The Mouse Gut Microbiome is Required for Response to Hepatis B Vaccine

The Mouse Gut Microbiome is Required for Response to Hepatis B Vaccine | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The Mouse Gut Microbiome is Required for Response to Hepatis B Vaccine Qiuxia Liu, Sha Zhang, Wei Fang, Bing Ruan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8936662/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Introduction Gut microbiome as an important impactor is directly linked to the humoral immune system. Whether and how the gut microbiota impacts the response to HBV vaccine remain unknown. We explored the role of gut microbiota on HBsAg-mediated humoral immune response in mice. The metabolites and cellular mechanism were further investigated between gut microbiota and humoral immune response to Hepatitis B vaccine. Methods Juvenile mice were treated with antibiotics and immunized with Hepatitis B vaccine. The plasma hepatitis B surface antibody (HBsAb) IgG level was detected, 16S rDNA gene sequencing and liquid chromatography-mass spectrometry (LC-MS) based metabolomics were used to profile the composition of the gut microbiota. Furthermore, antibiotic treated mice were gavaged with the fecal contents of age-matched antibiotic-untreated, or antibiotic-treated mice. The level of HBsAb IgG, IgG1, IgG2a and the B cell subsets such as the plasmablasts, plasma cells and GC B cells in the spleen and blood were analyzed after immunization to profile the B cell response. Results Early-life antibiotic treatment induced an impaired response to the Hepatitis B vaccine, as adult mice exposed to the same dose antibiotics did not have impaired vaccine antibody response. The relative abundance of Firmicutes, Proteobacteria, Antinobacteria and Lactobacillus decreased. Meanwhile, a significantly decreased fecal metabolites were observed with the perturbed gut microbiota, especially compounds vitamins, amino acids, and fatty acids. Antibiotic-treated mice received a fecal microbiota transfer (FMT) from antibiotic-untreated mice showed an increased humoral response, as the plasmablats, plasma cells and GC B cells in the spleen and blood were significantly increased after reconstituting the gut microbiota. Conclusion Early life exposure to antibiotic impaired the humoral immunity response to HBsAg, while the impaired antibody response could be rescued by the FMT. The disturbed gut microbiota with reduced metabolites and the levels of plasmablast, plasma cell and GC B cell changes could be one of the mechanisms of how gut microbiota impacts humoral immunity. However, how the gut microbiota or its metabolites contribute to the impaired humoral immunity remain unknown. gut microbiota humoral immunity hepatitis B vaccine FMT LC-MS Figures Figure 1 Figure 2 Figure 3 INTRODUCTION Hepatitis B caused by hepatitis B virus (HBV) infection remains a major global public health issue. Individuals with chronic HBV infection are at risk of developing severe liver diseases, such as chronic hepatitis B, liver cirrhosis, and hepatocellular carcinoma (HCC). Among over 900,000 new cases of liver cancer worldwide each year, 60% are associated with chronic HBV infection, with East Asian countries bearing a particularly heavy burden. International guidelines highly recommend HBV immunization, and the hepatitis B vaccine is an effective approach to reduce HBV infection and block HBV transmission [ 1 ]. While approximately 5%-10% of individuals fail to produce effective protective antibodies (hepatitis B surface antibody (HBsAb) ≥ 10 mIU/mL) [ 2 ]. These individuals after completing the standard 3-dose hepatitis B vaccination schedule are defined as hepatitis B vaccine non-responders and are at risk of HBV infection. The mechanism underlying non-response to hepatitis B vaccine has not been fully elucidated. Non-response to hepatitis B vaccine may be associated with factors such as injection site, gender, age, body weight, use of immunosuppressive drugs, immunodeficiency, and genetic factors [ 3 , 4 ]. Studies suggest that the gut microbiota can act as a natural vaccine adjuvant to influence the response of mice to influenza vaccines, and administration of probiotics enhances the production of influenza vaccine-specific antibodies in mice [ 5 ]. Germ-free (GF) mice treated with an antibiotic cocktail exhibit impaired lymphoid tissue development and dysregulated immune cell homeostasis due to gut microbiota depletion, which also alters the susceptibility of the gastrointestinal tract to infectious or inflammatory diseases [ 6 ]. The gut microbiota can generate and release important immunomodulatory molecules to influence the immune system, and signals derived from commensal bacteria in the gut can serve as immunomodulatory signal. Therefore, the goal of our present study was to investigate whether the gut microbiota is essential for the host to produce hepatitis B vaccine-specific antibodies and to explore the mechanisms underlying the impact of gut microbiota on hepatitis B vaccine response. To do so, we treated mice with an antibiotic cocktail, followed by hepatitis B vaccine injection. The titer of HBsAb IgG antibodies in the peripheral blood was measured, and we comprehensively analyzed the fecal via 16S rDNA sequencing and liquid chromatography-mass spectrometry (LC-MS). Additionally, fecal microbiota transplantation (FMT) was performed in antibiotic-treated juvenile mice and flow cytometry was used to detect the proportions of immune cells in the spleen and peripheral blood. MATRIALS AND METHODS Animals The animal experiment protocol was approved by the Laboratory Animal Management and Ethics Committee of Shandong Provincial Hospital (SZRJJ: NO.2021 − 182). Adult mice and litters of newborn BALB/C mice purchased from the SLAC Laboratory Animal Co., Ltd (Shanghai, China) were housed under specific pathogen-free conditions. The pups were weaned on postnatal day 21(P21). All mice had free access to food and water and were maintained in a temperature-controlled colony room on a 12h light/dark cycle. The mice were randomly divided into six groups (n = 10 each): adult antibiotics-treated mice immunized with hepatitis B vaccine (ABX1 group); antibiotics -untreated adult mice immunized with hepatitis B vaccine (CON1 group); untreated and unimmunized adult mice (unvaccined1 group); juvenile antibiotics-treated mice immunized with hepatitis B vaccine (ABX2 group); antibiotic-untreated juvenile mice immunized with hepatitis B vaccine (CON2 group); and untreated and unimmunized juvenile mice (unvaccined2 group). For antibiotic treatment, mice were gavaged daily with 100 µl of water containing 10 µg vancomycin (Sigma-Aldrich), 20 µg neomycin sulfate (Sigma-Aldrich), 20 µg metronidazole (Sigma-Aldrich) and 20 µg ampicillin (Sigma-Aldrich) for five consecutive days [ 7 ], followed by a 3-day rest period. Mice in the unvaccined1 and unvaccined2 groups received 100 µL of normal saline. Mice in the ABX1, ABX2, CON1 and CON2 groups were administered a three-dose series of hepatitis B vaccine. According to the study design, all immunized mice were intramuscularly injected in the right hind thigh with 100 µL of recombinant HBV vaccine (Saccharomyces cerevisiae) (Kangtai Biological Pharmaceutical Company, Shenzhen, China) containing 2 µg of hepatitis B virus surface antigen (HBsAg) on postnatal days P26, P33, and P47. ELISA The plasma samples were obtained after three immunizations. The concentration of HBsAb in the plasma was measured using anti-HBs ELISA kits from LanTu Bio-Tech (Quanzhou, China) according to the manufacturer’s protocols. In brief, 50 µl of plasma samples was added to each wells of the microtiter plate coated with HBsAg. Horseradish peroxidase-conjugated antibodies against IgG, IgG1 and IgG2a were added to the wells and incubated for 30 min at 37℃. After five washes, 50 µl of substrate solution was added to each well, followed by incubation for 15 min at 37 ℃. Next, 50 µl of stop solution was added to each well to terminate the reaction. The OD value was determined using a microplate reader at 450 nm. 16S rDNA Sequencing For microbiota profiling, genomic DNA was extracted from fecal pellets using the FastDNA Spin Kit for Soil (MPbio) following the manufacturer’s instructions. The V3-V4 region of the16S ribosomal DNA was amplified using the following primers: 338F (5’-ACTCCTACGGGAGGCAGCAG-3’), and 806R (5’-GGACTACHVGGGTWTCTAAT-3’). The libraries were quantified with a NEXTFLEX Rapid DNA-Seq Kit and sequenced on the Miseq PE300 platform. Sequence analyses were performed using UPARSE Version 7.1( http://drive5.com/uparse/ ). Operational taxonomic unit (OTUs) and taxonomy was assigned based on the Silva database. Significantly differential features at each level were identified using linear discriminant analysis (LDA) coupled with effect size measurement (LEfSe). Taxa were filtered according to the default criteria (P 2). Untargeted Metabolomics Analysis Fecal samples were thawed at 4°C, weighed, and then placed in a freeze dryer for drying followed by reweighing. The dried samples were homogenized (30 cycles per min, 7 min), and then cooled for 10 min. After centrifugation (12,000 × g, 4°C, 10 min), the supernatant was resuspended in ultrapure water at a ratio of 1 mg:50 µL, followed by vortexing for 5 min. An equal volume of the suspension was pipetted from each sample. Pre-chilled 100% methanol (1:4, v/v) was added, and the mixture was vortexed for 5 min. After centrifugation (15,000 × g, 4°C ,10 min), the supernatant was collected and dried. The dried samples were dissolved in 300 µL of 