Robust Innate Immune Response Research Articles

Lack of Association of TLR1 and TLR5 Coding Variants with Mortality in a Large Multicenter Cohort of Melioidosis Patients.

Melioidosis, infection caused by Burkholderia pseudomallei, is characterized by robust innate immune responses. We have previously reported associations of TLR1 single nucleotide missense variant rs76600635 with mortality and of TLR5 nonsense variant rs5744168 with both bacteremia and mortality in single-center studies of patients with melioidosis in northeastern Thailand. The objective of this study was to externally validate the associations of rs76600635 and rs5744168 with bacteremia and mortality in a large multicenter cohort of melioidosis patients. We genotyped rs76600635 and rs5744168 in 1,338 melioidosis patients enrolled in a prospective parent cohort study conducted at nine hospitals in northeastern Thailand. The genotype frequencies of rs76600635 did not differ by bacteremia status (P = 0.27) or 28-day mortality (P = 0.84). The genotype frequencies of rs5744168 did not differ by either bacteremia status (P = 0.46) or 28-day mortality (P = 0.10). Assuming a dominant genetic model, there was no association of the rs76600635 variant with bacteremia (adjusted odds ratio [OR], 0.75; 95% CI, 0.54-1.04, P = 0.08) or 28-day mortality (adjusted OR, 0.96; 95% CI, 0.71-1.28, P = 0.77). There was no association of the rs5744168 variant with bacteremia (adjusted OR, 1.24; 95% CI, 0.76-2.03, P = 0.39) or 28-day mortality (adjusted OR, 1.22; 95% CI, 0.83-1.79, P = 0.21). There was also no association of either variant with 1-year mortality. We conclude that in a large multicenter cohort of patients hospitalized with melioidosis in northeastern Thailand, neither TLR1 missense variant rs76600635 nor TLR5 nonsense variant rs5744168 is associated with bacteremia or mortality.

Expanding the Potential of Neoantigen Vaccines: Harnessing Bacille Calmette-Guérin Cell-Wall-Based Nanoscale Adjuvants for Enhanced Cancer Immunotherapy.

Personalized antitumor immunotherapy utilizing neoantigen vaccines holds great promise. However, the limited immunogenicity of existing recognized neoantigens and the inadequate stimulation of antitumor immune responses by conventional adjuvants pose significant challenges. To address these limitations, we developed a nanovaccine that combines a BCG bacterial cell wall skeleton (BCG-CWS) based nanoscale adjuvant (BCNA) with peptide neoantigens (M27 and M30). This integrated approach provides an efficient translational strategy for cancer immunotherapy. The BCNA nanovaccine, formulated with PLGA as an emulsifier, exhibits excellent biocompatibility and superior antigen presentation compared with conventional BCG-CWS adjuvants. Subcutaneous immunization with the BCNA-based nanovaccine effectively targets lymph nodes, eliciting robust innate and tumor-specific immune responses. Importantly, our findings demonstrate that BCNAs significantly enhance neoantigen immunogenicity while minimizing acute systemic toxicity. Furthermore, when combined with a mouse PD-L1 antibody, our strategy achieves complete tumor elimination in 60% of cases and prevents 25% of tumor growth in a melanoma mouse model. In conclusion, our BCNA-based nanovaccine represents a promising avenue for advancing personalized therapeutic neoantigen vaccines and holds significant implications for enhancing personalized immunotherapy and improving patient outcomes in the field of cancer treatment.

Immunostimulation of Asian elephant (Elephas maximus) blood cells by parapoxvirus ovis and CpG motif-containing bacterial plasmid DNA upregulates innate immune gene expression.

