Biological preparation of bifidobacterium adolescentis for enhancing immune response of hepatitis b vaccine

Abstract

This invention discloses a biological agent for enhancing the immune response to hepatitis B vaccine using *Bifidobacterium adolescentis*, comprising: a preparation of *Bifidobacterium adolescentis* and a hepatitis B vaccine. The *Bifidobacterium adolescentis* preparation is a live *Bifidobacterium adolescentis* capsule. The live *Bifidobacterium adolescentis* capsule uses *Bifidobacterium adolescentis* live capsules from Lizhu Chang Le. The hepatitis B vaccine is a recombinant yeast hepatitis B vaccine. The recombinant yeast hepatitis B vaccine uses a recombinant hepatitis B vaccine from *Saccharomyces cerevisiae*. In high-responder populations receiving hepatitis B vaccine booster vaccination, the intestinal flora is rich in *Bifidobacterium adolescentis* 8 weeks post-vaccination. KEGG function analysis indicates that the intestinal flora function of high-responder populations is stronger than that of low / non-responder populations to hepatitis B vaccine. Bifidobacterium is positively correlated with various metabolic functions. Oral administration of *Bifidobacterium adolescentis* can enhance the immune response to hepatitis B vaccine.

Description

Technical Field

[0001] This invention relates to the field of biological agents, specifically to a biological agent containing Bifidobacterium adolescentis that enhances the immune response to hepatitis B vaccine. Background Technology

[0002] Chronic hepatitis B virus (HBV) infection is a global public health threat [MaL, Xing HC. Elimination of hepatitis B as a public health threat: Addressing the challenge and taking action[J]. World Journal of Hepatology, 2024, 16(11). DOI: 10.4254 / wjh.v16.i11.1361.]. Literature reports that without effective intervention, the number of deaths due to HBV infection will continue to rise, peaking in 2034 [Seto WK, Lo YR, Pawlotsky JM, et al. Chronic hepatitis B virus infection[J]. Lancet, 2018, 392(10161):2313-2324. DOI: 10.1016 / s0140-6736(18)31865-8.]. It is estimated that the current prevalence of hepatitis B surface antigen positivity in the general population of my country is 5%–6%, with approximately 70 million chronic HBV infections [Liu J, Liang W, Jing W, et al. Countdown to 2030: eliminating hepatitis B disease, China[J]. Bull WorldHealth Organ, 2019, 97(3): 230-238. DOI: 10.2471 / blt.18.219469.]. HBV is transmitted from mother to child, through blood, and through sexual contact. In adults, transmission mainly occurs through blood and sexual contact, including transfusions of blood and blood products that have not undergone rigorous screening and unprotected sexual activity. HBV can also be transmitted through broken skin or mucous membranes, such as through occupational exposure, pedicures, tattoos, ear piercings, and sharing dental tools [Yeo YH, Nguyen M H. Review article: current gaps and opportunities in HBV prevention, testing and linkage to care in the United States-a call for action[J]. Aliment Pharmacol Ther, 2021, 53(1): 63-78. DOI:10.1111 / apt.16125.]. This indicates that susceptible populations still face the threat of HBV infection in daily life.

