CN113398261A

CN113398261A - Application of poly-sarcosine in preparing biological preparation for improving HBsAg positive mother infant hepatitis B vaccine response level

Info

Publication number: CN113398261A
Application number: CN202110808222.6A
Authority: CN
Inventors: 冯永亮; 武丽娜; 王素萍; 李雁笛; 扆琳珠; 晋聪; 陈文鑫; 王婷
Original assignee: Shanxi Medical University
Current assignee: Shanxi Medical University
Priority date: 2021-07-16
Filing date: 2021-07-16
Publication date: 2021-09-17

Abstract

The invention provides application of poly-sarcosine (PolyI: C) in preparation of a biological preparation for improving HBsAg positive mother infant hepatitis B vaccine response level, belonging to the technical field of biological preparations. Application of poly-sarcosine in preparing biological preparation for raising HBsAg positive mother baby hepatitis B vaccine response level. HBeAg of HBsAg positive mother inhibits TLR3 signal pathway, affects placenta immunity to reduce infant immunity level and results in no/or no/in hepatitis B vaccineA weak response. Polysarcosine activating TLR3 signaling pathway of trophoblast cells to increase IL-2 secretion and increase CD4 in PBMC⁺T cell proportion, Treg cell proportion reduction, improvement organism immunity level, rHBsAg stimulation can improve IL-6 expression quantity, marrow dendritic cell (mDC) proportion and reduce IL-10 expression, improve anti-HBs level. Therefore, the poly-sarcosine is beneficial to improving the response level of the organism to the hepatitis B vaccine.

Description

Application of poly-sarcosine in preparing biological preparation for improving HBsAg positive mother infant hepatitis B vaccine response level

Technical Field

The invention belongs to the technical field of biological preparations, and particularly relates to application of poly-inosinic acid (PolyI: C) in preparing a biological preparation for improving the hepatitis B vaccine response level of an HBsAg positive mother infant.

Background

Hepatitis B Virus (HBV) infection is a major public health problem worldwide, hepatitis B vaccine inoculation is an effective means for preventing HBV infection, the hepatitis B vaccine is brought into planned immunization of children in the last 90 th century in China, the carrying rate of hepatitis B virus surface antigen (HBsAg) of people is reduced by 3 percent, the obtained results attract attention of the world, but the HBsAg positive rate of China still reaches 6.1 percent at present, the hepatitis B vaccine is a popular area of HBV infection and more than 8000 ten thousand chronic HBV infectors exist. The HBsAg positive mother baby hepatitis B vaccine has high non/weak response rate (18.5% -40%), HBV infection is easy to occur, nearly 90% of perinatal HBV infected persons will develop chronic HBV infected persons, 15% -25% of chronic HBV infected babies die of HBV infection related diseases, HBV infection of HBsAg positive mother baby babies becomes a main cause of HBV chronic infected person accumulation in China, and health of HBsAg positive mother babies is seriously damaged. How to improve the success rate of the immune response of the hepatitis B vaccine of the population and reduce the HBV susceptibility is the key for preventing and controlling the HBV infection of infants of HBsAg positive mothers and adults and the occurrence of hepatitis B related diseases.

Different from common infants, HBsAg positive mother infants are under the HBV biological environment of the mother from inoculation to birth, the natural immunity and the specific immunity of the HBsAg positive mother infants are influenced by HBV to different degrees, the hepatitis B vaccine immune response effect of the HBsAg positive mother infants is further influenced, and the natural immunity possibly plays an important role in the hepatitis B vaccine immune response of the HBsAg positive mother infants. Toll-like receptors (TLRs) are important components of natural immunity and are bridges for communicating the natural immunity and adaptive immunity, and damage of TLRs can weaken the natural immunity and specific immunity and influence the immune effect of hepatitis B vaccines. Among all TLR, TLR3 is the only receptor which can specifically bind dsRNA and is closely related to HBV infection and clearance; TLR3 is mainly expressed in endosomes of immune cells such as dendritic cells, B cells, T cells and NK cells, and the interaction of TLR3 and ligand molecules is an important mechanism for recognizing HBV dsRNA and activating immune cells and an important way for activating immune response. TRIF is a unique ligand at the downstream of TLR3, a TLR3 signal can activate NF-kappa B through a TRIF/TRAF6 path, mediate the generation of chemotactic factors and proinflammatory cytokines and have a wide regulation effect on body immunity; IRF3 may also be activated via the TRIF/TRAF3 pathway to trigger IFN-induced gene expression. The TLR3 signal channel plays an important role in HBsAg positive mother infant hepatitis B vaccine immune response.

