Tertiary lymphoid structures (TLSs) have emerged as critical prognostic and immunotherapeutic indicators in cancer, with their clinical significance modulated by spatial distribution patterns and density. Here, we performed integrated single-cell and spatial transcriptomic profiling of 30 gastric cancer (GC) specimens stratified by TLS spatial localization patterns. Comparative analysis shows pronounced enrichment of CXCL13⁺ T lymphocytes (TLCs), CXCR5⁺ germinal center B lymphocytes (gc_B cells), LAMP3⁺CD80⁺ activated dendritic cells (DCs), and SELP⁺ACKR1⁺ high endothelial venule (HEV) cells within intratumoral-TLS (iTLS) rich tumors compared to peritumoral-TLS (pTLS) and desert-TLS (dTLS) tumor subtypes. Multimodal cell-cell interaction analysis and functional experiments demonstrate that HEV expressed VCAM1 and ICAM1 recruits and activates CXCL13⁺ TLC through the CXCL13-ACKR1 pathway, which promotes TLS formation via CXCL13-CXCR5-dependent crosstalk with B lymphocytes. We further develop a single-cell/spatial TLS signature that captures the cellular ecosystem of iTLS-containing tumor, demonstrating predictive value for immunotherapy outcomes in GC patients.

Helicobacter pylori (H. pylori) is a gram-negative pathogen classified as a class I carcinogen. The H. pylori urease B subunit (UreB) and heat shock protein A (HspA) are two important vaccine candidate antigens. In this study, we evaluated the immunogenicity and immunoprotective effect of the attenuated Shigella vector vaccine SH02 expressing the UreB-HspA fusion protein of H. pylori in a mouse model. Oral SH02 with or without subcutaneous injection of rUreB-HspA induced antigen-specific serum IgG, mucosal sIgA, and T cells immune response. Subcutaneous injection of the candidate antigen rUreB-HspA enhanced the level of serum antigen-specific IgG antibodies (p < 0.0001) and the levels of IgG1/IgG2a/IgG2b subtypes. In addition, injection boost also increased the proportion of spleen antigen-specific CD4+CD154+ T cells (p < 0.001), and the proportion of CD4+CD154+ T cells that secrete IFN-γ and IL-17A. Following the H. pylori challenge, the levels of H. pylori colonization in the two experimental groups (Groups A and B) significantly reduced compared with the control group (p < 0.001), indicating that the candidate vaccine yielded a preventive effect of anti-H.pylori infection. Compared with the non-subcutaneous booster injection group (Group A), the subcutaneous booster injection group (Group B) exhibited less gastric inflammation, but there was no significant difference in the level of colonization (p > 0.05). These results lay a foundation for the development of a vaccine against H. pylori and the optimization of immunization methods and procedures to prevent H. pylori infection.

Bacterial surface display platforms have been developed for applications such as vaccine delivery and peptide library screening. The type V secretion system is an attractive anchoring motif for the surface expression of foreign proteins in gram-negative bacteria. SadA belongs to subtype C of the type V secretion system derived from Salmonella spp. and promotes biofilm formation and host cell adherence. The inner membrane lipoprotein SadB is important for SadA translocation. In this study, SadA was used as an anchoring motif to expose heterologous proteins in Salmonella typhimurium using SadB. The ability of SadA to display heterologous proteins on the S. typhimurium surface in the presence of SadB was approximately three-fold higher than that in its absence of SadB. Compared to full-length SadA, truncated SadAs (SadA⁸⁷⁷ and SadA²⁶⁹) showed similar display capacities when exposing the B-cell epitopes of urease B from Helicobacter pylori (UreB158–172aa and UreB349–363aa). We grafted different protein domains, including mScarlet (red fluorescent protein), the urease B fragment (UreBm) from H. pylori SS1, and/or protective antigen domain 4 from Bacillus anthracis A16R (PAD4), onto SadA⁸⁷⁷ or SadA¹²⁹². Whole-cell dot blotting, immunofluorescence, and flow cytometric analyses confirmed the localization of Flag×3-mScarlet (~30 kDa) and Flag×3-UreBm-mScarlet (~58 kDa) to the S. typhimurium surface using truncated SadA⁸⁷⁷ or SadA¹²⁹² as an anchoring motif. However, Flag×3-UreBm-PAD4-mScarlet (~75 kDa) was displayed on S. typhimurium using SadA¹²⁹². The oral administrated pSadBA¹²⁹²-FUM/StmΔygeAΔmurI and pSadBA⁸⁷⁷-FUM/StmΔygeAΔmurI could elicit a significant mucosal and humoral immunity response. SadA could thus be used as an anchoring motif for the surface expression of large heterologous proteins as a potential strategy for attenuated bacterial vaccine development.

