Abstract Severe pneumonia caused by respiratory virus infection is one of the main threats to the lifespan of the aged population and physiological aging fundamentally drives poor disease outcomes in respiratory viral infections, necessitating investigation of the underlying mechanisms and the development of effective countermeasures. Pulmonary transcriptomic profiling reveals that diffuse cell death, inadequate antiviral responses, myeloid-driven excessive inflammation and immune-thrombosis dictate the pulmonary microenvironment are typical molecular pathology characteristics of the SARS-CoV-2-infected aged hamsters rather than the adult controls. The elevated pathological baseline and dysregulated immune responses are demonstrated as the key host factors of lethal viral pneumonia in the aged hamsters. Meanwhile, SARS-CoV-2 infection in the adult hamsters usually resulted in an aged-like pulmonary transcriptomic signature, suggesting a potential link among aging, dysregulated immune responses and severe illness. To reverse the progression of lethal severe pneumonia in the aged hamsters, we initiated a multidimensional combination therapy of dexamethasone, heparin and the broad-spectrum viral decoy CoVR-MV at three days after infection. Although single- and dual-drug therapies were insufficient to achieve functional cure, the three-drug combination therapy resulted in a potent reduction of high mortality, body weight loss, viral load, lung pathology and cytokine storm through the synergism of immunoregulatory, anticoagulant and antiviral effects, effectively intercepting the multifaceted pathogenic network. This study highlights physiological aging as a core driver of critical COVID-19 pathogenesis and provides valuable clues for the rational design of multi-drug and multi-target therapies against respiratory viral infections and lethal severe pneumonia in the elderly population.

Sexual hormones play an important role in modulating disease outcome of COVID-19. The interplay between viral replication, host immune responses, pathology process and sexual hormone levels are complicated, and the underlying mechanisms remain exclusive. Here, we reveal the dose-dependent manner and multi-faceted role of the male hormone testosterone in hamster model of COVID-19 by evaluations of manifestations including survival rate, body weight loss, viral load, immune responses and lung injury. Both low and high doses of testosterone treatment cause more severe illness in male hamsters. Low dose of testosterone is beneficial for female hamsters, but high dose is harmful. Therefore, we evaluate the therapeutic effect of the testosterone inhibitor finasteride in male hamsters and demonstrate that it is sufficient to prevent death and severe pneumonia caused by different SARS-CoV-2 strains. Moreover, pulmonary transcriptome data reveals key clues for the mechanisms of testosterone-mediated disease enhancement and finasteride therapy.

The continued evolution of SARS-CoV-2 has reduced the protective effectiveness of first-generation vaccines and underscores the need for broadly reactive next-generation vaccine candidates. Here, we evaluated STFKB, an alum-adjuvanted bivalent recombinant protein vaccine composed of the monomeric Spike (STFKprototype) and the engineered Spike (STFK1628X). We assessed the immunogenicity, tolerability, and protective efficacy of STFKB in mice, rats, guinea pigs, rhesus macaques, and Syrian hamsters. STFKB induced robust STFK- and STFK1628X-specific antibody responses and broadly reactive neutralizing antibodies against multiple SARS-CoV-2 variants in the tested animal models. In hamster challenge studies, STFKB vaccination protected animals from Omicron BA.1 and BA.5 challenge, as shown by reduced body-weight loss, lower viral RNA loads in respiratory tissues, and improved gross lung pathology. Across the tested preclinical models, STFKB was well tolerated, with no vaccine-related overt toxicity observed under the study conditions. These findings support the rationale and translational potential of the bivalent vaccine strategy proposed in this study.

Over the past decades, respiratory virus infections have led to millions of cases of critical illness and deaths in humans. However, the high mutation rate of respiratory viruses greatly reduces the effectiveness of virus-target countermeasures such as vaccines and antibodies, necessitating the development of host-target immunotherapies. In a hamster model of SARS-CoV-2 infection, we observed diverse disease outcomes, delineated the variant-specific lung transcriptome landscape and demonstrated that a rational combination of pharmacological targeting of innate and adaptive immune responses is sufficient to reduce mortality and severe illness caused by beta, delta and EG.1 variants. The synergism of Poly IC-mediated activation of the innate immune response and FK506-mediated inhibition of the adaptive immune response resulted in significant suppression of both viral load and lung injury. Notably, this strategy is also available in humanized mice infected with SARS-CoV-2, H1N1 and H3N2 influenza viruses, suggesting a potent cross-virus broad-spectrum therapeutic effect.