The placenta is an organ in which differentiation has already begun in the early embryonic development stage, and plays an important role in protecting the growth of the fetus and maintaining the long-term health of the offspring. Researchers found that TLRs were expressed in placental tissues, with the highest level of TLR3, suggesting that the placenta may exert its antiviral immune function primarily through the TLR3 signaling pathway. In previous researches, the applicant finds that compared with normal puerperae, the expression of TLR3 mRNA and protein in placenta of an HBsAg positive maternal plant is reduced by detecting placenta tissues of the HBsAg positive maternal plant after delivery, and suggests that HBV infection of the placenta of the HBsAg positive maternal plant can influence the placenta immune response by inhibiting a TLR3 signal channel and further influence the immune response of children, so that the children are easy to have no/weak response of hepatitis B vaccine.

Disclosure of Invention

In view of the above, the present invention aims to provide an application of polyinosinic acid in preparing a biological agent for increasing the response level of hepatitis B vaccine in an infant with an HBsAg positive mother.

The invention provides application of polyinosinic acid in preparing a biological preparation for improving HBsAg positive mother infant hepatitis B vaccine response level.

Preferably, the use of said polyinosinic acid in combination with an agent that activates the placental TLR3 signalling pathway in the preparation of a biological agent for increasing the level of HBsAg positive maternal infant hepatitis b vaccine response.

Preferably, the use of the polyinosinic acid combined with a cytokine for the preparation of a biological agent for increasing the level of HBsAg positive maternal infant hepatitis B vaccine response.

Preferably, the cytokine includes IL-2 and/or IL-6.

Preferably, the polyinosinic acid is combined with immune cells to prepare a biological preparation for improving the hepatitis B vaccine response level of an infant with an HBsAg positive mother.

Preferably, the immune cells comprise myeloid dendritic cells and/or CD4⁺T cells.

Preferably, the biological agent comprises a vaccine adjuvant;

the vaccine adjuvant comprises a hepatitis B vaccine adjuvant.

The invention provides a biological preparation for improving HBsAg positive mother and infant hepatitis B vaccine response level, which comprises polyinosinic acid and medically acceptable auxiliary materials.

Preferably, the kit further comprises an agent for activating the placental TLR3 signaling pathway, a cytokine, and/or an immune cell;

the cytokine comprises IL-2 and/or IL-6;

the immune cells comprise myeloid dendritic cells and/or CD4⁺T cells.

The invention provides application of polyinosinic acid in preparing a biological preparation for improving HBsAg positive mother infant hepatitis B vaccine response level. According to the invention, through population research and in vitro experiments, important signal molecule proteins of a TLR3 signal channel in placenta tissues are highly expressed, and the fact that the TLR3 signal channel is closely related to placenta immunity is shown. HBeAg of an HBsAg positive mother has a certain inhibiting effect on a placental TLR3 signal channel, and placental immunity is influenced, so that the infant has no/weak response to the hepatitis B vaccine. The invention shows that the poly-inosinic acid is utilized to activate the fetus through in vitro experimentsTLR3 signaling pathway in discotrophoblasts increases IL-2 secretion and increases CD4 in PBMCs⁺T cell ratio, reduce the proportion of Treg cells, improve the immune response of the infant. Whether HBV is exposed or not, after a TLR3 signal channel of a placental trophoblast cell is activated by poly-sarcosine, rHBsAg is used for stimulating, so that the level of IL-6 can be obviously increased compared with the level of IL-6 which is not activated; in the absence of HBV exposure, stimulation with rHBsAg after activation of the TLR3 signaling pathway in placental trophoblasts can significantly reduce IL-10 levels compared to when not activated; when HBV is exposed, the proportion of mDC can be increased compared with that when the mDC is not activated by activating a TLR3 signal channel of placental trophoblast cells and then stimulating the cells with rHBsAg; stimulating with rHBsAg after activating TLR3 signaling pathway of placental trophoblast cells regardless of HBV exposure, and generating anti-HBs at a higher level than when not activated; after no HBV exposure and activation of a TLR3 signal channel of placental trophoblast cells, the cells are stimulated by rHBsAg, and the anti-HBs production level is highest; TLR3 signaling pathway with HBV exposure and inactivated placental trophoblasts was stimulated with rHBsAg with minimal production of anti-HBs. This indicates that polyinosinic acid activates the TLR3 signal pathway, which is beneficial to improving the response level of HBsAg positive mother and infant to hepatitis B vaccine.

Detailed Description

The invention provides application of polyinosinic acid in preparing a biological preparation for improving HBsAg positive mother infant hepatitis B vaccine response.

