Rapid Immune Response Research Articles

Acute-phase innate immune responses in SIVmac239-infected Mamu-B*08+ Indian rhesus macaques may contribute to the establishment of elite control

IntroductionSpontaneous control of chronic-phase HIV/SIV viremia is often associated with the expression of specific MHC class I allotypes. HIV/SIV-specific CD8+ cytotoxic T lymphocytes (CTLs) restricted by these MHC class I allotypes appear to be critical for viremic control. Establishment of the elite controller (EC) phenotype is predictable in SIVmac239-infected Indian rhesus macaques (RMs), with approximately 50% of Mamu-B*08+ RMs and 20% of Mamu-B*17+ RMs becoming ECs. Despite extensive characterization of EC-associated CTLs in HIV/SIV-infected individuals, the precise mechanistic basis of elite control remains unknown. Because EC and non-EC viral load trajectories begin diverging by day 14 post-infection, we hypothesized that hyperacute innate immune responses may contribute to viremic control.MethodsTo gain insight into the immunological factors involved in the determination of EC status, we vaccinated 16 Mamu-B*08+ RMs with Vif and Nef to elicit EC-associated CTLs, then subjected these 16 vaccinees and an additional 16 unvaccinated Mamu-B*08+ controls to repeated intrarectal SIVmac239 challenges. We then performed whole-blood transcriptomic analysis of all 32 SIVmac239-infected Mamu-B*08+ RMs and eight SIVmac239-infected Mamu-B*08– RMs during the first 14 days of infection.ResultsVaccination did not provide protection against acquisition, but peak and setpoint viremia were significantly lower in vaccinees relative to controls. We did not identify any meaningful correlations between vaccine-induced CTL parameters and SIVmac239 acquisition rate or chronic-phase viral loads. Ultimately, 13 of 16 vaccinees (81%) and 7 of 16 controls (44%) became ECs (viremia ≤ 10,000 vRNA copies/mL plasma for ≥ 4 weeks). We identified subsets of immunomodulatory genes differentially expressed (DE) between RM groupings based on vaccination status, EC status, and MHC class I genotype. These DE genes function in multiple innate immune processes, including the complement system, cytokine/chemokine signaling, pattern recognition receptors, and interferon-mediated responses.DiscussionA striking difference in the kinetics of differential gene expression among our RM groups suggests that Mamu-B*08-associated elite control is characterized by a robust, rapid innate immune response that quickly resolves. These findings indicate that, despite the association between MHC class I genotype and elite control, innate immune factors in hyperacute SIV infection preceding CTL response development may facilitate the establishment of the EC phenotype.

Self-assembling nanoparticle engineered from the ferritinophagy complex as a rabies virus vaccine candidate

Over the past decade, there has been a growing interest in ferritin-based vaccines due to their enhanced antigen immunogenicity and favorable safety profiles, with several vaccine candidates targeting various pathogens advancing to phase I clinical trials. Nevertheless, challenges associated with particle heterogeneity, improper assembly and unanticipated immunogenicity due to the bulky protein adaptor have impeded further advancement. To overcome these challenges, we devise a universal ferritin-adaptor delivery platform based on structural insights derived from the natural ferritinophagy complex of the human ferritin heavy chain (FTH1) and the nuclear receptor coactivator 4 (NCOA4). The engineered ferritinophagy (Fagy)-tag peptide demonstrate significantly enhanced binding affinity to the 24-mer ferritin nanoparticle, enabling efficient antigen presentation. Subsequently, we construct a self-assembling rabies virus (RABV) vaccine candidate by noncovalently conjugating the Fagy-tagged glycoprotein domain III (GDIII) of RABV to the ferritin nanoparticle, maintaining superior homogeneity, stability and immunogenicity. This vaccine candidate induces potent, rapid, and durable immune responses, and protects female mice against the authentic RABV challenge after single-dose administration. Furthermore, this universal, ferritin-based antigen conjugating strategy offers significant potential for developing vaccine against diverse pathogens and diseases.

T cell landscape in the microenvironment of human solid tumors.