Rapid advances in vaccine technology are becoming increasingly important in tackling global health crises caused by respiratory virus infections. While traditional vaccines, primarily administered by intramuscular injection, have proven effective, they often fail to provide the broad upper respiratory tract mucosal immunity, which is urgently needed for first-line control of respiratory viral infections. Furthermore, traditional intramuscular vaccines may not adequately address the immune escape of emerging virus variants. In contrast, respiratory mucosal vaccines developed using the body's mucosal immune response mechanism can simultaneously establish both systemic and mucosal immunity. This dual action effectively allows the respiratory mucosal immune system to function as the first line of defense, preventing infections at the entry points. This review highlights the efficacy of respiratory mucosal vaccines, including innovative delivery methods such as nasal and oral formulations, in enhancing local and systemic immune barriers. Notably, respiratory mucosal vaccines offer potential advantages in protecting against emerging virus variants and maintaining long-term and multidimensional immune memory in the upper respiratory tract. In addition, a combination of intramuscular and respiratory mucosal delivery of vaccines largely improves their coverage and effectiveness, providing valuable insights for future vaccine development and public inoculation strategies.

Viruses have evolved multiple mechanisms to counteract the stimulator of the interferon genes (STING) pathway, resulting in the suppression of antiviral responses. Accordingly, in addition to developing STING agonist analogs with enhanced stability and deliverability, overcoming the defective STING function in virus-infected cells is essential for defense against viral infections. In this study, we developed STING pathway-activating complexes (SPAC) based on bioinspired vesicles that display and multimerize STING molecules with a specific affinity for agonist binding. As a broad-spectrum antiviral agent, this universal STING mimic triggers IFN-I signaling independently of endogenous STING. In infectious models, including CMV and SARS-CoV-2 infection, both prophylactic and therapeutic regimens of SPAC can reduce viral load and disease severity. These results indicate that SPAC, functioning as a host-targeted immune modulator, provides the distinct advantage of broad-spectrum therapy against infectious diseases caused by both DNA and RNA viruses, particularly those with strong STING antagonistic functions. The endogenous STING-independent activation mechanisms of SPAC may provide a universal therapy for infectious diseases, potentially serving as a candidate option to defend against future pandemics of "Disease X".

Trimerization motifs play pivotal roles in structural biology and therapeutic protein engineering. Here, we engineered a novel short trimerization motif (rFd1303) derived from the Reoviridae family reovirus σ1 protein. Compared with the widely used T4-Foldon, the rFd1303 enhanced thermal stability and 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 murine models. Leveraging the new tag, we engineered the trimeric ACE2-Ig (TriACE2-Ig), which exhibits exceptional stability (room-temperature storage for 30 days) and broad neutralization against multiple SARS-CoV-2 variants (average IC50 of 2.9 ng/mL), showing a 20.9-fold potency improvement over 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.

BACKGROUND:Liver involvement is a common complication of coronavirus disease 2019 (COVID-19), especially in hospitalized patients. However, the underlying mechanisms involved are not fully understood. METHODS:Immunohistochemistry (IHC) staining of SARS-CoV-2 spike (S) and nucleocapsid (N) proteins was conducted on liver tissues from six patients with COVID-19. The 10x Genomics Visium CytAssist Spatial Gene Assay was designed to analyze liver transcriptomics. TCR CDR3 sequences were analyzed in DNA from liver tissues. Liver function indicators were retrospectively studied in 650 hospitalized patients with COVID-19. FINDINGS:SARS-CoV-2 proteins were initially detected in the livers of naturally infected golden (Syrian) hamsters, prompting us to investigate the situation in clinical cases. Thus, we collected liver tissues from patients with abnormal liver biochemical values. Viral S and N proteins were detected in the livers of severe and deceased patients but not in those of moderate patients. We further demonstrated that hepatocytes and erythroid cells in hepatic sinusoids are major cells targeted by SARS-CoV-2. Immune cells, especially T cells, were enriched in surviving severe patients, characterized by enhanced CDR3α clonality and novel CDR3β recombination of the T-cell receptor. In contrast, hepatocyte apoptosis was triggered, and the transcription of albumin (ALB) was obviously impaired in the deceased patients. We then performed a retrospective study including patients with COVID-19. Serum aspartate aminotransferase (AST) and ALB levels at baseline significantly differed in the deceased cohort. However, AST regression did not decrease the risk of death. ALB recovery indicated clinical improvement, and declining or low serum ALB concentrations were associated with death. INTERPRETATION:This study provides clinical evidence for liver infection with SARS-CoV-2, insight into the impact of SARS-CoV-2 on the liver, and a potential way to evaluate the risk of death via assessing serum ALB concentration fluctuations in patients with COVID-19. FUNDING:National Key R&D Program of China (2021YFC2300602), National Natural Science Foundation of China (92369110), National Natural Science Foundation of China (U23A20474), Shanghai Municipal Science and Technology Major Project (ZD2021CY001), Shanghai Jinshan District Medical and Health Technology Innovation Fund Project (2023-WS-31).