Background: An emerging trend of mutual convergence between drug-resistant and highly virulent strains of K. pneumoniae has been identified, highlighting the urgent need for the development of novel vaccines. Methods: To delete the target genes and eliminate the plasmids carrying antibiotic resistance genes, CRISPR-Cas9 technology was employed to perform genome editing on a clinically isolated O2 serotype of K. pneumoniae. Subsequently, this strain was utilized as a host to express genes associated with the synthesis of O1 serotype LPSs to construct the recombinant strain capable of simultaneously expressing LPSs of both O1 and O2 serotypes. This recombinant strain was then used as the production strain for the preparation of vaccines based on GMMAs (Generalized Modules for Membrane Antigens), and its biological characteristics were characterized. Finally, the safety and immunogenicity of the vaccine were evaluated using mice as the model animals. Result: a GMMA vaccine characterized by a high yield and low toxicity was gained. Importantly, the lipopolysaccharides (LPSs) of both O1 and O2 serotypes of K. pneumoniae were successfully expressed on the surface of the outer membrane vesicles. Following immunization with the GMMA vaccine, mice were capable of producing antibodies against the GMMA and demonstrated resistance to the invasion of both serotypes of clinically isolated K. pneumoniae. Conclusions: The GMMA vaccine showed significant promise as a bivalent vaccine against K. pneumoniae.

Helicobacter pylori (H. pylori), heat-shock protein A (HspA), is a bacterial heat-shock chaperone that serves as a nickel ion scavenging protein. Ni²⁺ is an important co-factor required for the maturation and enzymatic activity of H. pylori urease and [NiFe] hydrogenase, both of which are key virulence factors for pathogen survival and colonization. HspA is an important target molecule for the diagnosis, treatment, and immune prevention of H. pylori. In this work, HspA was truncated into five fragments to determine the location of an antigen immunodominant peptide. A series of overlapping, truncated 11-amino-acid peptides in immunodominant peptide fragments were synthesized chemically and screened by ELISA. The immunogenicity and antigenicity of the screened epitope peptides were verified by ELISA, Western blot, and lymphocyte proliferation tests. Two novel B-cell epitopes were identified, covering amino acids 2–31 of HspA, which are HP11 (2–12; KFQPLGERVLV) and HP19 (18–28; ENKTSSGIIIP). The antiserum obtained from HP11-KLH and HP19-KLH immunized mice can bind to naive HspA in H. pylori SS2000, rHspA expressed in E. coli, and the corresponding GST fusion peptide. Among HspA seropositive persons, the seropositive rates of HP11 and HP19 were 21.4% and 33.3%, respectively. Both of the B-cell epitopes of HspA are highly conserved epitopes with good antigenicity and immunogenicity.

Most of the human gastric cancer (GC) worldwide are ascribed to Helicobacter pylori infections, which have a detrimental effect on the immunotherapy’s efficacy. Comprehensively dissecting the key cell players and molecular pathways associated with cancer immunotherapies is critical for developing novel therapeutic strategies against H. pylori infection-associated human GC. We performed a comprehensive single-cell transcriptome analysis of nine GC patients with current H. pylori infection (HpGC), three GC patients with previous H. pylori infection (ex-HpGC), six GC patients without H. pylori infection (non-HpGC), and six healthy controls (HC). We also investigated key cell players and molecular pathways associated with GC immunotherapy outcomes. We revealed the molecular heterogeneity of different cell components in GC, including epithelium, immune cells, and cancer-associated fibroblasts (CAFs) at the single-cell level. The malignant epithelium of HpGC exhibited high expression level of inflammatory and epithelial–mesenchymal transition signature, HpGC and ex-HpGC were enriched with VEGFA+ angiogenic tumor-associated macrophages (Angio-TAM) and IL11+ inflammatory CAF (iCAF), characterized by high expression levels of NECTIN2 and VEGFA/B. Additionally, we found significant correlations between the abundance of iCAF with Angio-TAM and TIGIT+ suppressive T cells, and iCAF interacted with Angio-TAM through the VEGF and ANGPTL angiogenic pathways. We also developed an immune signature and angiogenic signature and demonstrated that the iCAF abundance and angiogenic signature could predict poor immunotherapy outcomes in GC. We revealed the transcriptome characteristics and heterogeneity of various cellular constituents of HpGC patients and demonstrated that a synergistic combination of immunotherapy and anti-angiogenic targeted therapy may be an effective therapeutic modality for HpGC patients.