[0003] The World Health Organization has set a goal of eliminating viral hepatitis as a public health hazard by 2030, which requires a 90% reduction in new infections of chronic hepatitis B [Smith S, Harmanci H, Hutin Y, et al. Globalprogress on the elimination of viral hepatitis as a major public health threat: An analysis of WHO Member State responses 2017[J]. JHEP Rep, 2019, 1(2): 81-89. DOI: 10.1016 / j.jhepr.2019.04.002.]. Data shows that high coverage of hepatitis B vaccination has a significant immune protection effect. Twenty years after the hepatitis B vaccination program, the incidence rate in children under 15 years old decreased by 80-90% [Wang Guiqiang, Zhao Xuming, Li Xiuyan, et al. The impact of high coverage of hepatitis B vaccine on the incidence rate in the vaccinated population [J]. International Journal of Virology, 2017, 24(05): 336-338. DOI: 10.3760 / cma.j.issn.1673-4092.2017.05.011.]. Vaccination effectively reduces HBV infection rates, but 5%-10% of healthy individuals still do not respond after vaccination [Poland GA, Jacobson R M. Clinical practice: prevention of hepatitis B with the hepatitis B vaccine[J]. N Engl J Med, 2004, 351(27): 2832-8. DOI:10.1056 / NEJMcp041507.].A low response is defined as a hepatitis B surface antibody (HBsAb) level below 100 mIU / ml after routine hepatitis B vaccination. This poor response accounts for approximately 7-10% of the population, but it has not received sufficient attention [Lyu JJ, Yin XW, Yan BY, et al. Anti-HBs persistence following revaccination with three doses of hepatitis B vaccine among low-responsive adults after primary vaccination: a 4-year follow-up study[J].Zhonghua Yu Fang Yi Xue Za Zhi, 2016, 50(6): 491-6. DOI: 10.3760 / cma.j.issn.0253-9624.2016.06.004.]. Even after a booster dose of 60ug recombinant yeast hepatitis B vaccine, 10-25% of individuals with poor response still exhibit persistently low responses [Ge Shen, Ma Jianxin, Liu Yaoyao, et al. Meta-analysis of the reimmunization effect in healthy adults who do not respond to hepatitis B vaccine [J]. Chinese Journal of Vaccines and Immunization, 2019, 25(05): 598-604. DOI: 10.19914 / j.cjvi.2019.05.025.]. Currently, there is still a gap between the current prevalence of hepatitis B and the goal of eradicating viral hepatitis. High-titer protective antibodies in the population are crucial; therefore, the problem of poor hepatitis B vaccine response urgently needs to be addressed. Summary of the Invention

[0004] This invention provides a biological agent that uses Bifidobacterium adolescentis to enhance the immune response to hepatitis B vaccine. A biological agent that enhances the immune response to hepatitis B vaccine using Bifidobacterium adolescentis, comprising: a preparation of Bifidobacterium adolescentis and a hepatitis B vaccine.

[0005] Eight weeks after hepatitis B vaccine booster vaccination, the gut microbiota of high-responder populations was rich in *Bifidobacterium adolescentis*. KEGG functional analysis indicated that the gut microbiota function of high-responder populations was stronger than that of low / non-responder populations. Bifidobacterium is positively correlated with various metabolic functions. Oral administration of *Bifidobacterium adolescentis* can enhance the immune response to hepatitis B vaccine.

[0006] In the aforementioned biological agent for enhancing the immune response to hepatitis B vaccine with Bifidobacterium adolescentis, the preparation of Bifidobacterium adolescentis is a live capsule of Bifidobacterium adolescentis.

[0007] In the aforementioned biological agent for enhancing the immune response to hepatitis B vaccine with Bifidobacterium adolescentis, the live bacteria capsules of Bifidobacterium adolescentis are Bifidobacterium live bacteria capsules from Lizhu Chang Le.

[0008] In the aforementioned biological agent for enhancing the immune response to hepatitis B vaccine with Bifidobacterium adolescentis, the hepatitis B vaccine is a recombinant yeast hepatitis B vaccine.

[0009] In the aforementioned biological agent for enhancing the immune response to hepatitis B vaccine using Bifidobacterium adolescentis, the recombinant yeast hepatitis B vaccine is a recombinant hepatitis B vaccine derived from Saccharomyces cerevisiae.

[0010] Compared with the prior art, the present invention has the following advantages: This invention recruits healthy Chinese adults who are hepatitis B surface antibody negative, boosts their hepatitis B vaccination, and collects stool and blood samples. A high-responder group to the hepatitis B vaccine is compared with a low / non-responder group. Metagenomic and single-cell transcriptomic analyses are used to explore the gut microbiota characteristics of the high-responder group and the possible mechanisms by which the gut microbiota affects immunity. Furthermore, a prospective cohort study is conducted to confirm the regulatory role of probiotics in the hepatitis B vaccine immune response.