The immune system of Asian elephants (Elephas maximus) is poorly studied, compared to that of livestock, rodents or humans. The innate immune response has become a focus of interest in relation to Elephant endotheliotropic herpesviruses (EEHVs). EEHVs cause a fatal hemorrhagic disease (EEHV-HD) and are a significant threat to captive Asian elephant populations worldwide. Similar to other herpesvirus infections, nearly all animals become infected, but only some develop disease. As progression to EEHV-HD is often acute, a robust innate immune response is crucial to control EEHV infections. This is invariably true of the host in the first instance, but it can also potentially be modulated by intervention strategies. Here, two immunostimulant veterinary medicinal products, authorized for use in domestic species, were tested for their ability to induce innate anti-viral immune responses in Asian elephant blood cells. Sequence data were obtained for a range of previously unidentified Asian elephant immune genes, including C-X-C motif chemokine ligand 10 (CXCL10), interferon stimulated gene 15 (ISG15) and myxovirus GTPase 1 (Mx1), and were employed in the design of species-specific qPCR assays. These assays were subsequently used in analyses to determine fold changes in gene expression over a period of 24 hours. This study demonstrates that both immunostimulant medications are capable of inducing significant innate anti-viral immune responses which suggests that both could be beneficial in controlling EEHV infections in Asian elephants.

Characterization of the ADP-β-d-manno-heptose biosynthetic enzymes from two pathogenic Vibrio strains

ADP-activated β-d-manno-heptoses (ADP-β-d-manno-heptoses) are precursors for the biosynthesis of the inner core of lipopolysaccharide in Gram-negative bacteria. Recently, ADP-d-glycero-β-d-manno-heptose (ADP-d,d-manno-heptose) and its C-6′′ epimer, ADP-l-glycero-β-d-manno-heptose (ADP-l,d-manno-heptose), were identified as potent pathogen-associated molecular patterns (PAMPs) that can trigger robust innate immune responses. Although the production of ADP-d,d-manno-heptose has been studied in several different pathogenic Gram-negative bacteria, current knowledge of ADP-β-d-manno-heptose biosynthesis in Vibrio strains remains limited. Here, we characterized the biosynthetic enzymes of ADP-d,d-manno-heptose and the epimerase that converts it to ADP-l,d-manno-heptose from Vibrio cholerae (the causative agent of pandemic cholera) and Vibrio parahaemolyticus (non-cholera pathogen causing vibriosis with clinical manifestations of gastroenteritis and wound infections) in comparison with their isozymes from Escherichia coli. Moreover, we discovered that β-d-mannose 1-phosphate, but not α-d-mannose 1-phosphate, could be activated to its ADP form by the nucleotidyltransferase domains of bifunctional kinase/nucleotidyltransferases HldEVC (from V. cholerae) and HldEVP (from V. parahaemolyticus). Kinetic analyses of the nucleotidyltransferase domains of HldEVC and HldEVP together with the E. coli–derived HldEEC were thus carried out using β-d-mannose 1-phosphate as a mimic sugar substrate. Overall, our works suggest that V. cholerae and V. parahaemolyticus are capable of synthesizing ADP-β-d-manno-heptoses and lay a foundation for further physiological function explorations on manno-heptose metabolism in Vibrio strains.Key points• Vibrio strains adopt the same biosynthetic pathway as E. coli in synthesizing ADP-β-d-manno-heptoses.• HldEs from two Vibrio strains and E. coli could activate β-d-mannose 1-phosphate to ADP-β-d-mannose.• Comparable nucleotidyltransfer efficiencies were observed in the kinetic studies of HldEs.

Innate immune response to SARS-CoV-2 infection contributes to neuronal damage in human iPSC-derived peripheral neurons.

Severe acute respiratory coronavirus 2 (SARS-CoV-2) causes neurological disease in the peripheral and central nervous system (PNS and CNS, respectively) of some patients. It is not clear whether SARS-CoV-2 infection or the subsequent immune response are the key factors that cause neurological disease. Here, we addressed this question by infecting human induced pluripotent stem cell-derived CNS and PNS neurons with SARS-CoV-2. SARS-CoV-2 infected a low number of CNS neurons and did not elicit a robust innate immune response. On the contrary, SARS-CoV-2 infected a higher number of PNS neurons. This resulted in expression of interferon (IFN) λ1, several IFN-stimulated genes and proinflammatory cytokines. The PNS neurons also displayed alterations characteristic of neuronal damage, as increased levels of sterile alpha and Toll/interleukin receptor motif-containing protein 1, amyloid precursor protein and α-synuclein, and lower levels of cytoskeletal proteins. Interestingly, blockade of the Janus kinase and signal transducer and activator of transcription pathway by Ruxolitinib did not increase SARS-CoV-2 infection, but reduced neuronal damage, suggesting that an exacerbated neuronal innate immune response contributes to pathogenesis in the PNS. Our results provide a basis to study coronavirus disease 2019(COVID-19) related neuronal pathology and to test future preventive or therapeutic strategies.