T cells are the main effectors involved in anti-tumor immunity, mediating most of the adaptive response towards cancer. After priming in lymph nodes, tumor antigens-specific naïve T lymphocytes proliferate and differentiate into effector CD4+ and CD8+ T cells that migrate from periphery into tumor sites aiming to eliminate cancer cells. Then while most effector T cells die, a small fraction persists and recirculates as long-lived memory T cells which generate enhanced immune responses when re-encountering the same antigen. A number of T (and non-T) cell subsets, stably resides in non-lymphoid peripheral tissues and may provide rapid immune response independently of T cells recruited from blood, against the reemergence of cancer cells. When tumor grows, however, tumor cells have evaded immune surveillance of effector cells (NK and CTL cells) which are exhausted, thus favoring the local expansion of T (and non-T) regulatory cells. In this review, the current knowledge of features of T cells present in the tumor microenvironment (TME) of solid adult and pediatric tumors, the mechanisms upregulating immune-checkpoint molecules and transcriptional and epigenetic landscapes leading to dysfunction and exhaustion of T effector cells are reviewed. The interaction of T cells with cancer- or TME non-neoplastic cells and their secreted molecules shape the T cell profile compromising the intrinsic plasticity of T cells and, therefore, favoring immune evasion. In this phase regulatory T cells contribute to maintain a high immunosuppressive TME thus facilitating tumor cell proliferation and metastatic spread. Despite the advancements of cancer immunotherapy, many tumors are unresponsive to immune checkpoint inhibitors, or therapeutical vaccines or CAR T cell-based adoptive therapy: some novel strategies to improve these T cell-based treatments are lastly proposed.

Within-Host Viral Growth and Immune Response Rates Predict Foot-and-Mouth Disease Virus Transmission Dynamics for African Buffalo.

AbstractInfectious disease dynamics operate across biological scales: pathogens replicate within hosts but transmit among populations. Functional changes in the pathogen-host interaction thus generate cascading effects across organizational scales. We investigated within-host dynamics and among-host transmission of three strains (SAT-1, -2, -3) of foot-and-mouth disease viruses (FMDVs) in their wildlife host, African buffalo. We combined data on viral dynamics and host immune responses with mathematical models to ask the following questions: How do viral and immune dynamics vary among strains? Which viral and immune parameters determine viral fitness within hosts? And how do within-host dynamics relate to virus transmission? Our data reveal contrasting within-host dynamics among viral strains, with SAT-2 eliciting more rapid and effective immune responses than SAT-1 and SAT-3. Within-host viral fitness was overwhelmingly determined by variation among hosts in immune response activation rates but not by variation among individual hosts in viral growth rate. Our analyses investigating across-scale linkages indicate that viral replication rate in the host correlates with transmission rates among buffalo and that adaptive immune activation rate determines the infectious period. These parameters define the virus's relative basic reproductive number (ℛ0), suggesting that viral invasion potential may be predictable from within-host dynamics.

Rapid high-throughput method for investigating physiological regulation of neutrophil extracellular trap formation

BackgroundNeutrophils, the most abundant white blood cells in humans, play pivotal roles in innate immunity, rapidly migrating to sites of infection and inflammation to phagocytose, neutralize, and eliminate invading pathogens. Neutrophil extracellular trap (NET) formation is increasingly recognized as an essential rapid innate immune response, but when dysregulated, it contributes to pathogenesis of sepsis and immunothrombotic disease. ObjectivesCurrent NETosis models are limited, routinely employing nonphysiological triggers that can bypass natural NET regulatory pathways. Models utilizing isolated neutrophils and immortalized cell lines do not reflect the complex biology underlying neutrophil activation and NETosis that occurs in whole blood. To our knowledge, we report the first human ex vivo model utilizing naturally occurring molecules to induce NETosis in whole blood. This approach could be used for drug screening and, importantly, inadvertent activators of NETosis. MethodsHere we describe a novel, high-throughput ex vivo whole blood–induced NETosis model using combinatorial pooling of native NETosis-inducing factors in a more biologically relevant Synthetic-Sepsis model. ResultsWe found different combinations of factors evoked distinct neutrophil responses in the rate of NET generation and/or magnitude of NETosis. Despite interdonor variability, similar sets of proinflammatory molecules induced consistent responses across donors. We found that at least 3 biological triggers were necessary to induce NETosis in our system including either tumor necrosis factor-α or lymphotoxin-α. ConclusionThese findings emphasize the importance of investigating neutrophil physiology in a biologically relevant context to enable a better understanding of disease pathology, risk factors, and therapeutic targets, potentially providing novel strategies for disease intervention and treatment.