In the invention, the poly-sarcosine is used as an activator of a TLR3 signal channel in the preparation of biological agents for improving HBsAg positive mother infant hepatitis B vaccine response. The human research shows that important signal molecule proteins (TLR3, TRIF, NF-kappa B and IRF3) of a TLR3 signal channel in placenta tissues are in a high expression state, and the TLR3 signal channel is closely related to placenta immunity; at the same time, HBsAg-positive mothers influence the immune level of their infants by inhibiting the placental TLR3 signaling pathway leading to a non/weak response of the infant hepatitis b vaccine. The invention uses HBV positive serum to stimulate placenta trophoblast cells in vitro, co-cultures the placenta trophoblast cells infected by HBV and PBMC, stimulates the stimulation by rHBsAg to simulate the condition of inoculating hepatitis B vaccine to HBsAg positive mother newborn, discusses the non/weak response of placenta TLR3 signal channel in HBsAg positive mother infant hepatitis B vaccineThe result shows that HBV infection inhibits a TLR3 signal channel of placental trophoblast cells, so that the response level of an organism to the hepatitis B vaccine is reduced. The invention utilizes poly-inosinic acid to activate the TLR3 signal channel of the placental trophoblast cells, can improve the expression of IL-2 in PBMC and increase CD4⁺The proportion of T cells is reduced, and the proportion of Treg cells is reduced; the poly-sarcosine can improve the IL-6 level, the mDC ratio, the anti-HBs level and the IL-10 level by stimulating rHBsAg after activating a TLR3 signal channel of placental trophoblast cells, thereby being beneficial to improving the immune response of an infant body and the response level of hepatitis B vaccine.

In the present invention, the use of said polyinosinic acid, preferably in combination with other agents that activate the placental TLR3 signalling pathway, for the preparation of a biological agent and/or biologic for increasing the hepatitis b vaccine response in an infant with an HBsAg positive mother.

The invention further researches the effect of the placenta TLR3 signal channel in hepatitis B vaccine non/weak response, and the research result of people finds that the protein expression of placenta TLR3 and NF-kappa B in the non/weak response group is obviously reduced compared with the strong response group; the Bayesian network model shows that placenta NF-kB, placenta IRF3 and infant IL-6 are directly related to hepatitis B vaccine non/weak response, and placenta TLR3 is related to hepatitis B vaccine non/weak response through placenta NF-kB and infant IL-6. It can be seen that HBsAg positive mothers participate in a no/weak response to hepatitis b vaccine by inhibiting the placental TLR3 signaling pathway affecting the immune level of their infants. Based on the factor, the invention also provides the application of the polyinosinic acid preferably combined with NF-kB and infant IL-6 in preparing a biological preparation for improving the hepatitis B vaccine response of an HBsAg positive mother infant.

In the present invention, the use of said polyinosinic acid, preferably in combination with a cytokine, for the preparation of a biological agent for increasing the hepatitis b vaccine response in an infant with an HBsAg-positive mother. The cytokine preferably includes IL-2 and IL-6. Experiments show that activation of a TLR3 signal channel of placental trophoblast cells can significantly improve the expression of IL-2 in PBMCs; after stimulation by adding rHBsAg, the expression level of TLR3 signaling pathway activated group IL-6 of placental trophoblast cells is obviously higher than that of an inactivated group regardless of the existence of HBV.

In the invention, the polyinosinic acid is combined with immune cells and used for preparing a biological preparation for improving the hepatitis B vaccine response level of an infant with an HBsAg positive mother. The immune cells preferably comprise myeloid dendritic cells and/or CD4⁺T cells. The experimental results show that activation of the TLR3 signaling pathway in placental trophoblasts in the presence of HBV exposure results in stimulation of mDC and CD4 by rHBsAg compared to control (inactivated group)⁺The T ratio becomes higher.

The invention provides a biological preparation for improving HBsAg positive mother and infant hepatitis B vaccine response, which comprises polyinosinic acid and medically acceptable auxiliary materials. The active ingredients of the biological agent preferably also include other agents, cytokines and/or immune cells that can activate the placental TLR3 signaling pathway; the cytokine comprises IL-2 and/or IL-6; the immune cells comprise myeloid dendritic cells and/or CD4⁺T cells.

The formulation of the biological agent is not particularly limited in the present invention, and may be those known in the art. The invention does not specifically limit the types of the auxiliary materials, and can select the conventional auxiliary materials according to different formulations of the biological preparation. The method for preparing the biological agent is not particularly limited in the present invention, and a method for preparing a biological agent well known in the art may be used.

The application of the polyinosinic acid provided by the invention in preparing the biological preparation for reducing the HBsAg positive maternal infant hepatitis B vaccine No/No response is explained in detail in the following with the examples, but the poly-sarcosine is not to be construed as limiting the scope of the invention.

Example 1

1. Population study

HBsAg positive mothers and their neonate 399 pairs were collected continuously as subjects, and all neonates completed standard passive-active immunization prophylaxis for hepatitis b and were followed up to 1 year of age ± 1 month. Before delivery, 3mL of non-anticoagulation blood of the elbow vein of the pregnant woman is collected, and placenta tissues are collected within 30 minutes after delivery; 3mL of femoral vein non-anticoagulated blood is collected before hepatitis B immunoglobulin and hepatitis B vaccine are injected in 24 hours of birth of a newborn and after 6 +/-1 months (1 year +/-1 month of an infant) after standard passive-active immunization of hepatitis B is completed. The following studies were performed:

(1) detecting the HBV DNA content of serum of HBsAg positive mothers and babies thereof by using fluorescent Quantitative polymerase Chain Reaction (FQ-PCR), and specifically detecting by using a hepatitis B virus nucleic acid Quantitative detection kit (purchased from Daan Gen. Gen.GmbH of Zhongshan university);

