Yixin Chen

Trimerization motifs play pivotal roles in structural biology and therapeutic protein engineering. Here, we describe a short trimerization motif (rFd1303) derived from the Reoviridae family reovirus σ1 protein. Compared with the widely used T4-Foldon, the rFd1303 increased recombinant protein yields. The rFd1303-fused immunogens of SARS-CoV-2 spike and influenza hemagglutinin elicited antibody responses comparable to T4-Foldon-fused controls in mice. Using this tag, we engineered a trimeric ACE2-Ig fusion protein (TriACE2-Ig) that remained stable after 30 days at room temperature and neutralized a broad range of ACE2-utilizing sarbecoviruses, including 19 SARS-CoV-2 variants, SARS-CoV, and pangolin coronaviruses GD and GX, with higher potency than monomeric ACE2-Ig. In the hamster model challenged with various SARS-CoV-2 variants, our data demonstrated that intranasal TriACE2-Ig administration markedly reduced viral loads, virus-induced body-weight loss, lung pathology, and decreased within-cage virus transmission. These findings highlight rFd1303 as a versatile trimerization platform for vaccine and therapeutic protein development.

The principles governing how the immune system integrates distal infectious histories to orchestrate long-term, heterologous defense at remote mucosal barriers remain poorly defined. While trained immunity is well-characterized as a myeloid-mediated mechanism for antibacterial protection, the determinants of multi-lineage antiviral resistance are not established. Here, we show that recovery from systemic Plasmodium yoelii infection induces an integrated circuit between the respiratory epithelium, inflammatory monocytes, and CD8+ effector memory T cells (Tem) to provide broad protection against influenza A and respiratory syncytial viruses. In contrast to myeloid-dependent models, this resistance is strictly dependent on the synergy between both compartments; genetic or cellular ablation of either inflammatory monocytes or CD8+ T cells completely abolishes protection. Spatial and single-cell transcriptomics reveal that infected epithelial niches selectively recruit imprinted monocytes, which subsequently direct the site-specific accumulation and acute expansion of CD8+ Tem a Ccl4/Ccl5–Ccr5 axis. Within these localized foci, activated Tem executed rapid viral clearance through a T cell-intrinsic, INF- dependent manner. These findings define a multi-lineage "triad" synergy as an alternative arm of trained immunity, shifting the paradigm from cell-autonomous myeloid reprogramming toward an integrated innate–adaptive circuit for niche-restricted mucosal defense.

The concurrent circulation of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza virus, and respiratory syncytial virus (RSV) has caused severe coinfections, posing significant public health threats. Developing individual vaccines for these respiratory viruses is hindered by waning immunity and rapid viral mutations. A combination mRNA vaccine streamlines development and administration, reduces healthcare burdens, and offers convenient, compliance-enhancing protection against seasonal illnesses. Here, we present SIR, an 8-valent combination mRNA vaccine designed to simultaneously target SARS-CoV-2, influenza, and RSV. SIR was further optimized into SIRC by incorporating the TLR-9 agonist CpG oligodeoxynucleotides (CpG-ODN) 1018. SIRC induces robust binding and neutralizing antibodies against all three viruses, stimulates Th1-biased antigen-specific T-cell responses, and generates CD4⁺ and CD8⁺ memory T cells. In mice model, SIRC effectively protects against four influenza subtypes (H1N1, H3N2, B/V, and B/Y) and the RSV. Overall, SIRC demonstrates superior antibodies and T cell responses, and provides greater protection in vivo compared to both the individual and SIR vaccines, without causing immune interference. It also maintains a favorable safety and development potential. These findings establish the SIRC as a promising candidate for preventing coinfections involving SARS-CoV-2, influenza, and RSV.