[0011] The oral supplementation with Bifidobacterium adolescentis (probiotic group) of this invention resulted in higher levels of hepatitis B surface antibody at 8 weeks and 24 weeks post-vaccination. This demonstrates that Bifidobacterium adolescentis enhances the protective efficacy and duration of the hepatitis B vaccine. Attached Figure Description

[0012] Figure 1 Use Venn diagrams to show the unique and common microbial genera in each group.

[0013] Figure 2 This refers to the Shannon index.

[0014] Figure 3 For genus-level beta diversity analysis based on PCoA and ADONIS.

[0015] Figure 4 The relative abundance at the family level within the gut microbiota.

[0016] Figure 5 The relative abundance at the genus level in the gut microbiota.

[0017] Figure 6 The relative abundance at the species level in the gut microbiota.

[0018] Figure 7 Differential genera were identified at the genus level using LEfSe differential discriminant analysis.

[0019] Figure 8Differentially identified bacterial species at the species level using LEfSe differential discrimination analysis.

[0020] Figure 9 This study analyzes the functional differences between PCoA and ADONIS.

[0021] Figure 10 The relative abundance of the KEGG pathway showed differential enrichment in Level 3. Detailed Implementation

[0022] Example 1. Materials and Methods 1.1 Recruitment of Participants All participants were recruited from the First Affiliated Hospital of Zhejiang University between January 2023 and January 2025. The study protocol was approved by the hospital. Due to the involvement of human specimens, ethical considerations, including informed consent and protection of personal information, were of paramount importance. Written informed consent was obtained from all participants before they participated in the study. Factors affecting vaccine immune response include demographic characteristics such as age and sex, with higher rates of non-response in older adults and men. Lifestyle factors such as obesity, smoking, and alcohol consumption can reduce vaccine response. Medical conditions, including diabetes, chronic kidney and liver disease, HIV / AIDS, celiac disease, and inflammatory bowel disease, can affect vaccine response [Tahir A, Shinkafi SH, Alshrari AS, et al. A Comprehensive Review of Hepatitis B Vaccine Nonresponse and Associated Risk Factors[J]. Vaccines (Basel), 2024, 12(7). DOI: 10.3390 / vaccines12070710.]. Therefore, we established relatively strict inclusion and exclusion criteria to control confounding factors and focused on exploring the correlation between gut microbiota and hepatitis B vaccine response.

[0023] Inclusion criteria: 1) Age ≥ 18 years; 2) Negative for hepatitis B surface antigen, hepatitis B surface antibody and hepatitis B DNA; 3) Previously received three doses of hepatitis B vaccine according to routine procedures without serious adverse reactions; 4) Able to understand the purpose of the clinical trial and sign the informed consent form.

[0024] Exclusion criteria: 1) Suffering from acute illness, severe chronic disease, or acute exacerbation of chronic disease with fever greater than 38.1 degrees Celsius; 2) Suffering from uncontrolled epilepsy, progressive neurological disease, or major mental illness; 3) Having used antibiotics within the past month; 4) Long-term smokers or drinkers; 5) Pregnant or breastfeeding women.

[0025] 1.2 Data Collection and Sample Collection Initial screening and enrollment were conducted through questionnaires and on-site interviews. The baseline clinical characteristics of the participants, including gender, age, medical history, health status, and lifestyle habits, were recorded. Peripheral blood samples were drawn from the participants for quantitative hepatitis B serology, hepatitis B DNA testing, liver and kidney function tests, and complete blood count. Peripheral blood and fresh stool samples were also collected.

[0026] 1.3 Enhanced Hepatitis B Vaccination: Peripheral blood was drawn from all subjects 8 weeks and 24 weeks after receiving a single dose of 60ug recombinant yeast hepatitis B vaccine to detect quantitative hepatitis B three-lineage and hepatitis B DNA, liver and kidney function, and complete blood count. Peripheral blood samples and fresh stool samples were also collected at the same time.