60% acetonitrile and divided into 3 aliquots, with one 75 µL aliquot reserved for HILIC column analysis. The remaining two 75µL (total 150 µL) aliquot was freeze-dried again and then redissolved in 150 µL of a mixed solution (acetonitrile: ultrapure water: formic acid = 50:50:0.1). Quality control samples were prepared by mixing equal volumes of aliquots from 5 samples in each group. FMT The fecal samples from antibiotic-treated (ABX group) or untreated (CON group) juvenile mice were collected as donors. After weighing, pre-prepared glycerol was mixed with the feces at a ratio of 1:1. The mixture was homogenized and then centrifuged to obtain a suspension. The suspension was collected and stored at -80°C. The antibiotic-treated juvenile mice were randomly divided into two groups (n = 10 each), which received fecal samples from the ABX group (ABX + ABX group) or the CON group (ABX + CON group). One day before each HBV vaccine injection, 0.1 mL of the suspension sample was delivered to the intestinal cavity at a depth of approximately 4–5 cm via an enema needle. Then, the suspension was slowly injected and retained for 1 minute, after which the needle was slowly withdrawn. Flow Cytometry and Antibodies At postnatal day 54 (P54), spleens and whole blood were collected to obtain single-cell suspensions. Spleens were ground in DMEM, and splenocytes were collected. Lysis buffer (BD) was used to eliminate red blood cells. Cells were incubated with anti-mouse CD16/CD32 monoclonal antibody (MAb) to block Fcγ receptors for 10 min and then stained on ice for 15 min with combinations of MAbs. The MAbs used in this study are Alexa fluor-labeled anti-mouse CD4, BB515-labeled anti-mouse CD25, PE-labeled anti-mouse FAS, AF647-labeled anti-mouse GL7, BV650-labeled CD138, PerCP-Cy7-labeled CD38, and BV510-labeled CD19. All MAbs were purchased from BD Biosciences. The flow cytometry assay was performed on a FACS-Canto Ⅱ (Becton Dickinson, Mountain View, CA) and analyzed using FlowJo software (Tree Star). Statistical Analysis Statistical analysis was performed using GraphPad Prism 7, and data are expressed as mean ± SEM. The statistical significance between two groups was analyzed by Student’s t-test and the Mann-Whitney test was used for data with non-normal distribution. A value of P < 0.05 was considered statistically significant. RESULTS The Impaired HBV-Specific Antibody Response Induced by Disturbed Gut Microbiota We sought to examine vaccine-induced antibody responsiveness in juvenile and adult mice treated with antibiotics or not. Following immunization with a trivalent hepatitis B virus vaccine, the ABX2 group had a significantly attenuated HBsAb IgG response compared to the ABX1 and CON2 groups (P < 0.05) (Fig. 1 A). These data establish that juvenile mice in the antibiotic-treated group possess a low-responder phenotype to immunization strategies. A 16S rDNA sequencing approach was used to identify gut microbiota that may contribute to the impaired antibody responsiveness between mice treated with antibiotics or not. A total of 477,360 sequences and 589 OTUs were detected. A total of 291 core OTUs were shared by the ABX1, ABX2, and CON2 groups, while the numbers of unique OTUs was 71, 29, and 34, respectively (Fig. 1 B). Alpha diversity, including the Shannon index and Simpson index, are shown in the Fig. 2 B, the ABX2 group exhibited a significantly higher Shannon index and a significantly lower Simpson index compared to the CON2 group (P < 0.05) (Fig. 1 C). Additionally, the differences between the ABX1 and ABX2 were not statistically significant. These results indicated that after gut microbiota perturbation with the antibiotic cocktail, the alpha diversity and intestinal species diversity of juvenile mice were increased. For beta diversity, as shown in the principal component analysis (PCA) plot based on Unweighted UniFrac (Fig. 1 D), the PC1 accounted for 22.88% and the PC2 accounted for 12.56%. The gut microbiota of the three groups was clearly distinguishable, reflecting low similarity between groups. We further analyzed the gut microbiota composition: at the phylum level (Fig. 1 E), the top five phyla ranked by relative abundance were Firmicutes, Bacteroidetes, Proteobacteria, Saccharibacteria, and Actinobacteria. The relative abundances of Firmicutes and Actinobacteria in the ABX2 group were significantly lower than those in the ABX1 and CON2 groups, with statistically significant differences (Fig. 1 F). At the genus level, the top 13 were shown in Fig. 1 G, Lactobacillus, Bacteroidales_S24-7_group, Bacteroides, Lachnospiraceae_NK4A136_group, Prevotellaceae_UCG-001, Parabacteroides, Alloprevotella, norank_f_Lachnospiraceae, unclassified_f_Lachnospiraceae, Alistipes, Rikenellaceae_RC9_gut_group, Ruminococcaceae_UCG_014, and Lachnoclostridium. Among these genera, the differences in the relative abundances of Lactobacillus, Parabacteroides, Alloprevotella, unclassified_f_Lachnospiraceae, Alistipes, and Lachnoclostridium were statistically significant (Fig. 1 H). Profiling of Gut Microbiota-Derived Metabolite Alterations To examine the impact of antibiotics on the gut metabolites of juvenile mice, an LC-MS/MS-based untargeted metabolomic analysis was conducted to determine the changes in fecal metabolomic profiles with antibiotic intervention. PCA analysis was conducted to confirm the system stability and data reliability in negative and positive modes during the entire experimental process (Fig. 2 A, B). Furthermore, PLS-DA models in negative and positive modes were constructed, and the two groups of samples could be clearly distinguished (Fig. 2 C, D). Negative mode detected 2021 metabolites, with 263 demonstrating statistically significant differences. A total of 1379 metabolites were quantified in positive mode, 167 of which were significantly different. The annotated metabolites in negative and positive modes are listed in sTable 1 and sTable 2, respectively. Differential metabolites between the two groups were identified using the VIP values, FC values, and P-values of the PLS-DA model. Compared to the CON2 group, a total of 9 metabolites were up-regulated and 254 metabolites were down-regulated in negative mode, while 2 metabolites were up-regulated, and 165 metabolites were down-regulated in positive mode in the ABX2 group (Fig. 2 E, F). KEGG pathway enrichment analysis of differential metabolites revealed differences in gut microbial function between the two groups of juvenile mice. Differential metabolites were mainly enriched in the following pathways: arginine biosynthesis, beta-alanine metabolism, amino sugar and nucleotide sugar metabolism, nicotinate and nicotinamide metabolism, tryptophan metabolism, 2-oxocarboxylic acid metabolism, metabolic pathways, biosynthesis of amino acids, fatty acid biosynthesis, caffeine metabolism, porphyrin and chlorophyll metabolism, pantothenate and CoA biosynthesis, primary bile acid biosynthesis, pyrimidine metabolism, and biosynthesis of unsaturated fatty acids. The key differential metabolites involved in the above metabolic pathways are: quinolinic acid, N-acetylneuraminic acid, N-acetyl-L-glutamic acid, oleic acid, 3,6,8-trimethylallantoin, uracil, urobilinogen, and 7α,25-dihydroxycholesterol. sTable 1. Annotated metabolites with significant differences in negative mode from mouse fecal samples. sTable 2. Annotated metabolites with significant differences in positive mode from mouse fecal samples. Restoration of the Gut Microbiota Can Partially Restore the Hepatitis B Vaccine Response FMT was performed to restore the gut microbiota, thereby regulating the interaction between the microbiota and the host [ 8 ]. To investigate whether the impaired hepatitis B vaccine response caused by gut microbiota dysbiosis can be improved by FMT with a healthy gut microbiota, antibiotic-treated juvenile mice were gavaged with fecal samples from mice that were either antibiotic-treated or not. The results showed that the levels of HBsAb IgG, IgG1 and IgG2a (Fig. 3 A, B, C) in mice of the ABX + CON group were significantly higher than those in the ABX group and ABX + ABX group, while the levels of HBsAb IgG and IgG1 were still lower than those in the CON group. This indicated that the attenuated hepatitis B vaccine response of juvenile mice induced by antibiotic mixture intervention could be partially restored by FMT from healthy control mice, but it still could not reach the level of fully healthy control mice. The Frequencies of Plasma cells and Plasmablasts were Altered Following Gut Microbiota Disturbance To investigate the cellular mechanisms underlying the different antibody responsiveness, we assessed plasma cell (CD4-CD19-CD138 + CD38-) and plasmablast (CD4-CD19 + CD138+CD38+) frequencies in peripheral blood and spleen (Fig. 3 D, E, F, G). The frequencies of plasma cells and plasmablasts in both the peripheral blood and spleen of the ABX group were significantly lower than those of the CON group (P < 0.05). After FMT, the frequencies of plasma cells and plasmablasts in both the peripheral blood and spleen of the ABX + CON group were increased. Specifically, the frequencies of plasmablasts in the spleen of the ABX + CON group were significantly higher than that of the ABX group and the ABX + ABX group; meanwhile, the frequencies of plasma cells and plasmablasts in the peripheral blood of the ABX + CON group were significantly higher than those of the ABX + ABX group (P < 0.05). These results indicated that the intervention of gut microbiota with an antibiotic treatment can reduce the levels of plasma cells and plasmablasts in the