Epstein-Barr virus (EBV) infects more than 90% of adults worldwide and causes a range of diseases, including multiple malignancies and autoimmune disorders. However, due to a host range restriction, EBV cannot infect commonly used experimental animals, posing a significant obstacle to developing EBV-specific prophylactic and therapeutic agents. Rhesus lymphocryptovirus (rhLCV), an ortholog of EBV, naturally infects rhesus macaques, which is a surrogate model for EBV research. In this study, we demonstrate that cynomolgus macaque (Macaca fascicularis), a primate closely related to rhesus macaque, is susceptible to rhLCV infection. rhLCV can immortalize B cells of cynomolgus macaques to develop cy-LCLs. We developed a high rhLCV-producing cy-LCL cell line, LCL111, and optimized the induction conditions to increase viral production, surpassing the original rhLCV producer LCL8664. Importantly, EBV gHgL-specific monoclonal antibody (mAb) AMMO1 and gB-specific mAb 3A5 can cross-react with rhLCV proteins and block the formation of cy-LCLs. Overall, we established an efficient rhLCV-producing cell line, and rhLCV infection of cynomolgus macaques represents a promising alternative surrogate model for efficiency evaluation of EBV vaccines and mAbs. IMPORTANCE: Epstein-Barr virus (EBV) naturally infects only humans, creating a major barrier to evaluating the efficiency of vaccines and therapies in vivo . As an EBV ortholog, rhesus lymphocryptovirus (rhLCV) offers a biologically relevant surrogate system. However, its application has been primarily limited to rhesus macaques. Here, we demonstrate that cynomolgus macaque lymphocytes are also susceptible to rhLCV in vitro, and the newly transformed cy-LCL111 shows superior and sustained rhLCV production ability. rhLCV infection of cynomolgus macaque lymphocytes can be efficiently neutralized by anti-EBV gH/gL nAbs AMMO1 and anti-EBV gB mAbs 3A5, highlighting the potential of cynomolgus macaques as an in vivo model to assess anti-EBV mAb and vaccine efficacy. Our findings support the use of cynomolgus macaques as an additional model for EBV research and offer a useful platform for evaluating EBV-specific prophylactic or therapeutic strategies.

Recurrence and metastasis are the main causes of death in ovarian cancer (OC). Long non-coding RNAs (lncRNAs) are considered as good prognostic models and potential therapeutic targets for cancer patients because of their easy detection and strong correlation. Our study identifies an OC-associated lncRNA with tumor progression and therapeutic implications. It’s found that lncRNA AC093895.1 is highly expressed in OC tissues and correlated with poor prognosis. AC093895.1 has a potentiating effect during the progression and metastasis of ovarian cancer. The effects of AC093895.1 on ovarian cancer cells are miR-1253 dependent. Results showed that by interacting with tumor-suppressive gene miR-1253 as competing endogenous RNA (ceRNAs), AC093895.1 significantly upregulated the downstream gene SOX4 of AC093895.1/ miR-1253 axis, leading to tumor metastasis. In addition, chromatin immunoprecipitation (ChIP) results further confirmed that SOX4 could bind to the AC093895.1 promoter, forming a positive feedback loop SOX4/AC093895.1/miR-1253/SOX4. Therapeutic strategy to break the loop through AC093895.1 knockdown exhibited attenuated OC growth and metastasis in vivo both in SK-OV-3 subcutaneous model and pulmonary metastatic model. Our study unveiled the potentiating effects of SOX4/AC093895.1/miR-1253/SOX4 on ovarian cancer cell survival, migration, and invasion. AC093895.1 may be a promising patient prognostic biomarker and therapeutic candidate.

The global prevalence and disease burden of alphaherpesviruses infections, including human-infecting viruses such as HSV-1, HSV-2, and VZV, as well as animal-infecting viruses like PRV, BHV, CHV, and FHV, highlight the unmet need for more effective and universal antiviral strategies. However, there has been no significant progress in developing broad-spectrum interventions against herpesvirus. Here we report the identification of a broadly neutralizing antibody against alphaherpesviruses, 16F9, which targets the glycoprotein B (gB) of alphaherpesviruses and offers cross-protection against multiple viruses such as HSV-1, HSV-2, and PRV in mice. 16F9 demonstrated robust therapeutic efficacy in various female mouse models of herpesvirus diseases including PRV-induced viral encephalitis, HSV-1-induced viral encephalitis, viral keratitis, cutaneous herpes, and neonatal herpesvirus infections. High-resolution cryo-electron microscopy structures revealed that 16F9 binds a conserved site of vulnerability on Domain I of gB. The binding of 16F9 disrupts the interaction between pre-gB and gHgL complex, thereby preventing viral membrane fusion and blocking viral infection. This study provides a foundation for advancing antiviral strategies and underscores the potential of gB-targeted interventions for combating herpesvirus infections.