(2) an Electrochemiluminescence method (ECLIA) for detecting HBV serum markers of HBsAg positive mothers and infants thereof comprises the following steps: HBsAg, HBeAg, anti-HBs, HBeAb and anti-HBc, and specifically adopts HBV marker detection kit (purchased from Roche diagnostics GmbH, Germany) for detection;

(3) immunohistochemistry EnVision two-step method for detecting important signal molecule proteins of placenta TLR3 signal path (including TLR3, TRIF, NF-kappa B and IRF3), wherein rabbit anti-human TLR3 polyclonal antibody is purchased from Abcam America (ab62556), rabbit anti-human TRIF polyclonal antibody is purchased from Abcam America (ab13810), rabbit anti-human NF-kappa B polyclonal antibody is purchased from Abcam America (ab86299), and mouse anti-human IRF3 monoclonal antibody is purchased from Cell Signaling America (D9J 5Q); a positive result for each protein of interest was the appearance of a brownish yellow particle at the predicted subcellular localization; and dividing the result into 4 grades, namely 0 (0), 1 (1-2), 2 (3-4) and 3 (more than 4) according to the comprehensive integral of the dyeing intensity (weak: 1, medium: 2, strong: 3) and the positive cell percentage (negative: 0; less than 25%: 1; 25-50%: 2; more than 50%: 3), and judging the result as positive when the comprehensive integral is more than or equal to 1.

(4) The Luminex multi-factor detection analyzes the Th1/Th2 type cytokines (including IL-2, IL-6, IL-10, TNF-alpha and IFN-gamma) of the HBsAg positive mother baby, and specifically adopts a Th1/Th2 type cytokine multi-factor detection kit (purchased from eBioscience USA) for detection.

Results

1. The invention collects 399 pairs of HBsAg positive mothers and newborns thereof, and 295 infants finish follow-up visits with 18.31 percent of no/weak response rate (54/295), wherein 15 cases of non-responders and 39 cases of weak responders.

2. Expression of signal molecule protein important for placenta TLR3 signal channel

The expression analysis of the TLR3 signal channel important signal molecule protein is carried out on 50 non/weak responders and 50 corresponding strong responders which are randomly selected in total 100 subjects in 50 subjects out of 54 non/weak responder groups of no/weak responder groups of 50 subjects in the test item of the experiment, and the result shows that the expression positive rate of the TLR3 signal channel important signal molecule in the placenta is high, and the expression positive rates of TLR3, TRIF, NF-kappa B and IRF3 are 100%, 99%, 95% and 91% respectively. The expression level of TLR3 is positively correlated with the expression levels of TRIF (r is 0.22 and P is 0.031) and NF-kappa B (r is 0.38 and P is less than 0.001).

Effect of HBsAg-positive maternal HBV serological markers on placental TLR3 signaling pathway

Placental TLR3 protein expression was negatively correlated with maternal HBV DNA content (r ═ 0.29, P ═ 0.003), maternal HBeAg status (r ═ 0.28, P ═ 0.005), and maternal HBV DNA + HBeAg dication status (r ═ 0.24, P ═ 0.018). HBsAg positive mother HBV DNA load below 200IU/ml, 200-2X 10⁵IU/ml and 2X 10⁵The ratios of placental TLR3 high expression (score ≥ 3) were 53.97%, 40.00% and 22.22% respectively (Z ═ 7.29, P ═ 0.026) for more IU/ml, placental TLR3 high expression (score ≥ 3) for more than mother HBeAg positive and negative were 29.55% and 55.36% respectively (Z ═ 2.45, P ═ 0.014), and the ratios of placental TLR3 high expression (score ≥ 3) for both mother HBeAg and HBV DNA positive and non-double positive were 26.47% and 53.03% respectively (Z ═ 2.17, P ═ 0.030), suggesting that when HBsAg positive HBV viral load is high, maternal HBeAg positive, or both mother HBeAg and HBV DNA positive, the expression level of placental TLR3 protein is low.

4. Effect of placental TLR3 signaling pathway on immunization of HBsAg-positive mothers and infants

The expression of IL-10(1.93pg/ml vs 1.48pg/ml) and IFN-gamma (0.93pg/ml vs 0.71pg/ml) of the infant with high placenta TLR3 protein expression is higher than that of the infant with low placenta TLR3 protein expression, the expression of IL-6(30.78pg/ml vs 26.81pg/ml) and IFN-gamma (0.88pg/ml vs 0.66pg/ml) of the infant with high placenta TRIF protein expression is higher than that of the infant with low placenta TLR3 protein expression, the expression level of IL-6(40.28pg/ml vs 15.13pg/ml), IL-10(1.93pg/ml vs 1.38pg/ml) and TNF-alpha (2.51/ml vs 2.12/ml) of the infant with high placenta NF-kappa B protein expression is higher than that of the infant with low placenta NF-kappa B protein expression, and the expression level of IL-6 (82/ml) of the infant with high placenta TLR3 protein expression (3663.82 pg/ml) is higher than that of the infant IRB protein expression group, The level of IL-10(2.05pg/ml vs 1.27pg/ml) and IFN-gamma (1.31pg/ml vs 0.66pg/ml) was higher than that in the IRF3 protein low expression group. The expression level of the placenta NF-kB protein is positively correlated with the secretion level of the IL-6(r is 0.25, P is 0.020) and TNF-alpha (r is 0.24, P is 0.026) of the infant.