#1181 The relationship between renal ischaemia-reperfusion injury and circadian rhythm

Abstract Background and Aims Kidney disease is a global health problem in which various aetiologies contribute to renal injury and dysfunction. Renal ischaemia-reperfusion injury (IRI) is a condition caused by early oxidative stress due to renal tissue ischaemia and a rapid immune response after reperfusion. Circadian rhythms are important regulators of metabolism and their disruption can lead to heart disease and fibrosis in various organs. Although recent reports have shed light on the impact of renal injury and disease on the renal circadian clock, the exact mechanisms by which circadian rhythm genes contribute to the progression of renal dysfunction in IRI and the onset of chronic kidney disease (CKD) are not fully understood. Method Male Wistar rats (8-9 weeks old, 250-280 g body weight) were used to establish the I/R model. To induce renal I/R injury, the left renal artery was clamped for 30 or 45 min and then removed to restore blood flow. Simultaneously, nephrectomy of the right kidney was performed; renal tissue and blood were collected 14 days after I/R. HE staining, Sirius red staining and immunostaining (F4/80 staining) were performed. Kidney tissue was RNA-seq'd and qPCR'd for circadian rhythm genes and fibrosis genes involved in kidney failure that were altered by I/R. Kidney tissue was also RNA-seq'd and qPCR'd for circadian rhythm genes and fibrosis genes involved in kidney failure. We also established an in vitro hypoxia-reoxygenation (H/R) model using single cell RPTECs and performed qPCR and Western blotting. Results In the IRI rat group, the expression of circadian repressor genes Nr1d1 and Nr1d2, which are important for reducing inflammation, decreased, while the expression of Bmal1 and Cry1, which regulate renal deterioration, increased. Using RNA-seq, we also identified the involvement of the TGFβ pathway in linking the circadian clock to kidney injury and fibrosis, and observed similar results in the H/R group using RPTEC cells in vivo. Conclusion Fibrosis and circadian rhythms were found to be closely related in renal IRI. The findings described here may help in the development of new therapeutic approaches to ameliorate the effects of renal IRI and improve the outcome of kidney disease, including kidney transplantation, offering hope to patients worldwide.

AlbuCatcher for Long-Acting Therapeutics.

Therapeutic proteins, pivotal for treating diverse human diseases due to their biocompatibility and high selectivity, often face challenges such as rapid serum clearance, enzymatic degradation, and immune responses. To address these issues and enable prolonged therapeutic efficacy, techniques to extend the serum half-life of therapeutic proteins are crucial. The AlbuCatcher, a conjugate of human serum albumin (HSA) and SpyCatcher, was proposed as a general technique to extend the serum half-life of diverse therapeutic proteins. HSA, the most abundant blood protein, exhibits a long intrinsic half-life through Fc receptor (FcRn)-mediated recycling. The SpyTag/SpyCatcher (ST/SC) system, known for forming irreversible isopeptide bonds, was employed to conjugate HSA and therapeutic proteins. Site-specific HSA conjugation to SC was achieved using an inverse electron-demand Diels-Alder (IEDDA) reaction, minimizing activity loss. Using urate oxidase (Uox) as a model protein with a short half-life, the small ST was fused to generate Uox-ST. Then, HSA-conjugated Uox (Uox-HSA) was successfully prepared via the Uox-ST/AlbuCatcher reaction. In vitro enzyme assays demonstrated that the impact of ST fusion and HSA conjugation on Uox enzymatic activity is negligible. Pharmacokinetics studies in mice revealed that Uox-HSA exhibits a significantly longer serum half-life (about 18 h) compared to Uox-WT (about 2 h). This extended half-life is attributed to FcRn-mediated recycling of HSA-conjugated Uox, demonstrating the effectiveness of the AlbuCatcher strategy in enhancing the pharmacokinetics of therapeutic proteins.

Tissue-resident memory T cells: decoding intra-organ diversity with a gut perspective.

Tissue-resident memory T cells (TRM) serve as the frontline of host defense, playing a critical role in protection against invading pathogens. This emphasizes their role in providing rapid on-site immune responses across various organs. The physiological significance of TRM is not just confined to infection control; accumulating evidence has revealed that TRM also determine the pathology of diseases such as autoimmune disorders, inflammatory bowel disease, and cancer. Intensive studies on the origin, mechanisms of formation and maintenance, and physiological significance of TRM have elucidated the transcriptional and functional diversity of these cells, which are often affected by local cues associated with their presence. These were further confirmed by the recent remarkable advancements of next-generation sequencing and single-cell technologies, which allow the transcriptional and phenotypic characterization of each TRM subset induced in different microenvironments. This review first overviews the current knowledge of the cell fate, molecular features, transcriptional and metabolic regulation, and biological importance of TRM in health and disease. Finally, this article presents a variety of recent studies on disease-associated TRM, particularly focusing and elaborating on the TRM in the gut, which constitute the largest and most intricate immune network in the body, and their pathological relevance to gut inflammation in humans.

Trivalent mRNA vaccine-candidate against seasonal flu with cross-specific humoral immune response.