spleen and peripheral blood. After reconstructing the gut microbiota via FMT using feces from healthy mice, the frequencies of plasma cells and plasma blasts in the spleen and peripheral blood can be partially restored, but they are still lower than those in normal control mice. Effects of Antibiotic Treatment and FMT on Splenic GC B Cells Germinal center B (GC B) cells are a subset of B cells that proliferate and differentiate within the germinal centers of the spleen. As key cellular components of humoral immune responses, they can eventually differentiate into plasma cells. The percentage of GC B cells (CD4-CD19 + FAS+GL7+) in the spleen was detected by flow cytometry. As shown in Fig. 3 (H, I), the frequencies of GC B cells in the spleen of the ABX group decreased signficantly after antibiotic treatment. After FMT from healthy mice, the frequencies of GC B cells in the spleen of the ABX + CON group were elevated and were significantly higher than those in both the ABX group and the ABX + ABX group. These results indicated that antibiotic-mediated gut microbiota depletion led to a reduction in splenic GC B cells in mice; restoration of the gut microbiota via FMT reversed this reduction and increased the level of splenic GCB cells, although the level remained lower than that in the normal control group. DISSCUSSION In this study, we investigated whether and how the gut microbiota impacts the response to HBV vaccine in mice. The results showed that antibiotic-treated juvenile mice exhibited an impaired response capacity to the hepatitis B vaccine. We found that the profile of gut microbiota and the metabolites were distinctly differentiated between the antibiotic-treated and untreated mice. In addition, FMT from healthy control mice can partially restore the vaccine response capacity. Furthermore, we demonstrated that the frequencies of plasma cells, plasmablasts and GC B cells were reduced with antibiotic use, whereas they were elevated with the implementation of FMT. Hence, we revealed that early-life exposure to antibiotics impaired the humoral immune response to HBsAg, while the impaired antibody response could be rescued by the FMT from healthy control mice. The disturbed gut microbiota with reduced metabolites and the altered levels of plasmablasts, plasma cells and GC B cells could be one of the mechanisms by which the gut microbiota impacts humoral immunity. However, how the gut microbiota metabolites contribute to the impaired immunity remain unknown. Gut microbiota plays an essential role in human nutrition, immunity, growth and development, and metabolism. Studies have revealed that the gut microbiota is directly associated with the development and function of the immune system [ 9 ] and it can act as a natural adjuvant for vaccination [ 10 ].Previous studies have demonstrated that the gut microbiota can affect the response to the influenza vaccine in mice, and probiotics can increase the production of influenza vaccine-specific antibodies in mice [ 5 ].In this study, we found that juvenile mice treated with an antibiotics to perturb the gut microbiota exhibited a marked decline in humoral immune responsiveness, along with a reduction in the production of specific protective antibodies against the hepatitis B vaccine. By transplanting feces from normal mice into those with impaired gut microbiota, we observed a partial restoration of the compromised humoral immune responsiveness in the recipient mice. Meanwhile, we found that antibiotic treatment impaired the humoral immune responsiveness of juvenile mice. In contrast, adult mice subjected to the same antibiotic treatment showed no impairment in their capacity to produce HBsAb IgG. This indicates that gut microbiota dysbiosis impairs the humoral immune function of immature mice, which is consistent with previous findings that germ-free (GF) mice reared in a sterile environment fail to achieve a state of normal immune maturation [ 11 ] and exhibit an impaired capacity to produce protective antibodies against ovalbumin [ 12 ]. Additionally, studies have shown that the composition of the gut microbiota in children is associated with their responsiveness to the rotavirus vaccine [ 13 ] and parenteral vaccines [ 14 ]. Lactobacillus (phylum Firmicutes) and Bifidobacterium (phylum Actinobacteria) are of great significance for the healthy growth of infants, whereas the phylum Bacteroidetes includes pathogenic bacteria. In this study, we found that the profile of the gut microbiota and its metabolites changed significantly in antibiotic-treated mice. At the phylum level, the relative abundances of Firmicutes and Actinobacteria were decreased, while that of Bacteroidetes was increased. This is consistent with the findings of that the relative abundances of Actinobacteria and Firmicutes are positively correlated with vaccine response, whereas that of Bacteroidetes is negatively correlated with this response [ 15 ]. At the genus level, the relative abundance of Lactobacillus was markedly reduced. Studies have indicated that supplementation with Lactobacillus casei 431 can elevate the secretion of influenza vaccine-specific antibodies [ 16 ]. Additionally, oral administration of Lactobacillus rhamnosus GG to infants can enhance the secretion of rotavirus vaccine-specific IgM [ 17 ]. Alterations in gut microbiota composition can induce shifts in the metabolites secreted by these microbes, which in turn modulates the functional properties of the gut microbiota. Non-targeted metabolomics analysis revealed a total of 430 statistically significant differential compounds, with metabolites such as vitamins, amino acids and fatty acids significantly reduced. The pathways involved include arginine biosynthesis, β-alanine metabolism, nicotinic acid and nicotinamide metabolism, tryptophan metabolism, 2-oxocarboxylic acid metabolism, fatty acid and unsaturated fatty acid biosynthesis, and primary bile acid biosynthesis. We found that acetate derivatives, propionate derivatives, and butyrate derivatives were significantly lower in the antibiotic- treated group than in the control group. Gut microbiota can break down indigestible dietary fiber to produce short-chain fatty acids (SCFAs), including acetate, propionate, and butyrate. SCFAs can serve as an energy source for epithelial cells and modulate the migration, cytolytic activity, cytokine-secreting capacity, and epigenetic regulation of Treg cells, neutrophils, and macrophages [ 18 ]. We found that tryptophan metabolism, indole-related derivatives and kynurenine were decreased following gut microbiota perturbation by antibiotics. Tryptophan is metabolized by intestinal microbiota to produce indole-containing metabolites such as the antioxidant indole-3-propionate, including indole-3-acetic acid (IAA); it can also be metabolized into kynurenine (Kyn) by host cells via indoleamine 2,3-dioxygenase-1 (IDO-1) [ 19 ]. Active IDO-1 can inhibit T-cell responses, promote the differentiation of Treg cells, and induce immune tolerance [ 20 ]. We found that gut microbiota dysbiosis affects the proportion of GC B cells in the spleen, which in turn leads to a reduction in the differentiation of B cells into plasma cells and plasmablasts, as well as a decrease in the secretion of IgG, which can be partially restored via FMT. Germinal centers are the primary sites for the functional maturation of B cells. B cells circulate throughout the body via the blood and lymphatic system to secondary lymphoid organs such as the spleen and lymph nodes. Following activation by antigens, follicular B cells differentiate into memory B cells or plasma cells within germinal centers, which are capable of secreting IgA, IgE, IgG, IgD, and IgM. Studies have revealed that the morphological structure of B and T cell zones in the spleen and lymph nodes was relatively impaired [ 21 ], and the IgG levels were significantly lower in GF mice than in SPF mice [ 22 ]. When GF mice were housed with SPF mice in the same cages, the IgG levels in GF mice could increase within several weeks [ 23 ]. Studies have shown that the depletion of Gram-positive or Gram-negative bacteria alone via antibiotic treatment impairs the vaccine response in mice [ 5 ]. Probiotic supplementation enhances the activity of Th1 and Th2 cells, thereby promoting the secretion of vaccine specific IgG1 and IgG3 by B cells [ 16 ]. Probiotics can also potentiate the production of influenza vaccine-specific antibodies by memory B cells [ 24 ]. The entry of soluble bacterial degradation products into the systemic circulation may account for the differences in secondary lymphoid tissue structure and serum immunoglobulin levels between GF and SPF mice [ 25 , 26 ]. This study did not elucidate the specific mechanisms by which alterations in the gut microbiota modulate B cell function. Additionally, the specific bacterial strains and/or bacterial metabolites associated with the immune response to the hepatitis B vaccine in mice warrant further investigation. Conclusions In conclusion, we found that gut microbiota dysbiosis impairs the immune response to the hepatitis B vaccine in juvenile mice, and reconstitution of the gut microbiota can ameliorate the despaired responsiveness to the hepatitis B vaccine induced by antibiotic intervention. Interventions targeting the gut microbiota, or its metabolites may serve as a novel strategy to enhance vaccine efficacy in the future. Declarations Conflicts of Interest The authors declare no conflicts of interest. Funding This work was supported by the Shandong Provincial Natural Science Foundation Youth Project (NO. ZR2021QH344). Author Contribution Qiuxia Liu: Conceptualization, Methodology, Investigation, Data curation, Writing – original draft, Funding acquisition. Sha Zhang: Investigation, Data curation, Formal analysis, Writing – review & editing. Wei Fang: Resources, Validation, Visualization, Writing – review & editing. Bing Ruan: Supervision, Funding acquisition, Project administration, Writing – review & editing. All authors have read and agreed to the published version of the manuscript. Data Availability The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher. References Schillie S et al. Prevention of Hepatitis B Virus Infection in the United States: Recommendations of the Advisory Committee on Immunization Practices. (1545–8601 (Electronic)). Goncalves L et al. Pattern of T cell activation in absence of protective immunity against hepatitis B virus. Review (0535–5133 (Print)). Zuckerman JN. Nonresponse to hepatitis B vaccines and the kinetics of anti-HBs production. (0146–6615 (Print)). Newport MJ et al. 