Fan Wu

Background: Tertiary lymphoid structures (TLSs) play a crucial role in regulating tumor invasion and metastasis and serve as a promising prognostic biomarker in immunotherapy, influencing survival and immune response in multiple cancers. However, existing studies rely on limited gene signatures to assess TLSs, and there remains a lack of comprehensive TLS-related features for pan-cancer prognosis or immunotherapy response prediction. Methods: Based on published TLS gene signatures, mutation data, and expression profiles from 33 tumor types in TCGA, along with data from 15 immune checkpoint blockade (ICB) cohorts, we first systematically evaluated six TLS gene signatures in relation to immune-related indicators and assessed their predictive and prognostic performance across tumors and immunotherapy. Subsequently, using meta-analysis, we constructed a de novo TLS-related gene feature set, termed predictTLS, designed to predict ICB efficacy and prognosis. The rationality and effectiveness of predictTLS were validated using internal validation sets, single-cell transcriptomic, and spatial transcriptomic data. Results: The evaluation revealed associations between TLS gene signatures and key immune-related indicators. The newly constructed predictTLS feature set demonstrated effectiveness in predicting both ICB therapy outcomes and patient prognosis across the analyzed cohorts. Validation across internal datasets, single-cell profiles, and spatial transcriptomics supported the robustness and biological relevance of predictTLS. Conclusions: This study provides a systematically validated, de novo TLS-related gene signature that can serve as a clinical biomarker for predicting immunotherapy response and prognosis in pan-cancer settings. These findings offer new tools for risk stratification and potential therapeutic targeting in tumor immunotherapy.

Severe COVID-19 is characterized by profound immune dysregulation and excessive inflammation. Aberrant myeloid responses drive the hyperinflammation, yet the precise mechanisms remain elusive. Here, we demonstrate that certain spike antibodies promote the formation of heterotypic syncytia between monocytes/macrophages and virus-infected pneumocytes, leading to excessive inflammatory cytokine release. This antibody-dependent syncytium formation requires FcγRI (CD64) and ADAM10, as well as the assembly of the six-helix bundle by spike S2 subunit. Notably, these syncytia exhibit a more potent pro-inflammatory signature compared to virus-infected epithelial cells. In vivo, sub-neutralizing antibody concentrations amplified inflammation and exacerbated disease in SARS-CoV-2-infected mice without increasing viral burden. Furthermore, scRNA-seq of postmortem lung tissues from COVID-19 patients implicated the monocytes/macrophages-derived syncytia as a key cellular source of inflammatory cytokines. Together, these findings define a novel mechanism of antibody-dependent enhancement of inflammation that helps explain the hyperinflammatory responses in severe COVID-19, and suggest new therapeutic opportunities targeting this pathway.

Objectives: Understanding the antibody response in monkeypox virus (MPXV)-infected and uninfected individuals is essential for developing next-generation MPXV vaccines. This study aimed to characterize neutralizing antibody (NAb) and antibody-dependent cellular cytotoxicity (ADCC) responses in both groups, providing insights into immune protection and vaccine design. Methods: A recombinant vaccinia Tian Tan (VTT) virus was utilized to develop high-throughput luciferase-reporter-based neutralization and ADCC assays. These assays were applied to evaluate the presence and levels of poxvirus-specific antibodies in MPXV-infected and uninfected individuals, including those vaccinated with vaccinia-based vaccines. Results: Poxvirus-specific NAbs were detected in MPXV-negative individuals with prior vaccinia vaccination. However, MSM individuals exhibited significantly lower pre-existing NAb levels than non-MSM individuals, potentially contributing to their higher susceptibility to MPXV infection. In individuals with mild MPXV infection, robust NAb and ADCC responses were observed, regardless of vaccination status. Additionally, HIV-positive individuals demonstrated comparable antibody responses following MPXV infection. Conclusions: These findings highlight the potential role of pre-existing NAbs in MPXV susceptibility and the strong immune response elicited by mild MPXV infection. Further research is needed to determine whether MPXV-specific antibodies mitigate disease progression, which could inform the development of effective MPXV vaccines.