[0027] 1.4 Fecal metagenomic analysis: Total DNA was extracted from human feces using a DNA extraction kit (Qiagen, Hilden, Germany). DNA integrity and concentration were determined by agarose gel electrophoresis and Quantus Fluorometer (Picogreen). Qualified DNA samples were fragmented using a Covaris M220 (Genetron Health, China), purified, and then used to construct libraries using the NEXTFLEX Rapid DNA-Seq (Bioo Scientific, USA) library construction kit. Sequencing was performed by Shanghai Meiji Biotechnology Co., Ltd. on the Illumina Novaseq X-plus sequencing platform. Species annotations were obtained from the taxonomic information database corresponding to the NR library. The abundance of each species was then calculated using the gene abundance of the corresponding species. Raw data were analyzed and visualized on the cloud platform https: / / www.majorbio.com / . Microbial names were searched at the China Center for Type Culture Collection (https: / / cctcc.whu.edu.cn / portal / dictionary / ).

[0028] 1.5 Statistical Analysis Normally distributed measurement data are expressed as mean and standard deviation, while count data are expressed as quantity and proportion. Statistical analysis was performed using StataSE 15.1 software. P < 0.05 indicated a significant difference. Graphs were generated using Adobe Illustrator 2025 software.

[0029] 2. Research Results 2.1 Characteristics of Hepatitis B Vaccination Cohort A total of 152 subjects who were hepatitis B surface antibody negative and did not have hepatitis B received a booster dose of 60ug hepatitis B vaccine (recombinant hepatitis B vaccine (Saccharomyces cerevisiae) (Shenzhen Kangtai Pharmaceutical Co., Ltd.)) and were divided into 4 groups according to their HBsAb levels: high response group (HBsAb >1000 mIU / ml), normal response group (HBsAb =100-1000 mIU / ml), low response group (HBsAb =10-99 mIU / ml), and non-responder group (HBsAb <10 mIU / ml). The demographic and clinical characteristics of the subjects are summarized in Table 1. There were no statistically significant differences in age and sex distribution among the groups. BMI, fasting blood glucose levels, and triglycerides were all within the normal range in both groups, and there were no statistically significant differences. A few subjects in each group had elevated serum uric acid levels, but there were no statistically significant differences between the groups. To explore the characteristics of the high response group, the low / non-responder group was used as a control.

[0030] Table 1. Basic characteristics of high-responder and low / non-responder groups to hepatitis B vaccine. 2.2 Gut Microbiota Composition of Different Response Groups in Hepatitis B Vaccine Vaccine Population To investigate the gut microbiota characteristics of high-responder populations after hepatitis B vaccine booster vaccination, metagenomic sequencing was performed on fecal samples. In the high-responder group (high, n=53, HBsAb >1000 mIU / ml) and the low / non-responder group (low, n=38, HBsAb <100 mIU / ml), 296 and 94 specific genera were identified at the genus level, respectively. Figure 1 In terms of species richness and alpha diversity as measured by the Chao index and the Shannon index, the Shannon index indicates a significant difference. Figure 2 , p <0.05. Meanwhile, PCoA and ADONIS analyses at the genus level, based on Bray-Curtis distance calculations, showed significant differences in gut microbiota composition between the two groups ( Figure 3 , p =0.002). To further investigate the composition of the major microorganisms in each group, we performed community bar chart analysis. At the family level, Bacteroidetes ( Bacteroidaceae ), family Trichophyceae ( Lachnospiraceae ), Vibriospirillumaceae ( Oscillospiraceae ) and Prevotellaceae ( Prevotellaceae ) accounted for a larger proportion in both groups ( Figure 4 At the genus level, we found a unique case: Bacteroides in the high group (…). Bacteroides ), Maritime City Cocci (Phocaeicola ) and Bifidobacterium spp. Bifidobacterium The proportion of ) was relatively higher in the low group, and the genus *Segastella* ( Segatella The percentage is relatively lower in the low group. Figure 5 At the species level, we observed that the high group was usually inhabited by *Maritime City Cocci* ( Phocaeicola plebeius ) and unidentified species of Bifidobacterium ( Bifidobacterium sp The proportion of fecal septicemia was relatively higher in the low group, and the proportion of fecal septicemia was higher in the high group. Segatella copri The percentage is relatively lower in the low group. Figure 6 ).