5. Function of placenta TLR3 signal channel in HBsAg positive mother infant hepatitis B vaccine non/weak response

The proportion of high expression (score is more than or equal to 3 points) of placenta TLR3 (28% vs 60%) and NF-kB (12% vs 30%) in a hepatitis B vaccine non/weak response group is obviously lower than that in a strong response group (Z ═ 2.46, P ═ 0.014; Z ═ 2.61, P ═ 0.009); the proportion of the high protein expression (score is more than or equal to 3 points) of placenta TRIF (24% vs 36%) and IRF3 (18% vs 36%) in the hepatitis B vaccine non/weak response group is lower than that in the strong response group (Z ═ 1.64, P ═ 0.101; Z ═ 1.94, P ═ 0.052).

The Bayesian network model shows that the placenta NF-kB, the placenta IRF3 and the infant IL-6 are directly linked with the hepatitis B vaccine no/weak response, and when the levels of the placenta NF-kB, the placenta IRF3 and the infant IL-6 are all lower than the median, the hepatitis B vaccine no/weak response probability is obviously increased when the levels of the placenta NF-kB, the placenta IRF3 and the infant IL-6 are all higher than the median (63.27% vs 10.00%). Placental TLR3 was associated with no/weak response to hepatitis B vaccine either by placental NF-. kappa.B or by placental NF-. kappa.B and infant IL-6.

When the placental TLR3 level is reduced, the probability of the reduced placental NF-kappa B expression is 69.15%, and the probability of hepatitis B vaccine no/weak response when the NF-kappa B is reduced is 47.62%; the probability of a decrease in infant IL-6 expression with decreased placental NF- κ B expression was 64.71%. When the mother HBeAg is positive, the expression of the placenta TLR3 is easy to reduce, the probability is 91.18%, and the mother HBeAg influences the expression of placenta TLR3, so that the mother HBeAg is associated with the hepatitis B vaccine non/weak response.

Example 2

In vitro experiments

(1) Normal human trophoblast cell strain (HTR8-Svneo cell) is cultured in vitro, and the effect of special intrauterine environment of HBsAg positive mother on placenta trophoblast cell is simulated by using HBV positive serum stimulation. HTR8-Svneo intracellular HBV DNA was detected by FQ-PCR (as described in example 1, lg conversion was performed when the result of intracellular HBV DNA (IU/ml) detection was statistically processed); the mRNA content of important signal molecules of a TLR3 signal channel after HTR8-SVneo cells are infected with HBV is detected by Fluorescence Quantitative Reverse Transcription polymerase Chain Reaction (RT-qPCR). The primers used were as follows:

TLR3 upstream primer TGATGCTCCGAAGGGTGG (SEQ ID NO: 1);

downstream primer GCCGTGCTAAGTTGTTATGCTG (SEQ ID NO: 2);

TRIF upstream primer GGCCCATCACTTCCTAGCG (SEQ ID NO: 3);

downstream primer GAGAGATCCTGGCCTCAGTTT (SEQ ID NO: 4);

NF-. kappa.B upstream primer AGGCTCCTGTGCGTGTCTCC (SEQ ID NO: 5);

downstream primer TCGTCTGTATCTGGCAGGTACTGG (SEQ ID NO: 6);

IRF3 upstream primer GCAGGAGGATTTCGGAATCTTC (SEQ ID NO: 7);

downstream primer GGAAATTCCTCTTCCAGGTTGG (SEQ ID NO: 8);

beta-actin upstream primer TGGCACCCAGCACAATGAA (SEQ ID NO: 9);

downstream primer CTAAGTCATAGTCCGCCTAGAAGCA (SEQ ID NO: 10);

each primer is synthesized by the Shanghai biological engineering company Limited.

The RT-qPCR reaction system is prepared according to a GoTaq qPCR MasterMix kit, and each reaction system accounts for 20 mu l, and specifically comprises the following components:

the RT-qPCR reaction conditions were as follows:

results determination adopted 2^-ΔΔCtIndicates the relative expression amount of mRNA in the sample.

Flow Cytometry (FCM) is adopted to detect the content of important signal molecule protein of a TLR3 signal channel and downstream cytokine of the TLR3 signal channel after HTR8-SVneo cells are infected with HBV.