severeacute respiratory syndrome coronavirus 2 (SARS-CoV-2) bivalent vaccines show potential against variants but lack a full understanding of the immunological mechanisms that drive broadly neutralizing antibodies (bnAbs). This study explored the immunogenicity of a bivalent vaccine in rhesus macaques, containing spike (S) proteins from the prototype (Sprototype) and chimeric S protein (S1628x). The vaccine induced bnAbs against multiple variants, including challenging subvariants like EG.1, BA.2.86, and JN.1. The monomeric S protein exposed less accessible regions within the receptor-binding domain (RBD) "inner face" and "NTD face" and subdomains 1, eliciting a diverse array of bnAbs against various Omicron subvariants. Notably, antibodies targeting the conserved RBD inner face, such as 4A5, showed potent neutralization across all tested variants. Structural analyses provide insights into the broad protectiveness of these vaccine-elicited nAbs. This study underscores the potential of bivalent vaccines with monomeric spike proteins to confer broad-spectrum immunity, offering a promising direction for future SARS-CoV-2 universal vaccine design.

Itaconate (ITA), an immunomodulatory metabolite with known anti-inflammatory properties, has underexplored therapeutic or prophylactic potential against coronavirus disease 2019 (COVID-19). Using an interanimal transmission golden hamster model of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced acute lung injury, we first assessed ITA changes in dNS1-RBD-vaccinated hamsters via metabolomic profiling. Then, we evaluated prophylactic intranasal (20 mg/kg at 9, 6, and 3 days before SARS-CoV-2 infection) and therapeutic intraperitoneal (100 mg/kg at 6, 24, and 48 hours post-infection) ITA administration, assessed by histopathology, transcriptomic, and metabolomic profiling, followed by multi-omics integration, including gene expression clustering, pathway enrichment, and cytokine/chemokine–metabolites correlation analyses. Public bronchoalveolar lavage fluid (BALF) single-cell RNA-sequencing (scRNA-seq) datasets from COVID-19 patients were re-analyzed to explore macrophage heterogeneity. Intranasal dNS1-RBD vaccine upregulated ITA levels, prompting further exploration of its immunomodulatory role. Both prophylactic and therapeutic ITA treatments significantly mitigated weight loss and improved lung pathology. Correlation analyses implied a potential regulatory crosstalk between fatty acid β-oxidation (FAO) and reduced inflammatory response. Re-analysis of BALF scRNA-seq dataset highlighted transcriptional networks involving PPARG, RARA, BHLHE41, TCF7L2, and ESRRA—genes linked to macrophage self-renewal and metabolic homeostasis, which appeared to be preserved in ITA-treated hamsters. These findings underscore ITA's role in modulating immunometabolic responses, particularly through FAO-driven macrophage reprogramming, to attenuate SARS-CoV-2-induced lung damage. Together, this study provides insights into host-directed therapies targeting metabolic reprogramming to mitigate COVID-19 severity.

The pandemics of respiratory viruses pose a worldwide public health problem and bio-safety threat. Therefore, the development of high-throughput and accurate infection models is crucial for elucidating viral pathogenesis and accelerating countermeasures to address the evolving respiratory viruses and the unexpected outbreaks of emerging variants. Compared to traditional 2D cultures, organoids exhibit pronounced intercellular interactions, extracellular matrix signaling, and tissue-specific multicellular cooperation, thereby more accurately recapitulating the in vivo microphysiological environment. However, research involving animal models typically requires prolonged experimental timelines, making it challenging to perform high-throughput screening or rapidly develop therapeutic strategies within the valuable timeframe. Since the outbreak of SARS-CoV-2, organoids have significantly advanced basic virology research and demonstrated potential in replicating the pathological and immunological characteristics in human patients. This review provides a comprehensive summary of the theoretical foundations, methodological framework, and complete procedures for identification and validation in organoid construction, along with their applications in the investigation of various respiratory viruses, such as coronaviruses, the influenza virus, respiratory syncytial virus, and others. Overall, the development of organoids, in conjunction with the integration of interdisciplinary technologies, has significantly advanced our fundamental understanding of the immunopathology process of respiratory viral infections, improved research efficiency, and provided precise tools for translational medical research.