[0031] 2.3 Analysis of differences in gut microbiota species and functions among different response groups in hepatitis B vaccine recipients using LEfSe differential discriminant analysis ( Figure 7 , 8 LDA>2 p <0.05) Further investigation was conducted into the differences in gut microbiota between high-responder and low / non-responder groups of hepatitis B vaccine. At the genus level ( Figure 7 Twelve bacterial genera were identified that showed statistically significant differences between the two groups. The top three significantly enriched bacterial genera in the high-response group (high group) of the hepatitis B vaccine were Bifidobacterium (Bifidobacterium). Bifidobacterium ), Anaerobic Butyric Acid Bacteria ( Anaerobutyricum ), Streptomyces fusiformis ( Fusicatenibacter At this level ( Figure 8 The top three bacterial species significantly enriched in the high-response group of hepatitis B vaccine were unidentified Bifidobacterium species (Bifidobacterium tumefaciens). Bifidobacterium sp ) 、 fecal Bacteroides ( Bacteroides stercoris ) and Bifidobacterium adolescentis ( Bifidobacterium adolescentis ).

[0032] To further investigate the role of the gut microbiota in individuals with poor hepatitis B vaccine response, we performed metagenomic functional analysis based on the KEGG pathway. This analysis can obtain functional annotations of genes and reveal related metabolic pathways. PCOA and ADONIS analyses showed significant changes in gut microbiota function between the high and low groups. Figure 9 , p =0.003. In the Low group, the function of multiple metabolism-related KEGG pathways was weaker than in the High group ( Figure 10 ).

[0033] 2.4 Prospective studies suggest that *Bifidobacterium adolescentis* enhances the immune response to hepatitis B vaccine. Our previous studies found that individuals with a high response to hepatitis B vaccine had gut microbiota rich in *Bifidobacterium adolescentis* at baseline and 8 weeks post-vaccination, and this was associated with enhanced metabolic function of the gut microbiota. Therefore, we conducted a prospective study on microecological intervention to verify whether *Bifidobacterium adolescentis* could enhance the immune response to hepatitis B vaccine. Using the same inclusion and exclusion criteria, we recruited healthy adults who were not responsive to hepatitis B vaccine and divided them into two groups. Both groups received one dose of the recombinant yeast 60ug hepatitis B vaccine recommended by the hepatitis B guidelines. The probiotic group received daily oral supplementation with *Bifidobacterium adolescentis* [Lizhu Chang Le (Bifidobacterium live bacteria capsules)] for 4 weeks before and 8 weeks after vaccination, while the control group did not receive oral probiotics.

[0034] By comparing the HBsAb levels of the two groups after hepatitis B vaccination, it was found that oral supplementation with Bifidobacterium adolescentis (probiotic group) resulted in lower HBsAb levels 8 weeks after vaccination. Z =2.102, P =0.0356, Wilcoxon rank-sum test) / 24 weeks post-vaccination ( Z =2.171, P =0.0299 (Wilcoxon rank-sum test) showed a higher level of immune response. Therefore, *Bifidobacterium adolescentis* enhanced the protective efficacy (Table 2) and durability (Table 3) of the hepatitis B vaccine.

[0035] Table 2. Distribution of Hepatitis B Surface Antibody Levels in Participants at 8 Weeks Post-Vaccination in the Microbial Ecology Intervention Study Table 3. Distribution of Hepatitis B Surface Antibody Levels in Participants 24 Weeks After Vaccination in the Microbial Ecology Intervention Study 3. Discussion The incidence of chronic hepatitis B remains high, necessitating the exploration of methods to enhance vaccine efficacy. This study recruited healthy Chinese adults who were non-responsive to the hepatitis B vaccine after routine vaccination, and administered booster doses. The population was divided into high-response and low / non-response groups. The results showed that the gut microbiota of the high-response group was enriched with *Bifidobacterium adolescentis* at baseline and 8 weeks post-vaccination. Further prospective studies combining oral administration of *Bifidobacterium adolescentis* with booster hepatitis B vaccination demonstrated that *Bifidobacterium adolescentis* can enhance the immune response to the hepatitis B vaccine.