Given their critical role in eliciting respiratory immunity, mucosal SARS-CoV-2 vaccines receive significant research attention. Our previous phase 1/2 trial revealed that aerosolized live-attenuated vectored influenza virus COVID-19 vaccine likely elicited specific cellular immunity and weak s-IgA responses in placebo recipients. Here, we demonstrated that viral vector vaccines undergo contact or airborne transmission in various animal models. Vaccines have difficulty spreading in the mouse model, but can effectively spread through the air in more sensitive ferrets. However, the virus transmission in ferrets can be effectively restricted by ventilation system. Furthermore, contact transmission of virus vaccine provided effective protection against lung pathology post-challenge with SARS-CoV-2 in hamsters. These findings suggest the potential to achieve herd immunity in specialized scenarios through airborne or contact transmission of dNS1-RBD, provided that appropriate delivery and control measures are implemented during the administration of live attenuated or viral vectored vaccines.

Influenza remains a significant global public health concern. Live-attenuated influenza vaccines (LAIVs) are recognized as effective interventions for influenza prevention. Currently, two types of LAIVs are licensed for human use: one developed through cold-adapted viral gene mutation and the other through the deletion of the viral NS1 gene. However, the similarities and differences in these two LAIVs’ efficacy, safety, and immune responses have not been thoroughly studied. This study constructed a gene-deficient live-attenuated vaccine strain, CA4-dNS1, and a gene locus-mutated attenuated vaccine strain, CA4-cold, to compare their in vivo and in vitro replication capacity, broad-spectrum protective efficacy, safety, and immunogenicity. The results showed that both LAIVs provide comparable broad-spectrum protection against lethal H1N1 and H5N1 influenza challenges in mice and induce similar humoral and mucosal immune responses. Notably, the CA4-cold vaccine strain induces superior influenza memory T-cell responses, while the CA4-dNS1 vaccine strain demonstrates greater safety. These findings underscore the importance of gene modification in LAIVs in striking a balance between their safety and efficacy. The NS1 gene-deficient CA4-dNS1 strain may offer a more advantageous approach for developing next-generation LAIVs and other intranasal influenza virus vectored vaccines due to enhanced safety.

Zoonotic H7N9 avian influenza virus infection remains a global concern because of its pandemic potential. Therefore, developing effective antibodies and vaccines against H7N9 is vital for preventing and controlling major outbreaks. Here, we isolated a human VH3-30 gene-encoded antibody, designated 6Y13, from a survivor of H7N9 infection. This antibody recognized the hemagglutinins (HAs) of the representative H7 subtype zoonotic viruses spanning two decades of antigenic evolution and potently neutralized epidemic H7N9 viruses in vitro. Moreover, 6Y13 conferred complete protection in mice against lethal H7N9 challenge in both prophylactic and therapeutic experiments. Structural analysis by cryoelectron microscopy indicated that 6Y13 binds to a unique conserved site on the HA head, distinct from the receptor-binding site and lateral patch. Nevertheless, 6Y13 efficiently blocked viral receptor binding without interfering with HA receptor binding, independent of Fc-mediated steric hindrance. Our findings provide a promising therapeutic candidate against pan-H7 subtype viruses and are beneficial for the design of H7 subtype influenza vaccine immunogens.

Combination vaccines promise to simplify immunization schedules and improve coverage, but remain technically challenging owing to antigen compatibility, immunogenic balance and formulation complexity. Here we report a modular strategy that uses a single-component nanobody binder to noncovalently attach diverse antigens to intact particles from the licensed hepatitis E vaccine. To identify a suitable binder, an alpaca was immunized with the vaccine, and nanobodies were screened via phage display. One nanobody, P1-5B, selectively bound recessed, non-immunodominant sites on the particle surface and enabled stable antigen display without disrupting native immunogenicity. Using this binder, we generated three vaccine formulations displaying five to eleven antigens, including variants from SARS-2 coronavirus, influenza virus and respiratory syncytial virus. These multivalent particles exhibited high-affinity assembly, preserved solubility and induced neutralizing titres up to three log units higher than soluble antigens. In mice, hamsters and non-human primates, the candidate vaccines conferred robust protection and showed a favourable safety profile. This approach introduces a scalable, plug-and-display system for rapid development of customizable combination vaccines.