Silent battles: immune responses in asymptomatic SARS-CoV-2 infection.

SARS-CoV-2 infections manifest with a broad spectrum of presentations, ranging from asymptomatic infections to severe pneumonia and fatal outcomes. This review centers on asymptomatic infections, a widely reported phenomenon that has substantially contributed to the rapid spread of the pandemic. In such asymptomatic infections, we focus on the role of innate, humoral, and cellular immunity. Notably, asymptomatic infections are characterized by an early and robust innate immune response, particularly a swift type 1 IFN reaction, alongside a rapid and broad induction of SARS-CoV-2-specific T cells. Often, antibody levels tend to be lower or undetectable after asymptomatic infections, suggesting that the rapid control of viral replication by innate and cellular responses might impede the full triggering of humoral immunity. Even if antibody levels are present in the early convalescent phase, they wane rapidly below serological detection limits, particularly following asymptomatic infection. Consequently, prevalence studies reliant solely on serological assays likely underestimate the extent of community exposure to the virus.

Advancements in innate immune regulation strategies in islet transplantation.

As a newly emerging organ transplantation technique, islet transplantation has shown the advantages of minimal trauma and high safety since it was first carried out. The proposal of the Edmonton protocol, which has been widely applied, was a breakthrough in this method. However, direct contact between islets and portal vein blood will cause a robust innate immune response leading to massive apoptosis of the graft, and macrophages play an essential role in the innate immune response. Therefore, therapeutic strategies targeting macrophages in the innate immune response have become a popular research topic in recent years. This paper will summarize and analyze recent research on strategies for regulating innate immunity, primarily focusing on macrophages, in the field of islet transplantation, including drug therapy, optimization of islet preparation process, islet engineering and Mesenchymal stem cells cotransplantation. We also expounded the heterogeneity, plasticity and activation mechanism of macrophages in islet transplantation, providing a theoretical basis for further research.

Development and Evaluation of a Promising Biomarker for Diagnosis of Latent and Active Tuberculosis Infection

Abstract Background Diagnosing latent tuberculosis (TB) infection (LTBI) and active TB (ATB) is crucial for preventing disease progression and transmission. However, current diagnostic tests have limitations in terms of accuracy and sensitivity, making it challenging to diagnose these different infection states. Therefore, this study intends to develop a promising biomarker for LTBI and ATB diagnosis to overcome the limitations of the current diagnostic tests. Methods We developed a novel multiepitope-based diagnostic biomarker (MEBDB) from LTBI region of differentiation antigens using bioinformatics and immunoinformatics. Immune responses induced by MEBDM were detected using enzyme-linked immunosorbent spot and cytometric bead assays. This study was conducted from April 2022 to December 2022 in the Senior Department of Tuberculosis at the 8th Medical Center of PLA General Hospital, China. Blood samples were collected from participants with ATB, individuals with LTBI, and healthy controls (HCs). The diagnostic efficacy of MEBDB was evaluated using receiver operating characteristic curves. Results A novel MEBDB, designated as CP19128P, was generated. CP19128P comprises 19 helper T lymphocyte epitopes, 12 cytotoxic T lymphocyte epitopes, and 8 B-cell epitopes. In silico simulations demonstrated that CP19128P possesses strong affinity for Toll-like receptors and elicits robust innate and adaptive immune responses. CP19128P generated significantly higher levels of tumor necrosis factor (TNF-α), interleukin 4 (IL-4), and IL-10 in ATB patients (n = 7) and LTBI (n = 8) individuals compared with HCs (n = 62) (P < 0.001). Moreover, CP19128P-induced specific cytokines could be used to discriminate LTBI and ATB from healthy subjects with high sensitivity and specificity. Combining IL-2 with IL-4 or TNF-α could differentiate LTBI from HCs (the area under the receiver operating characteristic curve [AUC], 0.976 [95% confidence interval [CI], 0.934–1.000] or 0.986 [0.956–1.000]), whereas combining IL-4 with IL-17A or TNF-α could differentiate ATB from HCs (AUC, 0.887 [0.782–0.993] or 0.984 [0.958–1.000]). Conclusions Our study revealed that CP19128P is a potential MEBDB for the diagnosis of LTBI and ATB. Our findings suggest a promising strategy for developing novel, accurate, and sensitive diagnostic biomarkers and identifying new targets for TB diagnosis and management.