Seasonal influenza remains a serious global health problem, leading to high mortality rates among the elderly and individuals with comorbidities. Vaccination is generally accepted as the most effective strategy for influenza prevention. While current influenza vaccines are effective, they still have limitations, including narrow specificity for certain serological variants, which may result in a mismatch between vaccine antigens and circulating strains. Additionally, the rapid variability of the virus poses challenges in providing extended protection beyond a single season. Therefore, mRNA technology is particularly promising for influenza prevention, as it enables the rapid development of multivalent vaccines and allows for quick updates of their antigenic composition. mRNA vaccines have already proven successful in preventing COVID-19 by eliciting rapid cellular and humoral immune responses. In this study, we present the development of a trivalent mRNA vaccine candidate, evaluate its immunogenicity using the hemagglutination inhibition assay, ELISA, and assess its efficacy in animals. We demonstrate the higher immunogenicity of the mRNA vaccine candidate compared to the inactivated split influenza vaccine and its enhanced ability to generate a cross-specific humoral immune response. These findings highlight the potential mRNA technology in overcoming current limitations of influenza vaccines and hold promise for ensuring greater efficacy in preventing seasonal influenza outbreaks.

Polymorphism of IL4 and IL13 anti-inflammatory cytokine genes in indigenous populations of the russian Arctic

Living conditions in the Arctic zone are extremely unfavorable for living and contribute to the development of a number of diseases due to suppression of the functions of the immune system. Cytokines are one of the main mediators of the immune system; they are encoded by genes with a high degree of polymorphism. This study examined the distribution of genetic variants in the cytokine genes (rs2243250 IL4, rs1800925 IL13) among the Nenets, Dolgan-Nganasan and Slavs. It was previously established that these mutations are associated with the level of expression of these interleukins and their production, which leads to changes in the impairment of the immune response to the influence of the pathogen. The study showed the predominance of genotypes CT and TT of the rs2243250 IL4 polymorphism, and genotype CC of the rs1800925 IL13 polymorphism in the Nenets and Dolgan-Nganasan populations. The results obtained suggest that the indigenous inhabitants of the Russian Arctic have a genetically determined rapid immune response and protection against the development of allergic diseases compared to the Slavs.

Decomposition of dynamic transcriptomic responses during effector-triggered immunity reveals conserved responses in two distinct plant cell populations

Rapid plant immune responses in the appropriate cells are needed for effective defense against pathogens. Although transcriptome analysis is often used to describe overall immune responses, collection of transcriptome data with sufficient resolution in both space and time is challenging. We reanalyzed public Arabidopsis time-course transcriptome data obtained after low-dose inoculation with a Pseudomonas syringae strain expressing the effector AvrRpt2, which induces effector-triggered immunity in Arabidopsis. Double-peak time-course patterns are prevalent among thousands of upregulated genes. We implemented a multi-compartment modeling approach to decompose the double-peak pattern into two single-peak patterns for each gene. The decomposed peaks reveal an “echoing” pattern: the peak times of the first and second peaks correlate well across most upregulated genes. We demonstrated that the two peaks likely represent responses of two distinct cell populations that respond either cell autonomously or indirectly to AvrRpt2. Thus, the peak decomposition has extracted spatial information from the time-course data. The echoing pattern also indicates a conserved transcriptome response with different initiation times between the two cell populations despite different elicitor types. A gene set highly overlapping with the conserved gene set is also upregulated with similar kinetics during pattern-triggered immunity. Activation of a WRKY network via different entry-point WRKYs can explain the similar but not identical transcriptome responses elicited by different elicitor types. We discuss potential benefits of the properties of the WRKY activation network as an immune signaling network in light of pressure from rapidly evolving pathogens.

Th2 cells in rapid immune responses and protective avoidance reactions.

Type 2 helper cells (Th2 cells) differentiate from CD4 helper T cells under the influence of IL-4 and conventional or monocyte-derived CD11b+ dendritic cells. Th2 cells are capable of generating IL-4, IL-5, and IL-13, as well as evoking immunoglobulin class-switch to IgE. Three types of rapid immune responses are Th2 cell-dependent: (1) mast cell-IgE mediated allergic reactions, (2) Th2 cell-derived cytokine-mediated reactions that complement allergic reactions and protect the host from toxins, xenobiotics, environmental irritants, and helminthic parasites, and (3) IgE-stimulated mast cell-derived cysteinyl-leukotriene mediated avoidance of toxins. The contributions of Th2 cell-derived cytokines to eosinophilia (IL-5), IgE class-switch, and epithelial barrier activation, mucous secretion, and metaplasia (IL-4 and IL-13) in asthma, allergic rhinitis with polyps and atopic dermatitis have led to anti-cytokine monoclonal antibody treatments. Anti-IL-5 neutralizing monoclonal antibody in asthma and anti-IL-4/IL-13 receptor neutralizing monoclonal antibody in asthma and atopic dermatitis are proven successful therapies in appropriately selected patients who are not sufficiently improved by conventional treatments.

