Antibody Production Research Articles

Construction of bispecific antibodies by specific pairing between the heavy chain and the light chain using removable SpyCatcher/SnoopCatcher units

During the production of bispecific antibodies (bsAbs), nonspecific pairing results in low yields of target bsAb molecules, an issue known as the “mispairing problem.” Several antibody engineering techniques have been developed to overcome mispairing issues. Here, we introduce “bsAb by external pairing and excision” (BAPE), a novel chain pairing method that induces specific chain pairing by fusing external SpyCatcher/Tag and SnoopCatcher/Tag units. These tags are then removed via protease cleavage. In this study, we applied this method to force the correct pairings of heavy and light chains while the heavy-chain pairing was achieved by the Knobs-into-Holes mutation. We then confirmed the formation of interchain bridges with covalent isopeptide bonds. Both anti-CD3/anti-Her2 and anti-CD3/anti-EGFR bsAbs displayed satisfactory target binding activities and in vitro cell-killing activity with activated T-cells.

Evaluation of a Low-Temperature Immersion Immunization Strategy for the Infectious Spleen and Kidney Necrosis Virus orf037l Gene-Deleted Attenuated Vaccine

Background: Infectious spleen and kidney necrosis virus (ISKNV) poses a significant threat to aquaculture sustainability, particularly affecting mandarin fish (Siniperca chuatsi) and causing significant economic losses. Methods: To address this challenge, this study developed an ISKNV Δorf037l vaccine strain, where the orf037l gene was knocked out. Infection assays conducted at 28 °C showed that the knocking out the orf037l gene decreased the virulence of ISKNV and reduced lethality against mandarin fish by 26.7% compared to wild-type ISKNV. To further diminish residual virulence, the effect of low-temperature (22 °C) immersion immunization was evaluated. Results: The results indicate that low temperature significantly diminished the virulence of the Δorf037l vaccine strain, elevating the survival rate of mandarin fish to 90%. Furthermore, the vaccine strain effectively triggered the expression of crucial immune-related genes, such as IFN-h, IL-1, IκB, Mx, TNF-α, and Viperin, while inducing the production of specific neutralizing antibodies. Low-temperature immersion with Δorf037l achieved a high relative percentage of survival of 92.6% (n = 30) in mandarin fish, suggesting the potential of Δorf037l as a promising immersion vaccine candidate. Conclusions: These findings contribute to advancing fish immersion vaccine development and demonstrate the importance and broad applicability of temperature optimization strategies in vaccine development. Our work carries profound implications for both the theoretical understanding and practical application in aquaculture disease control.

A mathematical model simulating the adaptive immune response in various vaccines and vaccination strategies

Vaccination has been widely recognized as an effective measure for preventing infectious diseases. To facilitate quantitative research into the activation of adaptive immune responses in the human body by vaccines, it is important to develop an appropriate mathematical model, which can provide valuable guidance for vaccine development. In this study, we constructed a novel mathematical model to simulate the dynamics of antibody levels following vaccination, based on principles from immunology. Our model offers a concise and accurate representation of the kinetics of antibody response. We conducted a comparative analysis of antibody dynamics within the body after administering several common vaccines, including traditional inactivated vaccines, mRNA vaccines, and future attenuated vaccines based on defective interfering viral particles (DVG). Our findings suggest that booster shots play a crucial role in enhancing Immunoglobulin G (IgG) antibody levels, and we provide a detailed discussion on the advantages and disadvantages of different vaccine types. From a mathematical standpoint, our model proposes four essential approaches to guide vaccine design: enhancing antigenic T-cell immunogenicity, directing the production of high-affinity antibodies, reducing the rate of IgG decay, and lowering the peak level of vaccine antigen-antibody complexes. Our study contributes to the understanding of vaccine design and its application by explaining various phenomena and providing guidance in comprehending the interactions between antibodies and antigens during the immune process.