(2) HBV infected HTR8-SVneo cells were co-cultured with PBMC to simulate the effect of HBsAg positive mother's special intrauterine environment on fetal immune function: FCM detects the content of cell factors and the change of PBMC subgroup in a co-culture system; the reagents used were as follows:

(3) after HBV infects HTR8-SVneo cells, co-culture with PBMC is stimulated by rHBsAg, and HBsAg positive mother infants are simulated to be inoculated with hepatitis B vaccine: ECLIA measures anti-HBs levels; FCM detects cytokine levels and PBMC subpopulation changes.

As a result:

HBV infection of human placental trophoblasts in vitro and Effect on the TLR3 Signaling pathway of trophoblasts

HTR8-SVneo cells were divided into HBV DNA low load group (10)⁵IU/ml), HBV DNA middle load group (10)⁶IU/ml), HBV DNA high load group (10)⁷IU/ml), with serum of different HBV DNA loads, HBV DNA was detected in cells at 24h, 48h and 72h in each group, and with the increase of virus stimulation time, the HBV DNA content in HTR8-SVneo cells in the HBV DNA high load group tended to increase (24h, 48h and 72h, respectively: 4.51 +/-2.04, 6.25 +/-1.32 and 7.27 +/-1.17); the increase of HBV DNA content in HTR8-SVneo cells with the increase of virus concentration appeared in each group at 48h and 72h (5.04 + -1.66, 5.57 + -2.20, 6.25 + -1.32 for the low HBV load group, 5.57 + -2.20 for the medium HBV load group, and 4.42 + -1.58, 4.89 + -1.64, and 7.27 + -1.17 for 72h, respectively); the HBV DNA low-load group has the lowest HBV DNA content (3.55 +/-2.13) in HTR8-SVneo cells at 24 h; the HBV DNA high-load group 72h has the highest HBV DNA content (7.27 +/-1.17) in HTR8-SVneo cells.

mRNA levels: although the mRNA content of the important signal molecule of TLR3 signal channel of each group tends to increase along with the prolonging of the action time of HBV, the mRNA content of the important signal molecule of TLR3 signal channel of HBV exposure group is slightly lower than that of the control group at 72 h: the HBV DNA low, medium and high load group and the control group have TLR3 mRNA of 2.10 +/-0.21, 1.74 +/-1.25, 2.69 +/-2.47 and 3.61 +/-3.82 respectively, and TRIF mRNA of 6.06 +/-3.75, 4.77 +/-3.17, 8.46 +/-10.06 and 9.40 +/-8.87 respectively; while NF-kappa B mRNA was 24.13 + -9.54, 13.28 + -3.06, 16.72 + -15.65 and 22.24 + -11.17 respectively, IRF3 mRNA was 25.87 + -17.89, 21.72 + -14.19, 21.93 + -14.53 and 24.52 + -10.82 respectively, and NF-kappa B and IRF3 were lower in the medium and high viral load exposed groups than in the control group. As can be seen, HTR8-SVneo cell TLR3 signaling pathway molecules are in a certain inhibition state after HBV infection.

Protein level: the proportion (%) of cells with positive expression of TLR3, NF-kappa B, IRF3 and pIRF3 proteins in a TLR3 signaling pathway of HTR8-SVneo cells of each HBV exposure group is lower than that of a control group at 48 h: the ratios of the cells with positive expression of the TLR3 protein in HBV DNA low, medium and high load groups and a control group are 91.00 +/-8.00, 91.67 +/-6.51, 91.33 +/-7.51 and 96.67 +/-1.53 respectively, the NF-kappa B is 45.33 +/-15.50, 66.00 +/-13.00, 56.00 +/-21.00 and 70.67 +/-3.51, the IRF3 is 89.00 +/-10.00, 94.67 +/-4.51, 86.00 +/-13.00 and 98.00 +/-1.00, the pIRF3 is 4.33 +/-3.51, 4.00 +/-3.00, 2.44 +/-1.50 and 6.67 +/-5.51; the proportion (%) of cells positively expressing TLR3 and TRIF protein in the HBV DNA high load group at 72h was still lower than that in the control group (TLR 3: 96.92 + -2.88 VS 98.92 + -0.88; TRIF: 95.67 + -4.04 VS 97.33 + -2.52). In general, the proportion of the TLR3 protein expression positive cells in the different-load HBV groups was significantly reduced compared to the control group at three time points.

The expression of IL-2 in each group of HBV infected HTR8-SVneo cells shows a descending trend in each cytokine downstream of TLR3 signal channel at different time points, and the expression of IL-2 in each HBV exposed group is lower than that in a control group. The IL-2 levels at 24h in the low, medium and high HBV DNA load and control groups were 2.66. + -. 0.15pg/ml, 2.76. + -. 0.12pg/ml, 2.73. + -. 0.16pg/ml and 2.83. + -. 0.12pg/ml, at 48h 2.60. + -. 0.18pg/ml, 2.58. + -. 0.15pg/ml, 2.59. + -. 0.18pg/ml and 2.67. + -. 0.21pg/ml, at 72h 2.63. + -. 0.47pg/ml, 2.61. + -. 0.65pg/ml, 2.60. + -. 0.08pg/ml and 2.65. + -. 0.26 pg/ml.