Crosstalk between keratinocytes and neutrophils shapes skin immunity against S. aureus infection.

Staphylococcus aureus (S. aureus) infection of the skin leads to a rapid initial innate immune response with keratinocytes in the epidermis as the initial sensors. Polymorphonuclear neutrophils (PMNs) are the first innate immune cells to infiltrate infection sites where they provide an effective first-line of defense. Previous work of our group showed that in inflamed skin a crosstalk between PMNs and keratinocytes results in enhanced S. aureus skin colonization. In this work, we used an in vitro co-culture model to studied the crosstalk between primary human keratinocytes (PHKs) and PMNs in a sterile environment and upon S. aureus infection. We investigated the influence of PHKs on PMN activation by analyzing PMN lifespan, expression of degranulation markers and induction of proinflammatory cytokines. Furthermore, we analyzed the influence of PMNs on the inflammatory response of PHKs. Finally, we investigated the influence of the skin microbiome on PMN-mediated skin inflammation. We show that co-culture of PMNs with PHKs induces activation and degranulation of PMNs and significantly enhances their lifespan compared to PMN cultivation alone by an IL-8 mediated mechanism and, furthermore, primes PMNs for enhanced activity after S. aureus infection. The prolonged incubation with PMNs also induces inflammatory responses in PHKs which are further exacerbated in the presence of S. aureus and induces further PMN recruitment thus fueling skin inflammation. Interestingly, infection of PHKs with the skin commensal S. epidermidis reduces the inflammatory effects of PMNs in the skin and exhibits an anti-inflammatory effect. Our data indicate that skin infiltrating PMNs and PHKs influence each other in such a way to enhance skin inflammation and that commensal bacteria are able to reduce the inflammatory effect.

Persistence of KIRneg NK cells after haploidentical hematopoietic stem cell transplantation protects from human cytomegalovirus infection/reactivation.

Haploidentical hematopoietic stem cell transplantation (h-HSCT) is a therapeutic option to cure patients affected by hematologic malignancies. The kinetics and the quality of immune-reconstitution (IR) impact the clinical outcome of h-HSCT and limit the onset of life-threatening Human Cytomegalovirus (HCMV) infection/reactivation. Natural Killer (NK) cells are the first lymphocytes that recover after h-HSCT and they can provide rapid innate immune responses against opportunistic pathogens. By performing a longitudinal single-cell analysis of multiparametric flow-cytometry data, we show here that the persistence at high frequencies of CD158b1b2jneg/NKG2Apos/NKG2Cneg/NKp30pos/NKp46pos (KIRneg) NK cells is associated with HCMV infection/reactivation control. These KIRneg NK cells are "unlicensed", and are not terminal-differentiated lymphocytes appearing early during IR and mainly belonging to CD56bright/CD16neg and CD56bright/CD16pos subsets. KIRneg NK cells are enriched in oxidative and glucose metabolism pathways, produce interferon-γ, and are endowed with potent antiviral activity against HCMV ex vivo. Decreased frequencies of KIRneg NK cells early during IR are associated with clinically relevant HCMV replication. Taken together, our findings indicate that the prolonged persistence of KIRneg NK cells after h-HSCT could serve as a biomarker to better predict HCMV infection/reactivation. This phenomenon also paves the way to optimize anti-viral immune responses by enriching post-transplant donor lymphocyte infusions with KIRneg NK cells.

Selective strengthening of lipid metabolism and the rapid immune response of Ostrinia furnacalis larvae parasitized by Macrocentrus cingulum

The Macrocentrus cingulum is a vital endoparasitic wasp that parasites in Ostrinia furnacalis larvae. It has been widely utilized for biological control of corn borer. Although previous studies have explored how the wasps suppress the host's immunity, further research is required to determine the impact of the wasps on the host's lipid metabolism and activated immune response. In this study, high-throughput transcriptome sequencing, quantitative real-time PCR, and physiological and biochemical methods were used to investigate the balance between energy metabolism and immunity in parasitic systems. The results showed that specific genes linked to lipid metabolism and transportation were up-regulated in parasitized O. furnacalis larvae, including AKHR, AKH, apoLpIII, and FAS. Additionally, M. cingulum triggered immune responses in the parasitized larvae, leading to rapid recognition and the production of immunity effectors, such as Lysozyme and antimicrobial peptides, to protect against the invading wasps. The phenoloxidase (PO) activity was inhibited as a result of parasitization. Parasitism selectively promoted lipid metabolism and activated the immune recognition response, significantly up-regulated immune pattern recognition receptors (PRRs) and effector genes, but suppressed the PPO cascade response in O. furnacalis larvae. This study can shed light on lipid metabolism, immune response, and activating the immune recognition response for parasitoid and host co-evolution and provide insight into the biological control of O. furnacalis.