Revealing disease subtypes and heterogeneity in common variable immunodeficiency through transcriptomic analysis

Common Variable Immunodeficiency (CVID) is a primary immunodeficiency characterized by reduced levels of specific immunoglobulins, resulting in frequent infections, autoimmune disorders, increased cancer risk, and diminished antibody production despite an adequate B cell count. With its clinical manifestations being highly variable, the classification of CVID, including the widely recognized Freiburg classification, is primarily based on clinical symptoms and genetic variations. Our study aims to refine the classification of CVID by analyzing transcriptomics data to identify distinct disease subtypes. We utilized the GSE51405 dataset, examining transcriptomic profiles from 30 CVID patients without complications. Employing a combination of clustering techniques—KMeans, hierarchical agglomerative clustering, spectral clustering, and Gaussian Mixture models—and differential gene expression analysis with R’s limma package, we integrated molecular findings with demographic data (age and gender) through correlation analysis and identified common genes among clusters. Three distinct clusters of CVID patients were identified using KMeans, Agglomerative Clustering, and Gaussian Mixture Models, highlighting the disease’s heterogeneity. Differential expression analysis unveiled 31 genes with variable expression levels across these clusters. Notably, nine genes (EIF5A, RPL21, ANP32A, DTX3L, NCF2, CDC42EP3, CHP1, FOLR3, and DEFA4) exhibited consistent differential expression across all clusters, independent of demographic factors. The study recommends categorizing patients based on the four genes, NCF2, CHP1, FOLR3, and DEFA4—as they may assist in prognostic prediction. Transcriptomic analysis of common variable immunodeficiency (CVID) patients identified three distinct clusters based on gene expression, independent of age and gender. Nine differentially expressed genes were identified across these clusters, suggesting potential biomarkers for CVID subtype classification. These findings highlight the genetic heterogeneity of CVID and provide novel insights into disease classification and potential personalized treatment approaches.

The function of serine/threonine-specific protein kinases in B cells.

The serine/threonine-specific protein kinases (STKs) are important for cell survival, proliferation, differentiation, and apoptosis. In B cells, these kinases play indispensable roles in regulating important cellular functions. Multiple studies on human and other animal cells have shown that multiple STKs are involved in different stages of B cell development and antibody production. However, how STKs affect B cell development and function is still not completely understood. Considering that B cells are clinically important in immunity and diseases, our understanding of STKs' roles in B cells is in great need of investigation with current technologies. Investigating serine/threonine kinases will not only deepen our insight into B cell-related disorders but also facilitate the identification of more effective drug targets for conditions like lymphoma and systemic lupus erythematosus.

Preparation and immunological study of Ichthyophthirius multifiliis β-tubulin DNA vaccine in grass carp (Ctenopharyngodon idella)

Grass carp, a common aquatic species in China, suffer significant annual losses due to infection by the parasitic ciliate Ichthyophthirius multifiliis (Ich), thus necessitating effective prevention and control measures. In the present study, the Ich β-tubulin gene was cloned and inserted into the eukaryotic expression vector pVAX1 plasmid to construct the Ich β-tubulin DNA vaccine (pVAX1-Bt). After two doses of the pVAX1-Bt vaccine, the relative percentage survival (RPS) reached 70.0 %, whereas the RPS after 2 doses of pVAX1 reached 10.0 %. The β-tubulin antibody, specific antibody IgM, and immune enzymes (MDA, SOD, CAT, ACP, AKP, and cortisol) were tested over13 weeks. The findings revealed a significant enhancement in the vaccinated fish compared to their unvaccinated counterparts, indicating that the vaccine can activate the humoral immune pathway and non-specific immunity in fish, thereby promoting the production of specific antibodies and memory B cells. Collectively, these results indicate that the Ich β-tubulin DNA vaccine has potential in treatment of Ich infection (ichthyophthiriasis).

Saurian-associated Leishmania tarentolae in dogs: Infectivity and immunogenicity evaluation in the canine model.