Salmonella T3SS-elicited inflammatory innate immune response inhibits type I IFN response in macrophages

As a gram-negative intracellular bacterial pathogen, Salmonella enterica serovar Typhimurium (S. Typhimurium) invades different cell types including macrophages. Its infection in macrophages induces robust innate immune responses that are featured by proinflammatory and type I interferon (IFN) responses. The type III secretion systems (T3SSs) of S. Typhimurium play a crucial role in activating host inflammasome pathways. It has been recognized that the inflammasome pathways inhibit the type I IFN cascade. However, the potential role of T3SS in regulating the type I IFN response and the underlying mechanisms are largely unknown. In this study, we showed that S. Typhimurium infection activated strong proinflammatory, type I IFN and IFN-stimulated genes (ISGs) expression in macrophages. Furthermore, we showed that T3SS-defective S. Typhimurium mutant ΔinvC elicited attenuated inflammatory response but enhanced type I IFN and ISGs expression. Additionally, the inhibition of caspase-1 by a specific inhibitor VX-765 resulted in increased type I IFN response. Moreover, cell-permeable pan-caspase inhibitor Z-VAD-FMK also enhanced the type I IFN response upon S. Typhimurium infection. Intriguingly, compared with exponential phase S. Typhimurium infection, stationary phase bacteria triggered higher levels of type I IFN responses. Finally, the inhibition of caspase-1 by VX-765 substantially increased the intracellular S. Typhimurium burden. In conclusion, we demonstrated that the proinflammatory response induced by S. Typhimurium T3SS can inhibit the type I IFN response, which provides insight into the role of T3SS in orchestrating innate immunity during S. Typhimurium infection.

Human Synovial Mesenchymal Stem Cells Expressed Immunoregulatory Factors IDO and TSG6 in a Context of Arthritis Mediated by Alphaviruses.

Infection by arthritogenic alphaviruses (aavs) can lead to reactive arthritis, which is characterized by inflammation and persistence of the virus; however, its mechanisms remain ill-characterized. Intriguingly, it has been shown that viral persistence still takes place in spite of robust innate and adaptive immune responses, characterized notably by the infiltration of macrophages (sources of TNF-alpha) as well as T/NK cells (sources of IFN-gamma) in the infected joint. Aavs are known to target mesenchymal stem cells (MSCs) in the synovium, and we herein tested the hypothesis that the infection of MSCs may promote the expression of immunoregulators to skew the anti-viral cellular immune responses. We compared the regulated expression via human synovial MSCs of pro-inflammatory mediators (e.g., IL-1β, IL6, CCL2, miR-221-3p) to that of immunoregulators (e.g., IDO, TSG6, GAS6, miR146a-5p). We used human synovial tissue-derived MSCs which were infected with O'Nyong-Nyong alphavirus (ONNV, class II aav) alone, or combined with recombinant human TNF-α or IFN-γ, to mimic the clinical settings. We confirmed via qPCR and immunofluorescence that ONNV infected human synovial tissue-derived MSCs. Interestingly, ONNV alone did not regulate the expression of pro-inflammatory mediators. In contrast, IDO, TSG6, and GAS6 mRNA expression were increased in response to ONNV infection alone, but particularly when combined with both recombinant cytokines. ONNV infection equally decreased miR-146a-5p and miR-221-3p in the untreated cells and abrogated the stimulatory activity of the recombinant TNF-α but not the IFN-gamma. Our study argues for a major immunoregulatory phenotype of MSCs infected with ONNV which may favor virus persistence in the inflamed joint.