[0036] Hepatitis B vaccination is the most effective way to prevent HBV infection. The full course of hepatitis B vaccination requires 3 doses, and the earlier the hepatitis B vaccine is administered, the better. [Han M, Huang Y, Gui H, et al. Dynamic changes in host immune system and gut microbiota are associated with the production of SARS-CoV-2 antibodies[J]. Gut, 2023, 72(10): 1996-1999. DOI: 10.1136 / gutjnl-2022-327561.][Chinese Society of Hepatology, Chinese Society of Infectious Diseases. Guidelines for the Prevention and Treatment of Chronic Hepatitis B (2022 Edition)[J]. Chinese Journal of Hepatology, 2022, 30(12): 1309-1331.]. WHO recommends that adolescents, family members and sexual partners of hepatitis B surface antigen positive patients, and people at risk of hepatitis B virus infection receive enhanced hepatitis B vaccination to improve vaccine protection [Peng Y, Zhang L, Mok CKP, et al. Baseline gutmicrobiota and metabolome predict durable immunogenicity to SARS-CoV-2vaccines[J]. Signal Transduct Target Ther, 2023, 8(1): 373. DOI: 10.1038 / s41392-023-01629-8.], [Guidelines for the Prevention, Diagnosis, Care and Treatment for People with Chronic Hepatitis B Infection (Text Extract): Executive Summary[J]. Infect Dis Immun, 2024, 4(3): 103-105. DOI: 10.1097 / id9.0000000000000128.]. However, a certain percentage of the population still does not respond well to the hepatitis B vaccine, so it is necessary to understand the characteristics of this group. Research has found that gut microbiota affects hepatitis B vaccine response, but published studies on the gut microbiota in hepatitis B vaccines are extremely limited. Only a handful of studies have mentioned the association between hepatitis B vaccine response and gut microbiota.In this invention, it was found that microorganisms that may be enriched in the poor response group include Prevotella and Segastella, which are significantly negatively correlated with carbohydrate and lipid metabolism.

[0037] The sample size of this invention is limited. Although confounding factors such as smoking, alcohol consumption, and antibiotic use are controlled for, the number of subjects enrolled is also limited. Further studies could expand the sample size to explore various influencing factors, including the impact of gut microbiota on vaccine response levels, and the possible pathways by which the microecology affects active immunization. Prospective studies could also be designed to investigate the impact of specific microbiota interventions on hepatitis B vaccine immune responses.

[0038] In summary, this study characterized the gut microbiota of high-responder populations to hepatitis B vaccine, adding real-world research findings to the field of hepatitis B vaccine microecology. It explored the possible association between microecology and the active immunization effect of hepatitis B vaccine, and demonstrated through a cohort of hepatitis B vaccine booster vaccination with Bifidobacterium adolescentis that Bifidobacterium adolescentis can enhance the protective efficacy and durability of hepatitis B vaccine.

Claims

1. A biological agent containing Bifidobacterium adolescentis to enhance the immune response to hepatitis B vaccine, characterized in that, include: Preparations of Bifidobacterium adolescentis and hepatitis B vaccine.

2. The biological agent for enhancing the immune response to hepatitis B vaccine with Bifidobacterium adolescentis according to claim 1, characterized in that, The aforementioned Bifidobacterium adolescentis preparation is a live bacterial capsule of Bifidobacterium adolescentis.

3. The biological agent for enhancing the immune response to hepatitis B vaccine with Bifidobacterium adolescentis according to claim 2, characterized in that, The live bacteria capsules containing Bifidobacterium adolescentis are the same as those from Lizhu Chang Le Bifidobacterium live bacteria capsules.

4. The biological agent for enhancing the immune response to hepatitis B vaccine with Bifidobacterium adolescentis according to claim 1, characterized in that, The hepatitis B vaccine mentioned is a recombinant yeast hepatitis B vaccine.

5. The biological agent for enhancing the immune response to hepatitis B vaccine with Bifidobacterium adolescentis according to claim 4, characterized in that, The recombinant yeast hepatitis B vaccine mentioned above uses recombinant hepatitis B vaccine made from Saccharomyces cerevisiae.

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