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Benef Microbes. 2019;10(3):279–91. Crabbé PA, et al. Immunohistochemical observations on lymphoid tissues from conventional and germ-free mice. Lab Invest. 1970;22(5):448–57. Benveniste J, Lespinats G, Salomon J. Serum and secretory IgA in axenic and holoxenic mice. J Immunol. 1971;107(6):1656–62. Additional Declarations No competing interests reported. Supplementary Files sTable1.xlsx sTable 1. Annotated metabolites with significant differencesin negative mode from mouse fecal samples. sTable2.xlsx sTable 2. Annotated metabolites with significant differences in positive mode from mouse fecal samples. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 03 Apr, 2026 Reviewers agreed at journal 20 Mar, 2026 Reviewers invited by journal 04 Mar, 2026 Editor assigned by journal 23 Feb, 2026 Submission checks completed at journal 23 Feb, 2026 First submitted to journal 21 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8936662","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":601809505,"identity":"56ed10af-3e3a-46b3-8409-f91504333cc2","order_by":0,"name":"Qiuxia Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7UlEQVRIie3PsWrCQBzH8ZPAufwb139IH+JCQDJY+ip3ZA1dXEUOAtnU2UVfQRfnKwdJB6Gr49m+QKc2QxAPutmSZHS4L/yX4/cZjhCX6x7z7HGSABnmr+arwd4EgUCZRuuiD/nNLjEbhw+yx9avYIxmho/sxFn4JJOXkfTOH6cWEmiIGS8R2NHwOGtwiorGcdZCmIbIcGpJxVWaURRSAQ07CFP8YokSUieWbHuQyIjCkrd0kBNLdl0k0HTKxBIhOJbeYFGg2OuOv/jv+hDU35Nnv1r91HUzF5sqP3+2EaL+vHht8/+Jy+VyuW66Aiz+S+SQizazAAAAAElFTkSuQmCC","orcid":"","institution":"Department of Critical Care Medicine, Shandong Provincial Hospital Affiliated to Shandong First Medical University","correspondingAuthor":true,"prefix":"","firstName":"Qiuxia","middleName":"","lastName":"Liu","suffix":""},{"id":601809506,"identity":"a5ff4841-9649-42bd-a2ae-bffabe2fe2d0","order_by":1,"name":"Sha Zhang","email":"","orcid":"","institution":"Department of Critical Care Medicine, Shandong Provincial Hospital Affiliated to Shandong First Medical University","correspondingAuthor":false,"prefix":"","firstName":"Sha","middleName":"","lastName":"Zhang","suffix":""},{"id":601809507,"identity":"5ba7b9d6-e9c7-452a-ac6c-c27ff60b9ab6","order_by":2,"name":"Wei Fang","email":"","orcid":"","institution":"Department of Critical Care Medicine, Shandong Provincial Hospital Affiliated to Shandong First Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Fang","suffix":""},{"id":601809508,"identity":"41520305-1a19-4bfe-9e47-fea037a1665c","order_by":3,"name":"Bing Ruan","email":"","orcid":"","institution":"Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Bing","middleName":"","lastName":"Ruan","suffix":""}],"badges":[],"createdAt":"2026-02-22 03:23:54","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8936662/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8936662/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104222953,"identity":"4aa259ea-24ad-4779-94e8-58272366f1f2","added_by":"auto","created_at":"2026-03-09 10:40:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":439624,"visible":true,"origin":"","legend":"\u003cp\u003eThe Impaired HBV-Specific Antibody Response Induced by Disturbed Gut Microbiota (A) Plasma HBsAb IgG antibody levels in peripheral blood (B) Venn diagram of OTU numbers (C) Alpha diversity based on the Shannon and Simpson indices of the OTU level (D) PCA plot based on the Unweighted UniFrac distance metric (E) Relative abundance of gut microbiota at the phylum level (F) Differential microbiota analysis at the phylum level (G) Relative abundance of gut microbiota at the genus level (H) Differential microbiota analysis at the genus level.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8936662/v1/384cdc80e3bcf97a70034c13.png"},{"id":104223390,"identity":"900cdc67-9237-4cbb-b960-b12cf251090c","added_by":"auto","created_at":"2026-03-09 10:41:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":138457,"visible":true,"origin":"","legend":"\u003cp\u003eThe Impact of Antibiotics on Gut Microbiota Metabolites of Juvenile Mice. (A, B) PCA score plots in negative and positive modes (C, D) PLS-DA ordination validation plots in negative and positive modes (E, F) Volcano plot analyses of differential metabolites in negative and positive modes.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8936662/v1/ae4f13e8c5d3e78d064f113e.png"},{"id":104222967,"identity":"3b8b2621-c994-4eb0-9a8a-9edc3ef637c7","added_by":"auto","created_at":"2026-03-09 10:40:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":529399,"visible":true,"origin":"","legend":"\u003cp\u003eThe Gut Microbiota Regulates B cell Maturation in Vaccine Response (A) Differences in Titers of HBsAb IgG (B) Differences in Titers of IgG1 (C) Differences in Titers of IgG2a (D) Proportion of Plasma Cells (CD4⁻CD19⁻CD138⁺CD38⁻) in the Spleen (E) Proportion of Plasma Cells (CD4⁻CD19⁻CD138⁺CD38⁻) in Peripheral Blood (F) Proportion of Plasmablasts (CD4⁻CD19⁺CD138⁺CD38⁺) in the Spleen (G) Proportion of Plasmablasts (CD4⁻CD19⁺CD138⁺CD38⁺) in Peripheral Blood. (H) Dot Plot Showing Germinal Center (GC B) cells (CD4-CD19+FAS+GL7+) in the Spleen (I) Proportion of GC B cells in the spleen. *P ＜ 0.05，** P ＜ 0.01.\u003c/p\u003e","description":"","filename":"FIgure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8936662/v1/62de5dce6cc6ee92b5c9c010.png"},{"id":104223551,"identity":"efd610e6-9372-4ca5-946d-9face9573ba9","added_by":"auto","created_at":"2026-03-09 10:42:08","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":62103,"visible":true,"origin":"","legend":"\u003cp\u003esTable 1. Annotated metabolites with significant differencesin negative mode from mouse fecal samples.\u003c/p\u003e","description":"","filename":"sTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8936662/v1/865d5ddbef0052e73b72da7e.xlsx"},{"id":104223145,"identity":"9ba82fd7-beab-43b0-89a3-55e2e5d63e7c","added_by":"auto","created_at":"2026-03-09 10:41:04","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":39825,"visible":true,"origin":"","legend":"\u003cp\u003esTable 2. Annotated metabolites with significant differences in positive mode from mouse fecal samples.\u003c/p\u003e","description":"","filename":"sTable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8936662/v1/6b0339a685f858d21f86a7c5.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Mouse Gut Microbiome is Required for Response to Hepatis B Vaccine","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eHepatitis B caused by hepatitis B virus (HBV) infection remains a major global public health issue. Individuals with chronic HBV infection are at risk of developing severe liver diseases, such as chronic hepatitis B, liver cirrhosis, and hepatocellular carcinoma (HCC). Among over 900,000 new cases of liver cancer worldwide each year, 60% are associated with chronic HBV infection, with East Asian countries bearing a particularly heavy burden. International guidelines highly recommend HBV immunization, and the hepatitis B vaccine is an effective approach to reduce HBV infection and block HBV transmission [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. While approximately 5%-10% of individuals fail to produce effective protective antibodies (hepatitis B surface antibody (HBsAb)\u0026thinsp;\u0026ge;\u0026thinsp;10 mIU/mL) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. These individuals after completing the standard 3-dose hepatitis B vaccination schedule are defined as hepatitis B vaccine non-responders and are at risk of HBV infection.\u003c/p\u003e \u003cp\u003eThe mechanism underlying non-response to hepatitis B vaccine has not been fully elucidated. Non-response to hepatitis B vaccine may be associated with factors such as injection site, gender, age, body weight, use of immunosuppressive drugs, immunodeficiency, and genetic factors [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Studies suggest that the gut microbiota can act as a natural vaccine adjuvant to influence the response of mice to influenza vaccines, and administration of probiotics enhances the production of influenza vaccine-specific antibodies in mice [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Germ-free (GF) mice treated with an antibiotic cocktail exhibit impaired lymphoid tissue development and dysregulated immune cell homeostasis due to gut microbiota depletion, which also alters the susceptibility of the gastrointestinal tract to infectious or inflammatory diseases [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The gut microbiota can generate and release important immunomodulatory molecules to influence the immune system, and signals derived from commensal bacteria in the gut can serve as immunomodulatory signal.