In canine leishmaniosis endemic areas, Leishmania infantum may occur in sympatry with the non-pathogenic Leishmania tarentolae, which is associated to reptiles. The potential infectivity of L. tarentolae for mammals raises questions about the interactions between the two Leishmania species, and the potential cross-immune protection in dogs. This study aimed to assess the outcome of experimental L. tarentolae infection in dogs, determining: i) the anti-L. tarentolae antibody production, ii) the duration of the immunity and cytokine expression, and iii) the possible pathogenic effect in the canine host. Twelve purpose-bred beagle dogs were randomly allocated to three groups (intravenous inoculation, G1; intradermal inoculation, G2; negative control, G3). G1 and G2 dogs were inoculated twice (day 0, day 28) with 108 promastigotes of L. tarentolae strain (RTAR/IT/21/RI-325) isolated from a Tarentola mauritanica gecko. The animals were followed until day 206. Blood, serum, conjunctival swabs and lymph node aspirate samples were collected monthly and bone marrow, liver and spleen biopsies on day 91. Hematological and biochemical parameters were assessed monthly, as well as serology (IFAT and ELISA) and molecular identification of L. tarentolae. Mononuclear cells (PBMC) were obtained to assess the cytokine expression through in vitro stimulation or (re-) infection. Data from this study demonstrated that DNA from L. tarentolae is detectable up to 3 months post-infection, with seroconversion after day 28. Moreover, the non-pathogenic nature of L. tarentolae was confirmed, with a neutral Th1/Th2 polarization, and a possible shift to Th1 phenotype after derived macrophages (re-) infection, as demonstrated by the expression of IFN-gamma. Therefore, L. tarentolae demonstrated a great potential as a surrogate pathogen and/or immune-prophylaxis/immune-therapy against Leishmania infections in dogs and humans.

B cell immune repertoire sequencing in tobacco cigarette smoking, vaping, and chronic obstructive pulmonary disease in the COPDGene cohort.

Cigarette smoking (CS) impairs B cell function and antibody production, increasing infection risk. The impact of e-cigarette use ('vaping') and combined CS and vaping ('dual-use') on B cell activity is unclear. To examine B cell receptor sequencing (BCR-seq) profiles associated with CS, vaping, dual-use, COPD-related outcomes, and demographic factors. BCR-seq was performed on blood RNA samples from 234 participants in the COPDGene study. We assessed multivariable associations of B cell function measures (immunoglobulin heavy chain (IGH) subclass expression and usage, class-switching, V-segment usage, and clonal expansion) with CS, vaping, dual-use, COPD severity, age, sex, and race. We adjusted for multiple comparisons using the Benjamini-Hochberg method, identifying significant associations at 5% FDR and suggestive associations at 10% FDR. Among 234 non-Hispanic white (NHW) and African American (AA) participants, CS and dual-use were significantly positively associated with increased secretory IgA production, with dual-use showing the strongest associations. Dual-use was positively associated with class switching and B cell clonal expansion, indicating increased B cell activation, with similar trends in those only smoking or only vaping. We observed significant associations between race and IgG antibody usage. AA participants had higher IgG subclass proportions and lower IgM usage compared to NHW participants. CS and vaping additively enhance B cell activation, most notably in dual-users. Self-reported race was strongly associated with IgG isotype usage. These findings highlight associations between B cell activation and antibody transcription, suggesting potential differences in immune and vaccine responses linked to CS, vaping, and race.

Adenoviral fiber-knob based vaccination elicits efficient neutralizing antibodies and T cell responses against adenovirus infection