Single-cell transcriptomics of human traumatic brain injury reveals activation of endogenous retroviruses in oligodendroglia

Traumatic brain injury (TBI) is a leading cause of chronic brain impairment and results in a robust, but poorly understood, neuroinflammatory response that contributes to the long-term pathology. We used single-nuclei RNA sequencing (snRNA-seq) to study transcriptomic changes in different cell populations in human brain tissue obtained acutely after severe, life-threatening TBI. This revealed a unique transcriptional response in oligodendrocyte precursors and mature oligodendrocytes, including the activation of a robust innate immune response, indicating an important role for oligodendroglia in the initiation of neuroinflammation. The activation of an innate immune response correlated with transcriptional upregulation of endogenous retroviruses in oligodendroglia. This observation was causally linked invitro using human glial progenitors, implicating these ancient viral sequences in human neuroinflammation. In summary, this work provides insight into the initiating events of the neuroinflammatory response in TBI, which has therapeutic implications.

Rotavirus capping enzyme VP3 inhibits interferon expression by inducing MAVS degradation during viral replication.

The binding of viral RNA to RIG-I-like receptors triggers the formation of mitochondrial antiviral signaling (MAVS) protein aggregates critical for interferon (IFN) expression. Several rotavirus strains have been shown to suppress IFN expression by inducing MAVS degradation. Relying on transient expression assays, previous studies reached different conclusions regarding the identity of the rotavirus protein responsible for MAVS degradation, suggesting it was an activity of the rotavirus capping enzyme VP3 or the interferon antagonist NSP1. Here, we have used recombinant SA11 rotaviruses to identify the endogenous viral protein responsible for MAVS degradation and to analyze how the attack on MAVS impacts IFN expression. The recombinant viruses included those expressing modified VP3 or NSP1 proteins deficient in the ability to induce the degradation of MAVS or interferon regulatory factor-3 (IRF3), or both. With these viruses, we determined that VP3 directs the proteasomal degradation of MAVS but plays no role in IRF3 degradation. Moreover, NSP1 was determined to induce IRF3 degradation but to have no impact on MAVS degradation. Analysis of rotavirus-infected cells indicated that IRF3 degradation was more efficient than MAVS degradation and that NSP1 was primarily responsible for suppressing IFN expression in infected cells. However, VP3-mediated MAVS degradation contributed to IFN suppression in cells that failed to produce functional NSP1, pointing to a subsidiary role for VP3 in the IFN antagonist activity of NSP1. Thus, VP3 is a multifunctional protein with several activities that counter anti-rotavirus innate immune responses, including capping of viral (+)RNAs, hydrolysis of the RNase L 2-5A (2'-5' oligoadenylate) signaling molecule, and proteasomal degradation of MAVS. IMPORTANCE Rotavirus is an enteric RNA virus that causes severe dehydrating gastroenteritis in infants and young children through infection of enterocytes in the small intestine. Timely clearance of the virus demands a robust innate immune response by cells associated with the small intestine, including the expression of interferon (IFN). Previous studies have shown that some rotavirus strains suppress the production of interferon, by inducing the degradation of mitochondrial antiviral signaling (MAVS) protein and interferon regulatory factor-3 (IRF3). In this study, we have used reverse genetics to generate recombinant rotaviruses expressing compromised forms of VP3 or NSP1, or both, to explore the function of these viral proteins in the degradation of MAVS and IRF3. Our results demonstrate that VP3 is responsible for MAVS depletion in rotavirus-infected cells, and through this activity, helps to suppress IFN production. Thus, VP3 functions to support the activity of rotavirus NSP1, the major interferon antagonist of the virus.

Diamino Allose Phosphates: Novel, Potent, and Highly Stable Toll-like Receptor 4 Agonists.

Most known synthetic toll-like receptor 4 (TLR4) agonists are carbohydrate-based lipid-A mimetics containing several fatty acyl chains, including a labile 3-O-acyl chain linked to the C-3 position of the non-reducing sugar known to undergo cleavage impacting stability and resulting in loss of activity. To overcome this inherent instability, we rationally designed a new class of chemically more stable synthetic TLR4 ligands that elicit robust innate and adaptive immune responses. This new class utilized a diamino allose phosphate (DAP) scaffold containing a nonhydrolyzable 3-amide bond instead of the classical 3-ester. Accordingly, the DAPs have significantly improved thermostability in aqueous formulations and potency relative to other known natural and synthetic TLR4 ligands. Furthermore, the DAP analogues function as potent vaccine adjuvants to enhance influenza-specific antibodies in mice and provide protection against lethal influenza virus challenges. This novel set of TLR4 ligands show promise as next-generation vaccine adjuvants and stand-alone immunomodulators.