Single-dose of hybrid eukaryotic-prokaryotic nanovesicles to drive a rapid and controllable immune response against Klebsiella pneumoniae induced nosocomial pneumonia

Klebsiella pneumoniae is the main pathogen of hospital-acquired infections causing acute pneumonia, which is characterized by drug resistance, virulence and mortality, and for which there is no clinically approved vaccine formulations. Here, we designed biomimetic hybrid membrane vesicles (HMVs), which consist of outer membrane vesicles of Klebsiella pneumoniae and cell membrane vesicles of alveolar macrophages. The HMVs were administered via intratracheal instillation to mimic the delivery of an inhaled formulation, where immune cells could be rapidly mobilized in the lungs to promote pathogen-specific immunity and to establish local mucosal immunity. In a mouse model of acute pneumonia with experimental strain 43,816 and clinical strain 7,29,088, immunization of HMVs via the lungs provided significant protection, reduced the number of colonies in deep tissues, improved survival, and reduced symptoms of acute inflammation. As expected, HMVs not only retained the immunogenicity of OMVs, but also effectively reduced the excessive immune response induced by OMVs. In conclusion, as a proof-of-concept for the design of antibacterial vaccines, the HMVs is an effective platform for activating bacteria-specific immunity, which can rapidly and effectively protect against nosocomial pneumonia caused by Klebsiella pneumoniae.

Nicotinamide-Expanded Allogeneic Natural Killer Cells with CD38 Deletion, Expressing an Enhanced CD38 Chimeric Antigen Receptor, Target Multiple Myeloma Cells.

Natural killer (NK) cells are a vital component of cancer immune surveillance. They provide a rapid and potent immune response, including direct cytotoxicity and mobilization of the immune system, without the need for antigen processing and presentation. NK cells may also be better tolerated than T cell therapy approaches and are susceptible to various gene manipulations. Therefore, NK cells have become the focus of extensive translational research. Gamida Cell's nicotinamide (NAM) platform for cultured NK cells provides an opportunity to enhance the therapeutic potential of NK cells. CD38 is an ectoenzyme ubiquitously expressed on the surface of various hematologic cells, including multiple myeloma (MM). It has been selected as a lead target for numerous monoclonal therapeutic antibodies against MM. Monoclonal antibodies target CD38, resulting in the lysis of MM plasma cells through various antibody-mediated mechanisms such as antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity, and antibody-dependent cellular phagocytosis, significantly improving the outcomes of patients with relapsed or refractory MM. However, this therapeutic strategy has inherent limitations, such as the anti-CD38-induced depletion of CD38-expressing NK cells, thus hindering ADCC. We have developed genetically engineered NK cells tailored to treat MM, in which CD38 was knocked-out using CRISPR-Cas9 technology and an enhanced chimeric antigen receptor (CAR) targeting CD38 was introduced using mRNA electroporation. This combined genetic approach allows for an improved cytotoxic activity directed against CD38-expressing MM cells without self-inflicted NK-cell-mediated fratricide. Preliminary results show near-complete abolition of fratricide with a 24-fold reduction in self-lysis from 19% in mock-transfected and untreated NK cells to 0.8% of self-lysis in CD38 knock-out CAR NK cells. Furthermore, we have observed significant enhancements in CD38-mediated activity in vitro, resulting in increased lysis of MM target cell lines. CD38 knock-out CAR NK cells also demonstrated significantly higher levels of NK activation markers in co-cultures with both untreated and αCD38-treated MM cell lines. These NAM-cultured NK cells with the combined genetic approach of CD38 knockout and addition of CD38 CAR represent a promising immunotherapeutic tool to target MM.