The TNF expression level and the IFN expression level of the HBV DNA low-load group, the HBV DNA medium-load group and the control group at 24h and 48h are respectively detected, and the result shows that compared with the control group, the TNF and the IFN expression in the HBV DNA low-load group, the TNF and the IFN expression in the HBV DNA medium-load group are not statistically different.

Effect of activation of the trophoblast TLR3 Signaling pathway on Peripheral Blood Mononuclear Cell (PBMC) immune function following HBV infection

After HBV infection, a TLR3 signaling pathway of HTR8-SVneo cells is activated and is cultured with PBMC, and the result shows that the IL-2 level is remarkably improved at 6h (6 h: 2.97 +/-0.23 pg/ml of an activation group and 2.74 +/-0.21 pg/ml of a control group). The expression of IL-4 in the activated group is reduced compared with that in the inactivated group at 6h and 24h (6 h: 2.85 + -0.17 pg/ml in the activated group, 3.12 + -0.20 pg/ml in the control group; 3.05 + -0.07 pg/ml in the activated group, 3.12 + -0.12 pg/ml in the control group at 24 h). The expression amounts of IL-6, TNF and IFN of the TLR3 activated group are slightly higher than those of a control group (IL-6: 6h activation group 40.52 +/-5.24 pg/ml, a control group 39.36 +/-3.10 pg/ml, a 12h activation group 47.09 +/-3.26 pg/ml, a control group 43.98 +/-3.29 pg/ml, a 24h activation group 50.60 +/-2.14 pg/ml, a control group 49.94 +/-1.16 pg/ml, TNF of a 6h activation group 7.17 +/-1.59 pg/ml, a control group 6.13 +/-0.60 pg/ml, a 12h activation group 4.49 +/-0.21 pg/ml, a control group 4.23 +/-0.27 pg/ml, a 24h activation group 3.41 +/-0.16 pg/ml, a control group 3.11 +/-0.25 pg/ml, a control group 3.23 +/-0.27 pg/ml, a control group 3.34 +/-0.34 g/ml, a control group 3.34 +/-0.23 g/ml, a control group 3.27 g/ml, control group 3.38. + -. 0.32 pg/ml).

Activation of TLR3 signaling pathway of HTR8-SVneo cells after HBV infection, and CD4 after co-culture with PBMC⁺The proportion (%) of T cells is remarkably increased, and the proportion (%) of Treg cells is remarkably decreased(CD4⁺T cell: the activating group is 20.09 +/-0.45, the control group is 18.67 +/-0.54, t is 3.48, and P is 0.025; treg cells: 0.19 ± 0.09 for the activated group, 0.53 ± 0.21 for the control group, t ═ 2.83, and P ═ 0.048). Activation of group B cells, plasma cells, mDCs, CD8⁺The proportion (%) of T cells was slightly higher than that of the control group (B cells: activated group 2.41. + -. 0.34, control group 2.05. + -. 0.28; plasma cells: activated group 1.93. + -. 0.28, control group 1.61. + -. 0.08; mDC: activated group 1.50. + -. 0.23, control group 1.33. + -. 0.50; CD 8)⁺T cell: activation group 36.21 ± 1.37, control group 35.80 ± 2.04).

3. Effect of activation of trophoblast TLR3 signaling pathway on immune response of hepatitis B vaccine

Defining trophoblast cells that did not receive HBV and PolyI: C intervention as a blank control; trophoblast cells that received only poly i: C intervention were defined as poly i: C group; trophoblast cells receiving dual intervention of HBV and PolyI: C were defined as HBV + PolyI: C group; trophoblast cells that only receive HBV intervention are defined as the HBV group.

The 4 groups were co-cultured with PBMC and stimulated with rHBsAg, and the anti-HBs levels produced by the blank control group, PolyI: C group, HBV + PolyI: C group and HBV group were, at 6 h: 3.243 + -0.974 mIU/mL, 3.963 + -0.521 mIU/mL, 2.243 + -0.565 mIU/mL and 1.057 + -1.198 mIU/mL; at 12h, the following are respectively: 3.073 +/-0.439 mIU/mL, 3.753 +/-0.767 mIU/mL, 0.977 +/-0.830 mIU/mL and 0.597 +/-1.394 mIU/mL; at 24h, the following are respectively: 3.043 ± 0.211mIU/mL, 3.337 ± 1.285mIU/mL, 1.423 ± 0.190mIU/mL, 0.503 ± 1.357mIU/mL, and the difference in anti-HBs production between the groups was statistically significant (F ═ 9.56, P ═ 0.005). The results of the pairwise comparisons show: the anti-HBs levels in group C were significantly higher than in group HBV + PolyI C and HBV (P <0.05), and the anti-HBs levels in group HBV were significantly lower than in the blank control and PolyI C (P <0.05), suggesting that in the absence of HBV exposure, the levels of anti-HBs production following stimulation with rHBsAg were higher than in the presence of HBV exposure; activating a TLR3 signaling pathway of HTR8-SVneo cells, wherein the level of anti-HBs produced after stimulation by rHBsAg is higher than that produced when the TLR3 signaling pathway of the HTR8-SVneo cells is not activated; the HBV exposure is not generated, the TLR3 signal path of HTR8-SVneo cells is activated, and the anti-HBs generation level is highest after the stimulation of rHBsAg; with HBV exposure and no activation of the TLR3 signaling pathway in HTR8-SVneo cells, anti-HBs production was minimal after stimulation with rHBsAg.