Lipid Nanoparticle Delivery Alters the Adjuvanticity of the TLR9 Agonist CpG by Innate Immune Activation in Lymphoid Tissue.

Pharmacological strategies to activate innate immune cells are of great relevance in the context of vaccine design and anticancer immune therapy, to mount broad immune responses able to clear infection and malignant cells. Synthetic CpG oligodeoxynucleotides (CpG-ODNs) are short single-stranded DNA molecules containing unmethylated CpG dinucleotides and a phosphorothioate backbone. Class B CpG ODNs activate robust innate immune responses through a TLR9-dependent NF-κB signaling pathway. This feature is attractive to exploit in the context of vaccine design and cancer immunotherapy. Soluble CpG-ODNs cause hepatic toxicity, which reduces its therapeutic applicability. The formulation of class B CpG ODN1826 in lipid nanoparticles (LNPs) containing an ionizable cationic lipid that complexes CpG through electrostatic interaction is reported. Upon local administration, LNP-formulated CpG drains to lymph nodes and triggers robust innate immune activation. Unformulated, soluble, CpG, by contrast, is unable to induce robust innate activation in draining lymph nodes and is distributed systemically. In a vaccination setting, LNP-formulated CpG, admixed with a protein antigen, induces higher antigen-specific antibody titers and T cell responses than antigen admixed with unformulated soluble CpG.

Trained immunity: a cutting edge approach for designing novel vaccines against parasitic diseases?

The preventive situation of parasitosis, a global public health burden especially for developing countries, is not looking that good. Similar to other infections, vaccines would be the best choice for preventing and controlling parasitic infection. However, ideal antigenic molecules for vaccine development have not been identified so far, resulting from the complicated life history and enormous genomes of the parasites. Furthermore, the suppression or down-regulation of anti-infectious immunity mediated by the parasites or their derived molecules can compromise the effect of parasitic vaccines. Comparing the early immune profiles of several parasites in the permissive and non-permissive hosts, a robust innate immune response is proposed to be a critical event to eliminate the parasites. Therefore, enhancing innate immunity may be essential for designing novel and effective parasitic vaccines. The newly emerging trained immunity (also termed innate immune memory) has been increasingly recognized to provide a novel perspective for vaccine development targeting innate immunity. This article reviews the current status of parasitic vaccines and anti-infectious immunity, as well as the conception, characteristics, and mechanisms of trained immunity and its research progress in Parasitology, highlighting the possible consideration of trained immunity in designing novel vaccines against parasitic diseases.

Betacoronaviruses SARS-CoV-2 and HCoV-OC43 infections in IGROV-1 cell line require aryl hydrocarbon receptor

ABSTRACT The emergence of novel betacoronaviruses has posed significant financial and human health burdens, necessitating the development of appropriate tools to combat future outbreaks. In this study, we have characterized a human cell line, IGROV-1, as a robust tool to detect, propagate, and titrate betacoronaviruses SARS-CoV-2 and HCoV-OC43. IGROV-1 cells can be used for serological assays, antiviral drug testing, and isolating SARS-CoV-2 variants from patient samples. Using time-course transcriptomics, we confirmed that IGROV-1 cells exhibit a robust innate immune response upon SARS-CoV-2 infection, recapitulating the response previously observed in primary human nasal epithelial cells. We performed genome-wide CRISPR knockout genetic screens in IGROV-1 cells and identified Aryl hydrocarbon receptor (AHR) as a critical host dependency factor for both SARS-CoV-2 and HCoV-OC43. Using DiMNF, a small molecule inhibitor of AHR, we observed that the drug selectively inhibits HCoV-OC43 infection but not SARS-CoV-2. Transcriptomic analysis in primary normal human bronchial epithelial cells revealed that DiMNF blocks HCoV-OC43 infection via basal activation of innate immune responses. Our findings highlight the potential of IGROV-1 cells as a valuable diagnostic and research tool to combat betacoronavirus diseases.

Lc-pPG-612-OmpU-CTB: A promising oral vaccine for protecting Carassius auratus against Vibrio mimicus infection.