The discovery of broadly neutralizing antibodies (bNAbs) that target conserved epitopes on the HIV-1 envelope glycoprotein (Env) has garnered significant attention for its potential in the development of effective therapeutic and vaccine strategies. In this study, we isolated and characterized a CD4 binding site (CD4bs) antibody, FD22, from an elite neutralizer in China who had been infected with a clade B virus through contaminated blood plasma for 23 years. The heavy chain of FD22 was derived from a rarely reported IGHV3-30 germline gene and exhibited an exceptionally high degree of somatic hypermutation (SHM) (37%), along with a long and unique CDRH3 loop of 20-amino acids. FD22 exhibited potent and broad neutralizing activity, comparable to that of the well-known bNAb VRC01. It effectively neutralized 82% of a panel of 145 diverse HIV-1 pseudoviruses, including the two major circulating strains in China, CRF01_AE and CRF07_BC. FD22 bound strongly to HIV-1-infected cell lines, efficiently engaged FcγRIIIa receptors, triggered NK cell degranulation and the release of key cytokines such as IFN-γ and β-chemokines, and robustly induced antibody-dependent cellular cytotoxicity (ADCC) against HIV-1-infected target cells. Structural prediction for FD22 and the HIV Env SOSIP trimer performed by AlphaFold3, site-mutagenesis, and autologous virus reverse mutation assays revealed that the epitope of FD22 spans key CD4 binding site, including Loop D, the CD4 binding loop (CD4 BLP), and the V5 Loop. The unique long CDRH3 loop of FD22 interacts with the CD4 binding site through its negatively charged residue R102, distinguishing it from other CD4bs antibodies. Our findings provide valuable insights into the mechanisms of FD22 in viral neutralization and ADCC. The dual functionality of FD22 enhances its potential as a promising therapeutic antibody and offers new avenues for designing CD4bs-targeting vaccines with enhanced ADCC capabilities.

In November 2023, there was a substantial increase in the incidence of Mycoplasma pneumoniae infections in China following waves of SARS-CoV-2 Omicron variant and influenza outbreaks. This study aimed to elucidate the epidemiological features and clinical implications of M. pneumoniae infections in children and explore the potential influence of SARS-CoV-2 Omicron variants and influenza A infections on the M. pneumoniae outbreak. Among 38,668 children with lower respiratory tract infections from January to December 2023, 11,919 tested positive for M. pneumoniae , predominantly between October and December. The majority of the children with M. pneumoniae were aged 5–10 years, with type 1 strains and macrolide-resistant M. pneumoniae strains having the highest prevalence rates. Statistical analysis revealed elevated C-reactive protein, neutrophil, and monocyte levels and decreased lymphocyte, basophil, and eosinophil counts in M. pneumoniae -positive children. M. pneumoniae -positive children also presented significantly increased neutralizing antibody levels against preceding influenza A (H3N2) but not against SARS-CoV-2 Omicron variants. A parallel trend was observed between M. pneumoniae and H3N2 prevalence from June to December 2023. The emergence of macrolide-resistant strains and prior influenza A (H3N2) epidemics notably contributed to the M. pneumoniae outbreak. These findings suggested that H3N2 infection facilitates M. pneumoniae infection through various mechanisms. This study underscores the complex interactions between respiratory pathogens and highlights the need for comprehensive surveillance and response strategies. IMPORTANCE: This study identified key factors contributing to an outbreak of Mycoplasma pneumoniae that affected 11,919 children. The influencing factors included a high prevalence of macrolide-resistant epidemic strains (94.2%) and significantly higher H3N2 neutralizing antibody levels ( P < 0.0001) stimulated by the preceding H3N2 influenza epidemic. These findings highlight the complex relationship between the prevalence of M. pneumoniae and H3N2 infection in children, indicating that it is necessary to consider pathogen interactions in respiratory disease management by continuously monitoring respiratory pathogens. The emergence of macrolide-resistant strains in China and the previous H3N2 influenza epidemic significantly exacerbated the severity of the M. pneumoniae outbreak. H3N2 infection potentially amplifies Mycoplasma transmission. This study elucidates the epidemiological and clinical aspects of M. pneumoniae infections in children, yields insights regarding the cause of the outbreak, and provides guidance for improving respiratory infection management.