\u003c/p\u003e \u003cp\u003eTherefore, the goal of our present study was to investigate whether the gut microbiota is essential for the host to produce hepatitis B vaccine-specific antibodies and to explore the mechanisms underlying the impact of gut microbiota on hepatitis B vaccine response. To do so, we treated mice with an antibiotic cocktail, followed by hepatitis B vaccine injection. The titer of HBsAb IgG antibodies in the peripheral blood was measured, and we comprehensively analyzed the fecal via 16S rDNA sequencing and liquid chromatography-mass spectrometry (LC-MS). Additionally, fecal microbiota transplantation (FMT) was performed in antibiotic-treated juvenile mice and flow cytometry was used to detect the proportions of immune cells in the spleen and peripheral blood.\u003c/p\u003e"},{"header":"MATRIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003e The animal experiment protocol was approved by the Laboratory Animal Management and Ethics Committee of Shandong Provincial Hospital (SZRJJ: NO.2021\u0026thinsp;\u0026minus;\u0026thinsp;182). Adult mice and litters of newborn BALB/C mice purchased from the SLAC Laboratory Animal Co., Ltd (Shanghai, China) were housed under specific pathogen-free conditions. The pups were weaned on postnatal day 21(P21). All mice had free access to food and water and were maintained in a temperature-controlled colony room on a 12h light/dark cycle.\u003c/p\u003e \u003cp\u003eThe mice were randomly divided into six groups (n\u0026thinsp;=\u0026thinsp;10 each): adult antibiotics-treated mice immunized with hepatitis B vaccine (ABX1 group); antibiotics -untreated adult mice immunized with hepatitis B vaccine (CON1 group); untreated and unimmunized adult mice (unvaccined1 group); juvenile antibiotics-treated mice immunized with hepatitis B vaccine (ABX2 group); antibiotic-untreated juvenile mice immunized with hepatitis B vaccine (CON2 group); and untreated and unimmunized juvenile mice (unvaccined2 group).\u003c/p\u003e \u003cp\u003eFor antibiotic treatment, mice were gavaged daily with 100 \u0026micro;l of water containing 10 \u0026micro;g vancomycin (Sigma-Aldrich), 20 \u0026micro;g neomycin sulfate (Sigma-Aldrich), 20 \u0026micro;g metronidazole (Sigma-Aldrich) and 20 \u0026micro;g ampicillin (Sigma-Aldrich) for five consecutive days [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], followed by a 3-day rest period. Mice in the unvaccined1 and unvaccined2 groups received 100 \u0026micro;L of normal saline. Mice in the ABX1, ABX2, CON1 and CON2 groups were administered a three-dose series of hepatitis B vaccine. According to the study design, all immunized mice were intramuscularly injected in the right hind thigh with 100 \u0026micro;L of recombinant HBV vaccine (Saccharomyces cerevisiae) (Kangtai Biological Pharmaceutical Company, Shenzhen, China) containing 2 \u0026micro;g of hepatitis B virus surface antigen (HBsAg) on postnatal days P26, P33, and P47.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eELISA\u003c/h3\u003e\n\u003cp\u003eThe plasma samples were obtained after three immunizations. The concentration of HBsAb in the plasma was measured using anti-HBs ELISA kits from LanTu Bio-Tech (Quanzhou, China) according to the manufacturer\u0026rsquo;s protocols. In brief, 50 \u0026micro;l of plasma samples was added to each wells of the microtiter plate coated with HBsAg. Horseradish peroxidase-conjugated antibodies against IgG, IgG1 and IgG2a were added to the wells and incubated for 30 min at 37℃. After five washes, 50 \u0026micro;l of substrate solution was added to each well, followed by incubation for 15 min at 37 ℃. Next, 50 \u0026micro;l of stop solution was added to each well to terminate the reaction. The OD value was determined using a microplate reader at 450 nm.\u003c/p\u003e \u003cp\u003e \u003cb\u003e16S rDNA Sequencing\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFor microbiota profiling, genomic DNA was extracted from fecal pellets using the FastDNA Spin Kit for Soil (MPbio) following the manufacturer\u0026rsquo;s instructions. The V3-V4 region of the16S ribosomal DNA was amplified using the following primers: 338F (5\u0026rsquo;-ACTCCTACGGGAGGCAGCAG-3\u0026rsquo;), and 806R (5\u0026rsquo;-GGACTACHVGGGTWTCTAAT-3\u0026rsquo;). The libraries were quantified with a NEXTFLEX Rapid DNA-Seq Kit and sequenced on the Miseq PE300 platform. Sequence analyses were performed using UPARSE Version 7.1(\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://drive5.com/uparse/\u003c/span\u003e\u003cspan address=\"http://drive5.com/uparse/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Operational taxonomic unit (OTUs) and taxonomy was assigned based on the Silva database. Significantly differential features at each level were identified using linear discriminant analysis (LDA) coupled with effect size measurement (LEfSe). Taxa were filtered according to the default criteria (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 by Kruskal-Walli\u0026rsquo;s test; LDA score\u0026thinsp;\u0026gt;\u0026thinsp;2).\u003c/p\u003e\n\u003ch3\u003eUntargeted Metabolomics Analysis\u003c/h3\u003e\n\u003cp\u003eFecal samples were thawed at 4\u0026deg;C, weighed, and then placed in a freeze dryer for drying followed by reweighing. The dried samples were homogenized (30 cycles per min, 7 min), and then cooled for 10 min. After centrifugation (12,000 \u0026times; g, 4\u0026deg;C, 10 min), the supernatant was resuspended in ultrapure water at a ratio of 1 mg:50 \u0026micro;L, followed by vortexing for 5 min. An equal volume of the suspension was pipetted from each sample. Pre-chilled 100% methanol (1:4, v/v) was added, and the mixture was vortexed for 5 min. After centrifugation (15,000 \u0026times; g, 4\u0026deg;C ,10 min), the supernatant was collected and dried. The dried samples were dissolved in 300 \u0026micro;L of 60% acetonitrile and divided into 3 aliquots, with one 75 \u0026micro;L aliquot reserved for HILIC column analysis. The remaining two 75\u0026micro;L (total 150 \u0026micro;L) aliquot was freeze-dried again and then redissolved in 150 \u0026micro;L of a mixed solution (acetonitrile: ultrapure water: formic acid\u0026thinsp;=\u0026thinsp;50:50:0.1). Quality control samples were prepared by mixing equal volumes of aliquots from 5 samples in each group.\u003c/p\u003e\n\u003ch3\u003eFMT\u003c/h3\u003e\n\u003cp\u003eThe fecal samples from antibiotic-treated (ABX group) or untreated (CON group) juvenile mice were collected as donors. After weighing, pre-prepared glycerol was mixed with the feces at a ratio of 1:1. The mixture was homogenized and then centrifuged to obtain a suspension. The suspension was collected and stored at -80\u0026deg;C. The antibiotic-treated juvenile mice were randomly divided into two groups (n\u0026thinsp;=\u0026thinsp;10 each), which received fecal samples from the ABX group (ABX\u0026thinsp;+\u0026thinsp;ABX group) or the CON group (ABX\u0026thinsp;+\u0026thinsp;CON group). One day before each HBV vaccine injection, 0.1 mL of the suspension sample was delivered to the intestinal cavity at a depth of approximately 4\u0026ndash;5 cm via an enema needle. Then, the suspension was slowly injected and retained for 1 minute, after which the needle was slowly withdrawn.\u003c/p\u003e\n\u003ch3\u003eFlow Cytometry and Antibodies\u003c/h3\u003e\n\u003cp\u003eAt postnatal day 54 (P54), spleens and whole blood were collected to obtain single-cell suspensions. Spleens were ground in DMEM, and splenocytes were collected. Lysis buffer (BD) was used to eliminate red blood cells. Cells were incubated with anti-mouse CD16/CD32 monoclonal antibody (MAb) to block Fcγ receptors for 10 min and then stained on ice for 15 min with combinations of MAbs. The MAbs used in this study are Alexa fluor-labeled anti-mouse CD4, BB515-labeled anti-mouse CD25, PE-labeled anti-mouse FAS, AF647-labeled anti-mouse GL7, BV650-labeled CD138, PerCP-Cy7-labeled CD38, and BV510-labeled CD19. All MAbs were purchased from BD Biosciences. The flow cytometry assay was performed on a FACS-Canto Ⅱ (Becton Dickinson, Mountain View, CA) and analyzed using FlowJo software (Tree Star).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using GraphPad Prism 7, and data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. The statistical significance between two groups was analyzed by Student\u0026rsquo;s t-test and the Mann-Whitney test was used for data with non-normal distribution. A value of P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eThe Impaired HBV-Specific Antibody Response Induced by Disturbed Gut Microbiota\u003c/h2\u003e \u003cp\u003eWe sought to examine vaccine-induced antibody responsiveness in juvenile and adult mice treated with antibiotics or not. Following immunization with a trivalent hepatitis B virus vaccine, the ABX2 group had a significantly attenuated HBsAb IgG response compared to the ABX1 and CON2 groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). These data establish that juvenile mice in the antibiotic-treated group possess a low-responder phenotype to immunization strategies.