tyr Arg Glu Met Phe Ala

465 470 475 480

His His Gly Lys Ala Lys Tyr Asp Ala Asn Ile Thr Phe Val Ser Gln

485 490 495

Ala Ala Tyr Asp Lys Gly Ile Lys Glu Glu Leu Gly Leu Glu Arg Gln

500 505 510

Val Leu Pro Val Lys Asn Cys Arg Asn Ile Thr Lys Lys Asp Met Gln

515 520 525

Phe Asn Asp Thr Thr Ala His Ile Glu Val Asn Ser Glu Thr Tyr His

530 535 540

Val Phe Val Asp Gly Lys Glu Val Thr Ser Lys Pro Ala Asn Lys Val

545 550 555 560

Ser Leu Ala Gln Leu Phe Ser Ile Phe

565

<210> 4

<211> 31

<212> DNA

<213> Artificial sequences

<220>

<223> Helicobacter pylori 16SrDNA upstream primer

<400> 4

tttgttagag aagataatga cggtatctaa c 31

<210> 5

<211> 28

<212> DNA

<213> Artificial sequences

<220>

<223> Helicobacter pylori 16SrDNA downstream primer

<400> 5

cataggattt cacacctgac tgactatc 28

<210> 6

<211> 18

<212> DNA

<213> Artificial sequences

<220>

<223> Fluorescent probes

<400> 6

cgtgccagca gccgcggt 18

<210> 7

<211> 22

<212> DNA

<213> Artificial sequences

<220>

<223> IFN-γ upstream primer

<400> 7

gatcctttgg accctctgac tt 22

<210> 8

<211> 20

<212> DNA

<213> Artificial sequences

<220>

<223> IFN-γ downstream primer

<400> 8

tgactgtgcc gtggcagtaa 20

<210> 9

<211> 22

<212> DNA

<213> Artificial sequences

<220>

<223> IL-17A upstream primer

<400> 9

ctccagaagg ccctcagact ac 22

<210> 10

<211> 18

<212> DNA

<213> Artificial sequences

<220>

<223> IL-17A downstream primer

<400> 10

gggtcttcat tgcggtgg 18

Claims (6)

1. the helicobacter pylori dominant antigen based on CD4+T cellular immunity combines, which is characterized in that include the following three types: secondary Huang Purine nucleosides acidohydrogenase, II type citrate synthase and urease B subunit；Its amino acid sequence respectively as SEQ ID NO.1, Shown in SEQ ID NO.2 and SEQ ID NO.3.

2. dominant antigen combination according to claim 1, which is characterized in that the mass ratio of three kinds of antigen is 1:1:1, vaccine Total antigenic content is 100 μ g.

3. dominant antigen combination of any of claims 1 or 2 is in the vaccine of preparation prevention or treatment helicobacter pylori infections Using.

4. application according to claim 3, which is characterized in that the vaccine is albumen or nucleic acid vaccine.

5. application according to claim 3, which is characterized in that the vaccine also includes medically acceptable immune assistant Agent.

6. application according to claim 5, which is characterized in that the immunologic adjuvant is Freund's adjuvant, aluminium adjuvant, CPG Any one or more of ODN 1826 or AddaVax.