The Omicron subvariants BQ.1.1, XBB.1.5, and XBB.1.16 of SARS-CoV-2 are known for their adeptness at evading immune responses. Here, we isolate a neutralizing antibody, 7F3, with the capacity to neutralize all tested SARS-CoV-2 variants, including BQ.1.1, XBB.1.5, and XBB.1.16. 7F3 targets the receptor-binding motif (RBM) region and exhibits broad binding to a panel of 37 RBD mutant proteins. We develop the IgG-like bispecific antibody G7-Fc using 7F3 and the cross-neutralizing antibody GW01. G7-Fc demonstrates robust neutralizing activity against all 28 tested SARS-CoV-2 variants and sarbecoviruses, providing potent prophylaxis and therapeutic efficacy against XBB.1 infection in both K18-ACE and BALB/c female mice. Cryo-EM structure analysis of the G7-Fc in complex with the Omicron XBB spike (S) trimer reveals a trimer-dimer conformation, with G7-Fc synergistically targeting two distinct RBD epitopes and blocking ACE2 binding. Comparative analysis of 7F3 and LY-CoV1404 epitopes highlights a distinct and highly conserved epitope in the RBM region bound by 7F3, facilitating neutralization of the immune-evasive Omicron variant XBB.1.16. G7-Fc holds promise as a potential prophylactic countermeasure against SARS-CoV-2, particularly against circulating and emerging variants.

The ectodomain of influenza matrix protein 2 (M2e) is a promising target for the development of universal prophylactic and therapeutic agents against influenza viruses of different subtypes. We constructed three M2e‐specific monoclonal antibody variants, M2A1‐1 (IgG1), M2A1‐2a (IgG2a), M2A1‐2b (IgG2b), which have the same Fab region targeting the M2e epitope but different isotypes, and compared their protective efficacy in influenza PR8‐infected mice. We found that anti‐M2e antibodies provided protection against influenza virus in a subtype‐dependent manner, with the IgG2a variant providing significantly better protection with lower virus titers and milder lung injury than IgG1 and IgG2b isotypes. Additionally, we observed that the protective efficacy was dependent on the administration routes, with intranasal administration of antibody providing better protection than intraperitoneal administration. The timing of administration was also critical in determining the protective efficacy; while all the antibody isotypes provided protection when administered before influenza challenge, only IgG2a provided minimal protection when the antibodies were administered after virus challenge. These results provide valuable information for optimizing the therapeutics usage of M2e‐based antibodies and furthering the development of M2e‐based universal influenza vaccines.

The highly mutated and transmissible Omicron (BA.1) and its more contagious lineage BA.2 have provoked serious concerns over their decreased sensitivity to the current COVID-19 vaccines and evasion from most anti-SARS-CoV-2 neutralizing antibodies (NAbs). In this study, we explored the possibility of combating the Omicron and BA.2 by constructing bispecific antibodies based on non-Omicron NAbs. We engineered 10 IgG-like bispecific antibodies with non-Omicron NAbs named GW01, 16L9, 4L12, and REGN10987 by fusing the single-chain variable fragments (scFvs) of two antibodies through a linker and then connecting them to the Fc region of IgG1. Surprisingly, 8 out of 10 bispecific antibodies showed high binding affinities to the Omicron receptor-binding domain (RBD) and exhibited extreme breadth and potency against pseudotyped SARS-CoV-2 variants of concern (VOCs) including Omicron and BA.2, with geometric mean of 50% inhibitory concentration (GM IC₅₀) values ranging from 4.5 ng/mL to 103.94 ng/mL, as well as the authentic BA.1.1. Six bispecific antibodies containing the cross-NAb GW01 not only neutralized Omicron and BA.2, but also neutralized the sarbecoviruses including SARS-CoV and SARS-related coronaviruses (SARSr-CoVs) RS3367 and WIV1, with GM IC₅₀ ranging from 11.6 ng/mL to 103.9 ng/mL. Mapping analyses of 42 spike (S) variant single mutants of Omicron and BA.2 elucidated that these bispecific antibodies accommodated the S371L/F mutations, which were resistant to most of the non-Omicron NAbs. A cryo-electron microscopy (cryo-EM) structure study of the representative bispecific antibody GW01-16L9 (FD01) in its native full-length IgG form in complex with the Omicron S trimer revealed 5 distinct trimers and one novel trimer dimer conformation. 16L9 scFv binds the receptor-binding motif (RBM), while GW01 scFv binds a epitope outside the RBM. Two scFvs of the bispecific antibody synergistically induced the RBD-down conformation into 3 RBD-up conformation, improved the affinity between IgG and the Omicron RBD, induced the formation of trimer dimer, and inhibited RBD binding to ACE2. The trimer dimer conformation might induce the aggregation of virions and contribute to the neutralization ability of FD01. These novel bispecific antibodies are strong candidates for the treatment and prevention of infection with the Omicron, BA.2, VOCs, and other sarbecoviruses. Engineering bispecific antibodies based on non-Omicron NAbs could turn the majority of NAbs into a powerful arsenal to aid the battle against the pandemic.