Vibrio mimicus (V. mimicus) is known to cause severe bacterial diseases with high mortality rates in fish, resulting in significant economic losses in the global aquaculture industry. Therefore, the objective of this study was to develop a safe and effective vaccine for protecting Carassius auratus (C. auratus) against V. mimicus infection. Recombinant Lactobacillus casei (L. casei) strains, Lc-pPG-612-OmpU and Lc-pPG-612-OmpU-CTB (surface-displayed), were constructed using a L. casei strain (ATCC 393) as an antigen delivery carrier and the cholera toxin B subunit (CTB) as an adjuvant. The two recombinant strains of L. casei were administered to C. auratus via oral immunization, and the protective efficacy of the oral vaccines was assessed. The results demonstrated that oral immunization with the two strains significantly increased the levels of nonspecific immune indicators in C. auratus, including alkaline phosphatase (AKP), lysozyme (LYS), acid phosphatase (ACP), complement 3 (C3), complement 4 (C4), lectin, and superoxide dismutase (SOD). Moreover, the experiment groups exhibited significant increases in specific immunoglobulin M (IgM) antibodies against OmpU, as well as the transcription of immune-related genes (ie., IL-1β, TNF-α, IL-10, and TGF-β), when compared to the control groups. Following infection of C. auratus with V. mimicus, the mortality rate of the recombinant L. casei-treated fish was observed to be lower compared to the control group. This finding suggests that recombinant L. casei demonstrates effective protection against V. mimicus infection in C. auratus. Furthermore, the addition of the immune adjuvant CTB was found to induce a more robust adaptive and innate immune response in C. auratus, resulting in reduced mortality after infection with V. mimicus.

Natural Killer Cells in SARS-CoV-2-Vaccinated Subjects with Increased Effector Cytotoxic CD56dim Cells and Memory-Like CD57+NKG2C+CD56dim Cells.

The infection and negative effects of the SARS-CoV-2 (severe acute respiratory syndrome coronavirus) virus are mitigated by vaccines. It is unknown whether vaccination has worked by eliciting robust protective innate immune responses with high affinity. Twenty healthy volunteers received three doses of Comirnaty (Pfizer Australia Pty Ltd.) and were evaluated 9 months after the second vaccination and 1 month after the booster dose. The exclusion criteria were the presence of adverse effects following the vaccination, a history of smoking, and heterologous immunization. The inclusion criteria were the absence of prior Coronavirus Disease (COVID)-19 history, the absence of adverse effects, and the absence of comorbidities. Specific phenotype and levels of CD107a and granzyme production by blood NK (natural killer) cells were analyzed after exposure to SARS-CoV-2 spike antigen (Wuhan, Alpha B.1.1.7, Delta B.1.617.2, and Omicron B1.1.529 variants), and related with anti-SARS-CoV-2 antibody production. The booster dose caused early NK CD56dim subset activation and memory-like phenotype. We report the relevance of the innate immune response, especially NK cells, to SARS-CoV-2 vaccines to guarantee efficient protection against the infection following a booster dose.

Abstract 5916: Weight loss intervention triggered gut microbiota modulation can positively impact obesity associated endocrine therapy resistance