Dynamic immune landscape in vaccinated-BA.5-XBB.1.9.1 reinfections revealed a 5-month protection-duration against XBB infection and a shift in immune imprinting

The impact of previous vaccination on protective immunity, duration, and immune imprinting in the context of BA.5-XBB.1.9.1 reinfection remains unknown. Based on a 2-year longitudinal cohort from vaccination, BA.5 infection and XBB reinfection, several immune effectors, including neutralizing antibodies (Nabs), antibody-dependent cellular cytotoxicity (ADCC), virus-specific T cell immunity were measured to investigate the impact of previous vaccination on host immunity induced by BA.5 breakthrough infection and BA.5-XBB.1.9.1 reinfection. In absence of BA.5 Nabs, plasma collected 3 months after receiving three doses of inactivated vaccine (I-I-I) showed high ADCC that protected hACE2-K18 mice from fatality and significantly reduced viral load in the lungs and brain upon BA.5 challenge, compared to plasma collected 12 months after I-I-I. Nabs against XBB.1.9.1 induced by BA.5 breakthrough infection were low at day 14 and decreased to a GMT of 10at 4 months and 28% (9/32) had GMT ≤4, among whom 67% (6/9) were reinfected with XBB.1.9.1 within 1 month. However, 63% (20/32) were not reinfected with XBB.1.9.1at 5 months post BA.5 infection. Interestingly, XBB.1.9.1 reinfection increased Nabsagainst XBB.1.9.1 by 24.5-fold at 14 days post-reinfection, which was much higher than that against BA.5 (7.3-fold) and WT (4.5-fold), indicating an immune imprinting shifting from WT to XBB antigenic side. Overall, I-I-I can provide protection against BA.5 infection and elicit rapid immune response upon BA.5 infection. Furthermore, BA.5 breakthrough infection effectively protects against XBB.1.9.1 lasting more than 5 months, and XBB.1.9.1 reinfection results in immune imprinting shifting from WT antigen induced by previous vaccination to the new XBB.1.9.1 antigen. These findings strongly suggest that future vaccines should target variant strain antigens, replacing prototype strain antigens. This study was supported by R&D Program of Guangzhou National Laboratory (SRPG23-005), National Key Research and Development Program of China (2022YFC2604104, 2019YFC0810900), S&T Program of Guangzhou Laboratory (SRPG22-006), and National Natural Science Foundation of China (81971485, 82271801, 81970038), Emergency Key Program of Guangzhou Laboratory (EKPG21-30-3), Zhongnanshan Medical Foundation of Guangdong Province (ZNSA-2020013), and State Key Laboratory of Respiratory Disease (J19112006202304).

Acute Inflammation Redirect Bone Marrow Hematopoietic Stem Cell Cycling and Differentiation Program By Reshaping Their Chromatin Architecture at Long-Term

Background: Acute and persistent inflammation induced by microbial infections/sepsis or by therapeutic interventions, such as whole-body irradiation and CAR-T therapy, triggers a rapid overwhelming immune response in the host. While these responses are well defined in the short-term, the long-term impact of the inflammatory injury on the hematopoietic system still needs to be unveiled. Using a murine model of severe inflammation induced by bacteria P. aeruginosa ( P. aer.) or LPS, our group has previously reported that bone marrow (BM) hematopoietic stem cells (HSC) respond to infections with an aberrant expansion associated with their transitory inability to generate the downstream myeloid progenitors. Such dysfunctions correlated with the suppression of transcription factors governing stem-cell self-renewal and myeloid differentiation. Furthermore, when transplanted into healthy mice, the “inflamed” HSC showed reduced ability to contribute to the myeloid lineage and to engraft at long-term, suggesting that they retain memory of the LPS-mediated insult and maintain long-lasting changes. Thus, we hypothesized that severe inflammation alters the HSC epigenome, reshaping their transcriptional programs and affecting cell fate decisions and differentiation. Methods: We performed time-course experiments in WT mice challenged with a single dose of LPS or PBS, as control, and investigated 1) the kinetics of hematopoietic recovery by flow-cytometric immunophenotypic characterization, including cell-cycle 2) changes in the chromatin landscape of Lin- sorted BM subpopulations identified by the expression of the CD150 and CD48 markers as Long-Term (LT-HSC), Short-Term (ST-HSC), and Multipotent Progenitors (MPP1/2 and MPP3/4) using ATAC-seq. Results: Our studies revealed that the LPS challenge resulted in a significant increase in the cell-cycle of LT-HSC, ST-HSC, MPPs, common myeloid progenitors (CMP), and granulocyte-monocyte progenitors (GMP), which persisted up to 3 days from the inflammatory injury. Immunophenotypic analysis showed that while HSC frequencies returned to baseline at 5 days from LPS, the abundance and cell-cycle of myeloid progenitors and mature cells remained altered. Surprisingly, at 15 days post LPS, the cell-cycle of HSC markedly decreased, and such dysregulation was maintained until our last observation at 30 days from LPS. The pool of BM HSC was severely depleted and associated with exhaustion of T-cells but increased B-cells. GMP and Gr1+Mac1+ cells were also significantly decreased in the BM whereas mature lineages were increased in the peripheral blood, exhibiting high WBC, lymphocytes, and platelet count, neutrophilia, monocytosis, basophilia, and eosinophilia. ATAC-seq analysis revealed prominent differences in the chromatin architecture of the four HSC subpopulations at steady state (PBS). In all of them, LPS induced both i) a transient reorganization of the chromatin in some loci, and ii) the formation of persistently reshaped regions. Deeper investigations into the long-term changed sites revealed that chromatic accessibility was altered in proximity of CTCF-enriched regions fundamental to mediate chromatin reorganization and drive gene expression. Moreover, LPS modified the accessibility of genomic loci involved in the maintenance of HSC stemness and quiescence as well as regulation of their cell-cycle, self-renewal, and cell fate decision. Conclusions: Taken together these results suggest that LPS-mediated acute inflammation has long-lasting effects on HSC epigenetic regulation, influencing their cell-cycle and multilineage decisions. Relevance: The association between the resulting phenotype and the persistency of epigenetically remodeled HSC loci identified here may provide potential insights for future therapeutic approaches to re-direct “inflamed” HSC to a normal differentiation program, thereby enabling an efficient innate host response. Disclosures: No relevant conflicts of interest to declare.