The evolution and mutation of SARS-CoV-2 is elusive. However, the diverse in vivo pathogenicity and transmissibility of different SARS-CoV-2 Omicron/XBB variants are not well understood. We compared virological attributes of two XBB variants, XBB.1.16 and XBB.1.9.2.1 (EG.1) in new-born, juvenile, adult, middle-aged and senescent Syrian hamsters. In particular, EG.1 has a specific Q613H mutation and causes fatal severe pneumonia in hamsters of all ages. In contrast, all hamsters infected with XBB.1.16 survived and showed milder symptoms. The XBB.1.16 infected hamsters lost significantly less body weight and exhibited lower respiratory viral loads, pro-inflammatory cytokines and lung injury than those with EG.1 infection. In addition, EG.1 is more transmissible than XBB.1.16 in close contact co-housing. Both EG.1 and XBB.1.16 are highly resistant to therapeutic antibodies and convalescent serum. Overall, the unpredictable evolution, global transmission and potential threat of emerging SARS-CoV-2 variants necessitate the updating of prophylactic and therapeutic countermeasures in all age groups.

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.

The rapid evolution of SARS-CoV-2 and the subsequent emergence of Omicron subvariants pose significant challenges to the efficacy of existing vaccines and therapeutics, including those previously reported most broad neutralizing antibodies (bnAbs). Here, we investigated the molecular basis of the altered neutralization profile of a bnAb, 1C4, against recent variants. 1C4 is effective against early variants from Alpha to Omicron BQ.1, but is circumvented by BQ.1.1, XBB and thereafter variants, primarily due to an additional R346T mutation that diminishes its binding affinity. Cryo-electron microscopy analysis revealed that despite the loss of neutralizing potency, 1C4 retained residual binding to the spike protein of immune-evasive variants such as XBB, which harbor altered receptor-binding domain (RBD). Furthermore, 1C4 exhibited a diminished capacity to inhibit ACE2 engagement with Omicron variants, amplifying the intricacies of viral immune evasion tactics. To address this, we employed the mi3-SpyCatcher-based nanoparticle to polymerize 1C4 (mi3-1C4), which reestablished the neutralization potency against recent variants by enhancing avidity via multivalent binding. Such multivalent binding can promote efficient spike aggregation as well as viral cross-linking, thereby providing enhanced protection against both the infection of Beta and XBB variants in a hamster model. Together, our findings delineate the molecular landscape of immune evasion by neutralizing antibodies and provide strategic insight for the adaptation of antibody engineering to keep pace with viral evolution.

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. A modular nanobody-based approach assembles combination vaccines by stably attaching diverse antigens to hepatitis E virus-like particles, preserving native immunogenicity and inducing broad protection against respiratory pathogens.

Despite remarkable achievements in antibody‒drug conjugates (ADCs), payloads remain limited. The identification of ADC payloads with novel mechanisms will increase therapeutic options and expand indications. Herein, we describe the use of dihydroorotate dehydrogenase inhibitors (DHODHi) as a novel payload class that provides highly potent ADCs for antitumor and antiviral therapies. Technical innovations include the development of stability-controllable linkers to meet the distinct requirements of acute viral infections and chronic tumor conditions. The antitumor ADC TH-C8H exhibited significant efficacy against gastric cancer in vivo as monotherapy and enhanced efficacy when combined with the ferroptosis inducer RSL3. The antiviral ADC HG-C3 showed broad-spectrum anti-SARS-CoV-2 activity in vitro and in vivo. Our study expands the types of ADC payloads and provides novel insights into the development of innovative broad-spectrum ADCs.