Abstract Background: 41.9% of adult US population is obese and every 5-unit BMI increase is associated with 12 percent increase in breast cancer risk; it is an independent predictor of poor outcomes in breast cancer. Microbiome has emerged as a central regulator of obesity and we recently discovered that microbes can not only potentiate breast cancer growth and metastasis but also affect therapeutic response. We showed weight-loss by Diet/Exercise/Behavioral intervention (POWER-remote trial) exerts favorable effects. Here, we examine molecular underpinnings of poor therapy response in obese state and explore the gut microbiome to mitigate the same. Methods: We co-implanted lineage determined human White preAdipocytes (hWAT) with MCF7 breast cancer cells in the mammary glands of mice. Changes in tumor microenvironment and tumor cells were evaluated ex situ by RNA sequencing, flow cytometry, Raman spectroscopy, Quantitative phase microscopy, PCR, immunoblotting and functional assays. To inspect the gut microbiome, 16 obese breast cancer patients were subjected to weight loss intervention over 14 weeks. Gut microbiome was analyzed at baseline and post-intervention by microbial shotgun sequencing. Patients were divided into responders and non-responders based on % weight-loss. Results: We found that human breast cancer cells co-implanted with hWAT formed more aggressive tumors compared to controls. RNA sequencing revealed significant upregulation of multiple oncogenic pathways along with robust innate immune response. Tumor dissociated cells from hWAT-MCF7 tumors showed extensive molecular reprogramming rendering them more proliferative, motile and stem-like along with dramatic physiological changes like increased intercellular lipid accumulation. hWAT-MCF7 cells attained resistance to multiple drugs including Tamoxifen, Fulvestrant, Letrozole and Palbociclib. Simultaneous targeting of multiple signaling pathways using pharmacological inhibitors is infeasible in clinical setting hence we explored the obesity-microbiome axis. Obese breast cancer patients underwent POWER-remote intervention and their gut microbiome analysis showed significant differential expression of many microbes. Based on abundance and metabolite profile, F. prausnitzii was selected as the candidate of interest. On treatment with F. prausnitzii growth media, acquired multi-drug resistance in hWAT-MCF7 cells could be reversed suggesting that increase in gut population of the bacterium could be helpful in sensitizing patients unresponsive to therapy. Conclusion: Direct interaction between breast cancer cells and WAT rendered them resistant to multiple drugs. Metabolites produced by weight-loss-associated bacterium F. prausnitzii reversed adiposity-induced multi-drug resistance, hence, can potentially have therapeutic benefits in obese breast cancer patients. Citation Format: Sheetal Parida, Sumit Siddharth, Nethaji Muniraj, Zhenhui Liu, Ishan Barman, Michael Smith, Jenni Sheng, Vered Stearns, Janelle W. Coughlin, Dipali Sharma. Weight loss intervention triggered gut microbiota modulation can positively impact obesity associated endocrine therapy resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5916.

Abstract 1212: Horizontal transfer of functional human papillomavirus genes in head and neck squamous cell carcinoma

Abstract Human papillomavirus (HPV) infections are currently associated with more than 70% of oropharyngeal squamous cell carcinomas (OPSCCs). HPV-positive OPSCCs display a distinct biologic behavior and treatment response and thus are now considered a distinct entity than HPV-negative OPSCCs. However, the underlying process of HPV-related OPSCC carcinogenesis is not entirely clear. HPV as a driver of cervical carcinogenesis is well-characterized with identification of a canonical mechanism dependent on HPV DNA integration, loss of HPV E2 gene expression, and increased expression of HPV E6 and E7 oncogenes with no active infection present in malignant lesions. However, whole genome sequencing found no HPV DNA integration in ~25% of HPV-associated OPSCC, suggesting an alternative mechanism of HPV carcinogenesis. Expression of all HPV genes, including self-assembling capsid proteins, in a substantial portion of HPV+ OPSCC, led us to hypothesize that tumors with episomal HPV are capable of transferring HPV genes to surrounding cells and that HPV gene transfer contributes to carcinogenesis in this subset of OPSCC. Our data support this hypothesis, showing that growth media from HPV+ OPSCC cell lines, as well as isolates from HPV+ tumors or nodal metastases, effectively transferred functional HPV genes to cells lacking HPV. The HPV DNA Transferring Agent (HDTA) derived from HPV+ OPSCC contained HPV L1 capsid protein and intact HPV genome protected from DNAase degradation. Initial characterization revealed that anti-L1 antibody blocked gene transfer and that exposure of cells to the HDTA initiated a robust innate immune response in recipient cells. Negative staining of HDTA isolate from metastatic lymph nodes revealed many pleomorphic, electron-dense particles with diameter ranging between 20 and 80nM, with approximately 40% of particles being between 50 and 60nM, consistent with the size of HPV. We propose that development and/or maintenance of OPSCCs depend on continuous production of HDTA capable of transferring HPV genes into neighboring cells. This paradigm of tumorigenesis/maintenance in OPSCC is most likely to apply to cancers lacking HPV integration, but may also apply to tumors with integration, since recent data suggested that integrated HPV may excise from the genome to recreate a transient episomal like structure, even containing cellular DNA. Our findings offer novel insights into oropharyngeal carcinogenesis and suggest targeting HDTA as a new therapeutic strategy for HPV-positive OPSCCs. Citation Format: Jason Tasoulas, Wendell Gray Yarbrough, Natalia Issaeva. Horizontal transfer of functional human papillomavirus genes in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1212.