\u003c/p\u003e \u003cp\u003eA 16S rDNA sequencing approach was used to identify gut microbiota that may contribute to the impaired antibody responsiveness between mice treated with antibiotics or not. A total of 477,360 sequences and 589 OTUs were detected. A total of 291 core OTUs were shared by the ABX1, ABX2, and CON2 groups, while the numbers of unique OTUs was 71, 29, and 34, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Alpha diversity, including the Shannon index and Simpson index, are shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, the ABX2 group exhibited a significantly higher Shannon index and a significantly lower Simpson index compared to the CON2 group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Additionally, the differences between the ABX1 and ABX2 were not statistically significant. These results indicated that after gut microbiota perturbation with the antibiotic cocktail, the alpha diversity and intestinal species diversity of juvenile mice were increased.\u003c/p\u003e \u003cp\u003eFor beta diversity, as shown in the principal component analysis (PCA) plot based on Unweighted UniFrac (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), the PC1 accounted for 22.88% and the PC2 accounted for 12.56%. The gut microbiota of the three groups was clearly distinguishable, reflecting low similarity between groups. We further analyzed the gut microbiota composition: at the phylum level (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), the top five phyla ranked by relative abundance were Firmicutes, Bacteroidetes, Proteobacteria, Saccharibacteria, and Actinobacteria. The relative abundances of Firmicutes and Actinobacteria in the ABX2 group were significantly lower than those in the ABX1 and CON2 groups, with statistically significant differences (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). At the genus level, the top 13 were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG, Lactobacillus, Bacteroidales_S24-7_group, Bacteroides, Lachnospiraceae_NK4A136_group, Prevotellaceae_UCG-001, Parabacteroides, Alloprevotella, norank_f_Lachnospiraceae, unclassified_f_Lachnospiraceae, Alistipes, Rikenellaceae_RC9_gut_group, Ruminococcaceae_UCG_014, and Lachnoclostridium. Among these genera, the differences in the relative abundances of Lactobacillus, Parabacteroides, Alloprevotella, unclassified_f_Lachnospiraceae, Alistipes, and Lachnoclostridium were statistically significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eProfiling of Gut Microbiota-Derived Metabolite Alterations\u003c/h2\u003e \u003cp\u003eTo examine the impact of antibiotics on the gut metabolites of juvenile mice, an LC-MS/MS-based untargeted metabolomic analysis was conducted to determine the changes in fecal metabolomic profiles with antibiotic intervention. PCA analysis was conducted to confirm the system stability and data reliability in negative and positive modes during the entire experimental process (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). Furthermore, PLS-DA models in negative and positive modes were constructed, and the two groups of samples could be clearly distinguished (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D). Negative mode detected 2021 metabolites, with 263 demonstrating statistically significant differences. A total of 1379 metabolites were quantified in positive mode, 167 of which were significantly different. The annotated metabolites in negative and positive modes are listed in sTable 1 and sTable 2, respectively. Differential metabolites between the two groups were identified using the VIP values, FC values, and P-values of the PLS-DA model. Compared to the CON2 group, a total of 9 metabolites were up-regulated and 254 metabolites were down-regulated in negative mode, while 2 metabolites were up-regulated, and 165 metabolites were down-regulated in positive mode in the ABX2 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, F).\u003c/p\u003e \u003cp\u003eKEGG pathway enrichment analysis of differential metabolites revealed differences in gut microbial function between the two groups of juvenile mice. Differential metabolites were mainly enriched in the following pathways: arginine biosynthesis, beta-alanine metabolism, amino sugar and nucleotide sugar metabolism, nicotinate and nicotinamide metabolism, tryptophan metabolism, 2-oxocarboxylic acid metabolism, metabolic pathways, biosynthesis of amino acids, fatty acid biosynthesis, caffeine metabolism, porphyrin and chlorophyll metabolism, pantothenate and CoA biosynthesis, primary bile acid biosynthesis, pyrimidine metabolism, and biosynthesis of unsaturated fatty acids. The key differential metabolites involved in the above metabolic pathways are: quinolinic acid, N-acetylneuraminic acid, N-acetyl-L-glutamic acid, oleic acid, 3,6,8-trimethylallantoin, uracil, urobilinogen, and 7α,25-dihydroxycholesterol.\u003c/p\u003e \u003cp\u003esTable 1. Annotated metabolites with significant differences in negative mode from mouse fecal samples.\u003c/p\u003e \u003cp\u003esTable 2. Annotated metabolites with significant differences in positive mode from mouse fecal samples.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eRestoration of the Gut Microbiota Can Partially Restore the Hepatitis B Vaccine Response\u003c/h2\u003e \u003cp\u003eFMT was performed to restore the gut microbiota, thereby regulating the interaction between the microbiota and the host [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. To investigate whether the impaired hepatitis B vaccine response caused by gut microbiota dysbiosis can be improved by FMT with a healthy gut microbiota, antibiotic-treated juvenile mice were gavaged with fecal samples from mice that were either antibiotic-treated or not. The results showed that the levels of HBsAb IgG, IgG1 and IgG2a (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B, C) in mice of the ABX\u0026thinsp;+\u0026thinsp;CON group were significantly higher than those in the ABX group and ABX\u0026thinsp;+\u0026thinsp;ABX group, while the levels of HBsAb IgG and IgG1 were still lower than those in the CON group. This indicated that the attenuated hepatitis B vaccine response of juvenile mice induced by antibiotic mixture intervention could be partially restored by FMT from healthy control mice, but it still could not reach the level of fully healthy control mice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eThe Frequencies of Plasma cells and Plasmablasts were Altered Following Gut Microbiota Disturbance\u003c/h2\u003e \u003cp\u003eTo investigate the cellular mechanisms underlying the different antibody responsiveness, we assessed plasma cell (CD4-CD19-CD138\u0026thinsp;+\u0026thinsp;CD38-) and plasmablast (CD4-CD19\u0026thinsp;+\u0026thinsp;CD138+CD38+) frequencies in peripheral blood and spleen (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, E, F, G). The frequencies of plasma cells and plasmablasts in both the peripheral blood and spleen of the ABX group were significantly lower than those of the CON group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). After FMT, the frequencies of plasma cells and plasmablasts in both the peripheral blood and spleen of the ABX\u0026thinsp;+\u0026thinsp;CON group were increased. Specifically, the frequencies of plasmablasts in the spleen of the ABX\u0026thinsp;+\u0026thinsp;CON group were significantly higher than that of the ABX group and the ABX\u0026thinsp;+\u0026thinsp;ABX group; meanwhile, the frequencies of plasma cells and plasmablasts in the peripheral blood of the ABX\u0026thinsp;+\u0026thinsp;CON group were significantly higher than those of the ABX\u0026thinsp;+\u0026thinsp;ABX group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These results indicated that the intervention of gut microbiota with an antibiotic treatment can reduce the levels of plasma cells and plasmablasts in the spleen and peripheral blood. After reconstructing the gut microbiota via FMT using feces from healthy mice, the frequencies of plasma cells and plasma blasts in the spleen and peripheral blood can be partially restored, but they are still lower than those in normal control mice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEffects of Antibiotic Treatment and FMT on Splenic GC B Cells\u003c/h2\u003e \u003cp\u003eGerminal center B (GC B) cells are a subset of B cells that proliferate and differentiate within the germinal centers of the spleen. As key cellular components of humoral immune responses, they can eventually differentiate into plasma cells. The percentage of GC B cells (CD4-CD19\u0026thinsp;+\u0026thinsp;FAS+GL7+) in the spleen was detected by flow cytometry. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e (H, I), the frequencies of GC B cells in the spleen of the ABX group decreased signficantly after antibiotic treatment. After FMT from healthy mice, the frequencies of GC B cells in the spleen of the ABX\u0026thinsp;+\u0026thinsp;CON group were elevated and were significantly higher than those in both the ABX group and the ABX\u0026thinsp;+\u0026thinsp;ABX group. These results indicated that antibiotic-mediated gut microbiota depletion led to a reduction in splenic GC B cells in mice; restoration of the gut microbiota via FMT reversed this reduction and increased the level of splenic GCB cells, although the level remained lower than that in the normal control group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISSCUSSION","content":"\u003cp\u003eIn this study, we investigated whether and how the gut microbiota impacts the response to HBV vaccine in mice. The results showed that antibiotic-treated juvenile mice exhibited an impaired response capacity to the hepatitis B vaccine. We found that the profile of gut microbiota and the metabolites were distinctly differentiated between the antibiotic-treated and untreated mice. In addition, FMT from healthy control mice can partially restore the vaccine response capacity. Furthermore, we demonstrated that the frequencies of plasma cells, plasmablasts and GC B cells were reduced with antibiotic use, whereas they were elevated with the implementation of FMT. Hence, we revealed that early-life exposure to antibiotics impaired the humoral immune response to HBsAg, while the impaired antibody response could be rescued by the FMT from healthy control mice. The disturbed gut microbiota with reduced metabolites and the altered levels of plasmablasts, plasma cells and GC B cells could be one of the mechanisms by which the gut microbiota impacts humoral immunity. However, how the gut microbiota metabolites contribute to the impaired immunity remain unknown.