Studies have reported variable effects of sex hormones on serious diseases. Severe disease and mortality rates in COVID-19 show marked gender differences that may be related to sex hormones. Sex hormones regulate the expression of the viral receptors ACE2 and TMPRSS2, which affect the extent of viral infection and consequently cause variable outcomes. In addition, sex hormones have complex regulatory mechanisms that affect the immune response to viruses. These hormones also affect metabolism, leading to visceral obesity and severe disease can result from complications such as thrombosis. This review presents the latest researches on the regulatory functions of hormones in viral receptors, immune responses, complications as well as their role in COVID-19 progression. It also discusses the therapeutic possibilities of these hormones by reviewing the recent findings of clinical and assay studies.

The poor prognosis observed in elderly individuals infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a serious clinical burden and the underlying mechanism is unclear, which necessities detailed investigation of disease characteristics and research for efficient countermeasures. To simulate lethal coronavirus disease 2019 (COVID-19) in senescent human patients, 80-week-old male hamsters are intranasally inoculated with different doses of SARS-CoV-2 Omicron BA.5 variant. Exposure to a low dose of the Omicron BA.5 variant results in early activation of the innate immune response, followed by rapid viral clearance and minimal lung damage. However, a high dose of BA.5 results in impaired interferon signaling, cytokine storm, uncontrolled viral replication, and severe lung injury. To decrease viral load and reverse the deterioration of COVID-19, a new bio-mimic decoy called CoVR-MV is used as a preventive or therapeutic agent. Administration of CoVR-MV as a preventive or therapeutic intervention in the early stages of infection can effectively suppress viral load, regulate the immune response, and rescue animals from death and critical illness. These findings underscore the risk associated with SARS-CoV-2 Omicron BA.5 exposure in senescent hamsters and highlight the importance of early intervention to prevent disease progression.

The XBB.1.5 subvariant has garnered significant attention due to its exceptional immune evasion and transmissibility. Significantly, the evolutionary trajectory of SARS-CoV-2 has shown continual progression, with a recent global shift observed from XBB to BA.2.86, exemplified by the emergence of the predominant JN.1 subvariant. This phenomenon highlights the need for vaccines that can provide broad-spectrum antigenic coverage. In this study, we utilized a NS1-deleted (dNS1) influenza viral vector to engineer an updated live-attenuated vectored vaccine called dNS1-XBB-RBD. This vaccine encodes the receptor-binding domain (RBD) protein of the XBB.1.5 strain. Our findings demonstrate that the dNS1-XBB-RBD vaccine elicits a similar systemic and mucosal immune response compared to its prototypic form, dNS1-RBD. In hamsters, the dNS1-XBB-RBD vaccine provided robust protection against the SARS-CoV-2 immune-evasive strains XBB.1.9.2.1 and Beta. Remarkably, nasal vaccination with dNS1-RBD, which encodes the ancestor RBD gene, also effectively protected hamsters against both the XBB.1.9.2.1 and Beta strains. These results provide valuable insights about nasal influenza-vectored vaccine and present a promising strategy for the development of a broad-spectrum vaccine against COVID-19 in the future.

Efficacy of small molecules against the severe acute respiratory syndrome coronavirus 2 XBB1.16 and XBB1.9.2.1Dear Editor, The Omicron variant (B.1.1.529) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and hundreds of its progenies have rapidly emerged as the predominant circulating viruses worldwide.2][3] This evasion mechanism stems from intensive mutations within the receptor binding domain (RBD) and N-terminal domain (NTD) of the spike protein, thereby bolstering the ability to circumvent neutralizing antibodies and heightening the incidence of breakthrough infections (BTIs).][3] These antiviral options present supplementary avenues for combating BTIs, facilitating in-house treatment, and alleviating the severity rate among hospitalized patients.However, the naturally occurring mutations of SARS-CoV-2 main protease confer drug resistance to nirmatrelvir, 4 which suggests the urgent need for evaluation of risk of drug resistant.Recently, several Omicron sublineages of SARS-CoV-2, specifically XBB1.16 and XBB1.9, have emerged prominently.Their subsequent generations, encompassing XBB1.16.1/1.16.6, XBB1.9.1/1.9.2, XBB1.9.2.1 (EG.1), and XBB1.9.2.5.1 (EG.5.1), have collectively contributed to more than 60% of the global SARS-CoV-2 infections.In contrast to the earlier prevailing sublineage XBB1.5, XBB.1.16showcases a dual substitution phenomenon in both nonstructural proteins (NSPs) and the spike protein.Specifically, T478R mutation within the RBD and E180V mutation within the NTD define its genetic makeup.As expected, XBB.1.16displays notable resistance to various