After the TLR3 signaling pathway of trophoblast cells was activated and stimulation was performed with rHBsAg, the differences in the expression levels of IL-6(F ═ 10.13, P ═ 0.004) and IL-10(F ═ 4.95, P ═ 0.005) were statistically significant in each group. The level of IL-6 expression at activation of the TLR3 signaling pathway in trophoblasts (PolyI: group C: 48.47. + -. 3.74pg/ml and HBV + PolyI: group C: 47.87. + -. 2.46pg/ml) was higher than that at non-activation (blank group: 43.14. + -. 3.09pg/ml and HBV group: 42.38. + -. 1.54pg/ml) with or without HBV exposure; in the absence of HBV, the level of IL-10 (4.51 + -1.03 pg/ml) when activating TLR3 signaling pathway in trophoblast cells was lower than that when not activated (6.16 + -0.10 pg/ml). TNF and IFN did not differ significantly when the TLR3 signaling pathway of trophoblasts was activated compared to the inactivated group, with or without HBV exposure.

The mDC ratio of the activated group without HBV action (polyI: C group) is significantly higher than that of the HBV group and HBV + polyI: C group (F ═ 4.64, P ═ 0.037) after the stimulation by activating TLR3 signaling pathway added with rHBsAg, while the mDC ratio of the activated group with HBV action (HBV + polyI: C group) is slightly higher than that of the inactivated HBV group (polyI: C group: 2.61. + -. 1.03, HBV + polyI: C group: 1.09. + -. 0.35; blank group: 1.75. + -. 0.16, HBV group: 1.07. + -. 0.37).

The above examples show that the invention starts from the TLR3 signal channel closely related to placental immunity, integrates various factors of mothers, placentas and babies, and comprehensively discusses the function of the placental TLR3 signal channel in HBsAg positive mothers and babies and hepatitis B vaccine non/weak response. The result shows that HBeAg of the HBsAg positive mother has certain inhibition effect on placenta TLR3 signal channel, and then placenta immunity is influenced. In vitro, by stimulating placental trophoblasts with HBV positive serum, culturing trophoblasts infected by HBV with PBMC together, and stimulating with rHBsAg, the conditions of HBsAg positive mother newborn infant inoculated with hepatitis B vaccine are simulated, and the possible action mechanism of HBsAg positive mother infant hepatitis B vaccine in no/weak response is preliminarily clarified. The invention utilizes Poly I: C to activate a placenta TLR3 signal channel, improves the immune cell and cytokine level in trophoblast cells, thereby improving the immune response level of HBsAg positive mother infant hepatitis B vaccine and improving the immunity of infants to hepatitis B virus.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

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Claims

1. Application of poly-sarcosine in preparing biological preparation for raising HBsAg positive mother baby hepatitis B vaccine response level.

2. The use of claim 1, wherein the polyinosinic acid is used in combination with an agent that activates the placental TLR3 signaling pathway, in the preparation of a biological agent that increases the level of HBsAg positive maternal infant hepatitis B vaccine response.

3. The use of claim 1, wherein the polyinosinic acid is used in combination with a cytokine for the preparation of a biological agent for increasing the level of HBsAg positive maternal infant hepatitis B vaccine response.

4. The use of claim 3, wherein the cytokine comprises IL-2 and/or IL-6.

5. The use of claim 1, wherein the polyinosinic acid is used in combination with immune cells in the preparation of a biological agent for increasing the level of HBsAg positive maternal infant hepatitis B vaccine response.

6. The use according to claim 5, wherein said immune cells comprise myeloid dendritic cells and/or CD4⁺T cells.

7. The use of any one of claims 1 to 6, wherein the biological agent comprises a vaccine adjuvant;

the vaccine adjuvant comprises a hepatitis B vaccine adjuvant.

8. A biological agent for increasing HBsAg positive mother infant hepatitis B vaccine response level, which comprises polyinosinic acid and medically acceptable auxiliary materials.

9. The biologic agent of claim 8, further comprising an agent, cytokine, and/or immune cell that activates the placental TLR3 signaling pathway.

10. The biologic agent of claim 9, wherein said cytokine comprises IL-2, and/or IL-6;

the immune cell comprises CD4⁺T cells and/or myeloid dendritic cells.