\u003c/p\u003e \u003cp\u003eGut microbiota plays an essential role in human nutrition, immunity, growth and development, and metabolism. Studies have revealed that the gut microbiota is directly associated with the development and function of the immune system [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] and it can act as a natural adjuvant for vaccination [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].Previous studies have demonstrated that the gut microbiota can affect the response to the influenza vaccine in mice, and probiotics can increase the production of influenza vaccine-specific antibodies in mice [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].In this study, we found that juvenile mice treated with an antibiotics to perturb the gut microbiota exhibited a marked decline in humoral immune responsiveness, along with a reduction in the production of specific protective antibodies against the hepatitis B vaccine. By transplanting feces from normal mice into those with impaired gut microbiota, we observed a partial restoration of the compromised humoral immune responsiveness in the recipient mice. Meanwhile, we found that antibiotic treatment impaired the humoral immune responsiveness of juvenile mice. In contrast, adult mice subjected to the same antibiotic treatment showed no impairment in their capacity to produce HBsAb IgG. This indicates that gut microbiota dysbiosis impairs the humoral immune function of immature mice, which is consistent with previous findings that germ-free (GF) mice reared in a sterile environment fail to achieve a state of normal immune maturation [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and exhibit an impaired capacity to produce protective antibodies against ovalbumin [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Additionally, studies have shown that the composition of the gut microbiota in children is associated with their responsiveness to the rotavirus vaccine [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and parenteral vaccines [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLactobacillus (phylum Firmicutes) and Bifidobacterium (phylum Actinobacteria) are of great significance for the healthy growth of infants, whereas the phylum Bacteroidetes includes pathogenic bacteria. In this study, we found that the profile of the gut microbiota and its metabolites changed significantly in antibiotic-treated mice. At the phylum level, the relative abundances of Firmicutes and Actinobacteria were decreased, while that of Bacteroidetes was increased. This is consistent with the findings of that the relative abundances of Actinobacteria and Firmicutes are positively correlated with vaccine response, whereas that of Bacteroidetes is negatively correlated with this response [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. At the genus level, the relative abundance of Lactobacillus was markedly reduced. Studies have indicated that supplementation with Lactobacillus casei 431 can elevate the secretion of influenza vaccine-specific antibodies [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Additionally, oral administration of Lactobacillus rhamnosus GG to infants can enhance the secretion of rotavirus vaccine-specific IgM [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlterations in gut microbiota composition can induce shifts in the metabolites secreted by these microbes, which in turn modulates the functional properties of the gut microbiota. Non-targeted metabolomics analysis revealed a total of 430 statistically significant differential compounds, with metabolites such as vitamins, amino acids and fatty acids significantly reduced. The pathways involved include arginine biosynthesis, β-alanine metabolism, nicotinic acid and nicotinamide metabolism, tryptophan metabolism, 2-oxocarboxylic acid metabolism, fatty acid and unsaturated fatty acid biosynthesis, and primary bile acid biosynthesis. We found that acetate derivatives, propionate derivatives, and butyrate derivatives were significantly lower in the antibiotic- treated group than in the control group. Gut microbiota can break down indigestible dietary fiber to produce short-chain fatty acids (SCFAs), including acetate, propionate, and butyrate. SCFAs can serve as an energy source for epithelial cells and modulate the migration, cytolytic activity, cytokine-secreting capacity, and epigenetic regulation of Treg cells, neutrophils, and macrophages [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. We found that tryptophan metabolism, indole-related derivatives and kynurenine were decreased following gut microbiota perturbation by antibiotics. Tryptophan is metabolized by intestinal microbiota to produce indole-containing metabolites such as the antioxidant indole-3-propionate, including indole-3-acetic acid (IAA); it can also be metabolized into kynurenine (Kyn) by host cells via indoleamine 2,3-dioxygenase-1 (IDO-1) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Active IDO-1 can inhibit T-cell responses, promote the differentiation of Treg cells, and induce immune tolerance [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe found that gut microbiota dysbiosis affects the proportion of GC B cells in the spleen, which in turn leads to a reduction in the differentiation of B cells into plasma cells and plasmablasts, as well as a decrease in the secretion of IgG, which can be partially restored via FMT. Germinal centers are the primary sites for the functional maturation of B cells. B cells circulate throughout the body via the blood and lymphatic system to secondary lymphoid organs such as the spleen and lymph nodes. Following activation by antigens, follicular B cells differentiate into memory B cells or plasma cells within germinal centers, which are capable of secreting IgA, IgE, IgG, IgD, and IgM. Studies have revealed that the morphological structure of B and T cell zones in the spleen and lymph nodes was relatively impaired [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], and the IgG levels were significantly lower in GF mice than in SPF mice [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. When GF mice were housed with SPF mice in the same cages, the IgG levels in GF mice could increase within several weeks [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Studies have shown that the depletion of Gram-positive or Gram-negative bacteria alone via antibiotic treatment impairs the vaccine response in mice [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Probiotic supplementation enhances the activity of Th1 and Th2 cells, thereby promoting the secretion of vaccine specific IgG1 and IgG3 by B cells [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Probiotics can also potentiate the production of influenza vaccine-specific antibodies by memory B cells [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The entry of soluble bacterial degradation products into the systemic circulation may account for the differences in secondary lymphoid tissue structure and serum immunoglobulin levels between GF and SPF mice [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study did not elucidate the specific mechanisms by which alterations in the gut microbiota modulate B cell function. Additionally, the specific bacterial strains and/or bacterial metabolites associated with the immune response to the hepatitis B vaccine in mice warrant further investigation.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, we found that gut microbiota dysbiosis impairs the immune response to the hepatitis B vaccine in juvenile mice, and reconstitution of the gut microbiota can ameliorate the despaired responsiveness to the hepatitis B vaccine induced by antibiotic intervention. Interventions targeting the gut microbiota, or its metabolites may serve as a novel strategy to enhance vaccine efficacy in the future.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflicts of Interest\u003c/h2\u003e \u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the Shandong Provincial Natural Science Foundation Youth Project (NO. ZR2021QH344).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eQiuxia Liu: Conceptualization, Methodology, Investigation, Data curation, Writing \u0026ndash; original draft, Funding acquisition. Sha Zhang: Investigation, Data curation, Formal analysis, Writing \u0026ndash; review \u0026amp; editing. Wei Fang: Resources, Validation, Visualization, Writing \u0026ndash; review \u0026amp; editing. Bing Ruan: Supervision, Funding acquisition, Project administration, Writing \u0026ndash; review \u0026amp; editing. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSchillie S et al. \u003cem\u003ePrevention of Hepatitis B Virus Infection in the United States: Recommendations of the Advisory Committee on Immunization Practices.\u003c/em\u003e (1545\u0026ndash;8601 (Electronic)).