2888. Safety and Immunogenicity of an Adjuvanted Chikungunya Virus (CHIKV) Virus-like Particle (VLP) Based Vaccine in Two Pivotal Phase 3 Trials, ≥12 Years of Age

Abstract Background CHIKV remains a significant public health concern globally. We report the results of two phase 3 trials evaluating an aluminum hydroxide adjuvanted CHIKV VLP vaccine. Methods Two multicenter, randomized, double-blind, placebo-controlled, parallel-group trials were conducted: an adult/adolescent trial (NCT05072080) in ages 12-64 years and an older adult trial in ages ≥ 65 years (NCT05349617). Participants received CHIKV VLP vaccine or placebo as a single intramuscular dose. Immunogenicity objectives assessed anti-CHIKV NT80 serum neutralizing antibody (SNA) titers at selected timepoints. Seroresponse rate (SRR) was the percentage of participants who achieved NT80 SNA titer ≥ 100 (FDA/EMA agreed threshold). Safety outcomes were evaluated through monitoring adverse events (AEs) through Day 183. Study overview. Results Adult/adolescent trial: 3254 participants (2790 CHIKV VLP vaccine, 464 placebo) were enrolled. Primary endpoints were met with a Day 22 SRR of 98% (2503/2559) for vaccine and 1% for placebo (5/424; p< 0.0001), as well as lot consistency, and superiority to placebo in geometric mean titer (GMT). A rapid antibody response was observed in the CHIKV VLP vaccine group with Day 8 SRR=47% (1169/2510) and Day 15 SRR=97% (2355/2434); responses were durable through Day 183 with SRR=86% (1967/2301). Older adult trial: 413 participants (206 CHIKV VLP vaccine, 207 placebo) were enrolled. Primary endpoints were met with a Day 22 SRR of 87% (167/191) for vaccine and 1% for placebo (2/183; p< 0.0001), as well as by GMT. At Day 15 a rapid antibody response was observed in the CHIKV VLP vaccine group with SRR=82% (150/182). CHIKV VLP vaccine demonstrated a favorable safety profile, and most AEs were mild to moderate in severity. The most common AEs were myalgia, fatigue, and headache. Conclusion CHIKV VLP vaccine is the only VLP-based vaccine in clinical development for active immunization against CHIKV disease. Results demonstrate that CHIKV VLP vaccine induces a rapid and robust immune response in most people by Day 15 and through Day 183. These findings support the potential of this VLP-based vaccine to help protect individuals 12 years and older from CHIKV. Disclosures Jason S. Richardson, PhD, Bavarian Nordic Canada Inc: Stocks/Bonds Debbie Anderson, MS, Bavarian Nordic: Employee|Emergent BioSolutions: Stocks/Bonds Sufia Muhammad, MD, Bavarian Nordic: Employee|Emergent BioSolutions: Stocks/Bonds Lauren Tindale, PhD, Bavarian Nordic: Employee Tobi Loreth, n/a, Emergent BioSolutions: Stocks/Bonds Sarah Royalty Tredo, MBA, Emergent Biosolutions: Employee|Emergent Biosolutions: Stocks/Bonds Victoria Jenkins, PhD, Bavarian Nordic: Employee Patrick Ajiboye, MD, Bavarian Nordic: Employee|Emergent BioSolutions: Stocks/Bonds Lisa Bedell, Director Biostatistic, Bavarian-Nordic: Advisor/Consultant|Emergent BioSolutions: Employee|Emergent BioSolutions: Stocks/Bonds