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoncalves L et al. Pattern of T cell activation in absence of protective immunity against hepatitis B virus. Review (0535\u0026ndash;5133 (Print)).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZuckerman JN. \u003cem\u003eNonresponse to hepatitis B vaccines and the kinetics of anti-HBs production.\u003c/em\u003e (0146\u0026ndash;6615 (Print)).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNewport MJ et al. \u003cem\u003eGenetic regulation of immune responses to vaccines in early life.\u003c/em\u003e (1466\u0026ndash;4879 (Print)).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOh JZ et al. TLR5-mediated sensing of gut microbiota is necessary for antibody responses to seasonal influenza vaccination. (1097\u0026ndash;4180 (Electronic)).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKamada N et al. \u003cem\u003eRole of the gut microbiota in immunity and inflammatory disease.\u003c/em\u003e (1474\u0026ndash;1741 (Electronic)).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBlock KE et al. Gut Microbiota Regulates K/BxN Autoimmune Arthritis through Follicular Helper T but Not Th17 Cells. (1550\u0026ndash;6606 (Electronic)).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKelly CR et al. \u003cem\u003eUpdate on Fecal Microbiota Transplantation 2015: Indications, Methodologies, Mechanisms, and Outlook.\u003c/em\u003e (1528-0012 (Electronic)).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee YK, Mazmanian SK. Has the microbiota played a critical role in the evolution of the adaptive immune system? Science. 2010;330(6012):1768\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePabst O, Hornef M. Gut microbiota: a natural adjuvant for vaccination. Immunity. 2014;41(3):349\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChung H, et al. Gut immune maturation depends on colonization with a host-specific microbiota. Cell. 2012;149(7):1578\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLamous\u0026eacute;-Smith ES, Tzeng A, Starnbach MN. The intestinal flora is required to support antibody responses to systemic immunization in infant and germ free mice. PLoS ONE. 2011;6(11):e27662.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarris VC, et al. Significant Correlation Between the Infant Gut Microbiome and Rotavirus Vaccine Response in Rural Ghana. J Infect Dis. 2017;215(1):34\u0026ndash;41.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuda MN, et al. Stool microbiota and vaccine responses of infants. Pediatrics. 2014;134(2):e362\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZimmermann P, Curtis N. The influence of the intestinal microbiome on vaccine responses. Vaccine. 2018;36(30):4433\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRizzardini G, et al. Evaluation of the immune benefits of two probiotic strains Bifidobacterium animalis ssp. lactis, BB-12\u0026reg; and Lactobacillus paracasei ssp. paracasei, L. casei 431\u0026reg; in an influenza vaccination model: a randomised, double-blind, placebo-controlled study. Br J Nutr. 2012;107(6):876\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIsolauri E, et al. Improved immunogenicity of oral D x RRV reassortant rotavirus vaccine by Lactobacillus casei GG. Vaccine. 1995;13(3):310\u0026ndash;2.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCox LM, et al. Altering the intestinal microbiota during a critical developmental window has lasting metabolic consequences. Cell. 2014;158(4):705\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKnip M, Siljander H. The role of the intestinal microbiota in type 1 diabetes mellitus. Nat Rev Endocrinol. 2016;12(3):154\u0026ndash;67.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKing NJ, Thomas SR. Molecules in focus: indoleamine 2,3-dioxygenase. Int J Biochem Cell Biol. 2007;39(12):2167\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBauer H, et al. The response of the lymphatic tissue to the microbial flora. Studies on germfree mice. Am J Pathol. 1963;42(4):471\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBenveniste J, et al. Immunoglobulins in intact, immunized, and contaminated axenic mice: study of serum IgA. J Immunol. 1971;107(6):1647\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMackie RI, Sghir A, Gaskins HR. Developmental microbial ecology of the neonatal gastrointestinal tract. Am J Clin Nutr. 1999;69(5):s1035\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVan den Elsen LWJ, et al. Prebiotic oligosaccharides in early life alter gut microbiome development in male mice while supporting influenza vaccination responses. Benef Microbes. 2019;10(3):279\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCrabb\u0026eacute; PA, et al. Immunohistochemical observations on lymphoid tissues from conventional and germ-free mice. Lab Invest. 1970;22(5):448\u0026ndash;57.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBenveniste J, Lespinats G, Salomon J. Serum and secretory IgA in axenic and holoxenic mice. J Immunol. 1971;107(6):1656\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"gut-pathogens","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gutp","sideBox":"Learn more about [Gut Pathogens](http://gutpathogens.biomedcentral.com/)","snPcode":"13099","submissionUrl":"https://submission.nature.com/new-submission/13099/3","title":"Gut Pathogens","twitterHandle":"@GutPathogens","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"gut microbiota, humoral immunity, hepatitis B vaccine, FMT, LC-MS","lastPublishedDoi":"10.21203/rs.3.rs-8936662/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8936662/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eIntroduction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGut microbiome as an important impactor is directly linked to the humoral immune system. Whether and how the gut microbiota impacts the response to HBV vaccine remain unknown. We explored the role of gut microbiota on HBsAg-mediated humoral immune response in mice. The metabolites and cellular mechanism were further investigated between gut microbiota and humoral immune response to Hepatitis B vaccine.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJuvenile mice were treated with antibiotics and immunized with Hepatitis B vaccine. The plasma hepatitis B surface antibody (HBsAb) IgG level was detected, 16S rDNA gene sequencing and liquid chromatography-mass spectrometry (LC-MS) based metabolomics were used to profile the composition of the gut microbiota. Furthermore, antibiotic treated mice were gavaged with the fecal contents of age-matched antibiotic-untreated, or antibiotic-treated mice. The level of HBsAb IgG, IgG1, IgG2a and the B cell subsets such as the plasmablasts, plasma cells and GC B cells in the spleen and blood were analyzed after immunization to profile the B cell response.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEarly-life antibiotic treatment induced an impaired response to the Hepatitis B vaccine, as adult mice exposed to the same dose antibiotics did not have impaired vaccine antibody response. The relative abundance of Firmicutes, Proteobacteria, Antinobacteria and Lactobacillus decreased. Meanwhile, a significantly decreased fecal metabolites were observed with the perturbed gut microbiota, especially compounds vitamins, amino acids, and fatty acids. Antibiotic-treated mice received a fecal microbiota transfer (FMT) from antibiotic-untreated mice showed an increased humoral response, as the plasmablats, plasma cells and GC B cells in the spleen and blood were significantly increased after reconstituting the gut microbiota.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEarly life exposure to antibiotic impaired the humoral immunity response to HBsAg, while the impaired antibody response could be rescued by the FMT. The disturbed gut microbiota with reduced metabolites and the levels of plasmablast, plasma cell and GC B cell changes could be one of the mechanisms of how gut microbiota impacts humoral immunity. However, how the gut microbiota or its metabolites contribute to the impaired humoral immunity remain unknown.\u003c/p\u003e","manuscriptTitle":"The Mouse Gut Microbiome is Required for Response to Hepatis B Vaccine","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-09 10:38:11","doi":"10.21203/rs.3.rs-8936662/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-04-03T08:11:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"324262376889313002721587921821487249145","date":"2026-03-20T15:16:39+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-04T08:16:53+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-23T06:12:48+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-23T06:11:43+00:00","index":"","fulltext":""},{"type":"submitted","content":"Gut Pathogens","date":"2026-02-22T03:18:14+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"gut-pathogens","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gutp","sideBox":"Learn more about [Gut Pathogens](http://gutpathogens.biomedcentral.com/)","snPcode":"13099","submissionUrl":"https://submission.nature.com/new-submission/13099/3","title":"Gut Pathogens","twitterHandle":"@GutPathogens","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b56406f6-1d1e-4162-9060-e7f802511a25","owner":[],"postedDate":"March 9th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-09T10:38:12+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-09 10:38:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8936662","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8936662","identity":"rs-8936662","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}