BackgroundHuman adenoviruses (HAdVs) frequently cause common respiratory or gastrointestinal infections among children, adults, individuals with immune deficiencies, and other vulnerable populations with varying degree of symptoms, ranging from mild to server, and in some cases, even fatalities. Despite the significant clinical impact of HAdVs, there is currently no approved vaccine available.MethodsThis study explores the potential of the adenovirus type 5 fiber knob (Ad5-FK) to stimulate the production of Ad-specific neutralizing antibodies and T-cell responses in mice. Based on structure predictions, we first expressed Ad5-FK in E. coli and confirmed the assembly of FK into its trimeric form. After testing the binding capability of the trimeric FK to susceptible cells, the immunogenicity of the protein in combination with the c-di-AMP adjuvant was assessed in BALB/c mice.ResultsThe purified Ad5-FK exhibited self-trimerization and maintained correct conformation akin to the authentic FK structure. This facilitated effective binding to susceptible HEK293 cells. Notably, the protein demonstrated significant inhibition of HEK293 cells infection by rAd5-GFP. Immunization of BALB/c mice with Ad5-FK, or Ad5-FK mixed with c-di-AMP yielded FK-specific antibodies with potent neutralization capacity. Significantly, Ad5-FK was found to elicit a vigorous CD4+ T-cell response in the immunized mice.ConclusionOur findings underscore the efficacy of FK-based vaccine in eliciting anti-Ad humoral immune response and CD4 T-cell immune reactions essential for protection against viral infections.

8150 CD1a Lipid Presentation Contributes to T2D Phenotype in DIO Mouse Model

Abstract Disclosure: J.A. Ayala Angulo: None. K.D. Wiggins: None. M. Lopez: None. D. Nicholas: None. Type 2 Diabetes’s complex etiology, progression, and comorbidities are not fully explained by current models. Elevation of inflammatory markers and immune cell infiltration into tissues of T2D patients has been described in the literature extensively, but no clear answers as to what antigens are driving this response and how these observations contribute to disease progression exist. Recently, CD1a has been shown to present lipids to T cells and induce a memory response. Our previous work shows that T2D patients produce anti-lipid antibodies likely derived with T cell help, not present in healthy patients. Together, these data suggest a potential role for lipid-specific T cell responses to drive T2D progression through inflammatory stress, antibody production, and tissue infiltration. Current work with human CD1a transgenic B6 mice points to elevated liver steatosis and hepatitis along with higher glucose levels relative to low fat diet controls. CD1a mice show smaller islets suggesting pancreatic atrophy. This validates the CD1a-T cell mechanism as a potential contributor to T2D but also introduces a more physiologically relevant mouse model of T2D. Presentation: 6/1/2024

6479 Acute Estradiol and Progesterone Therapy In Hospitalized Adults With Moderate COVID-19: A Randomized Control Trial

Abstract Disclosure: D. Lovre: Research Investigator; Self; Novo Nordisk, Corcept Therapeutics. K.M. Bateman: None. L.Y. Saltzman,: None. M. Qadir: None. F. Mauvais-Jarvis: None. Background and Objective: COVID-19 outcomes are less severe in women vs men. Experimental studies show that estradiol (E2) and progesterone (P4) produce anti-inflammatory immune responses and immune tolerance and enhance antibody production. The effect of E2 and P4 combination on COVID-19 severity is unknown. The aim of the study was to evaluate the efficacy of a short systemic E2/P4 combination in mitigating COVID-19 severity in hospitalized men and women. Methods: Phase 2, double blind, placebo-equivalent controlled, single center trial of ten participants (men and women) who were hospitalized for COVID-19, with scores 3 to 5 on the 9-point World Health Organization (WHO) ordinal scale for clinical improvement. Participants were randomized into two arms and treated for five days: 1) E2 Cypionate (5 mg IM once) and micronized P4 (200mg, PO daily); and 2) Placebo-equivalent (normal saline (1ml, IM once) and folate (400mcgs, PO daily), in addition to standard of care (SOC). The primary outcome was the proportion of patients improving to scores 1 or 2 on the WHO scale on the day of discharge. Secondary outcomes included length of hospital stay (LOS), days on oxygen therapy (DOT), readmission rates (RR), adverse events (AEs) and change in inflammatory biomarkers assessed by untargeted proteomics. Findings: There were no significant differences between the treatment groups in COVID-19 severity by WHO score, LOS, DOT, RR or AEs. Patients in the E2/P4 arm exhibited a decrease in inflammatory biomarkers of respiratory and gastrointestinal disease (GI) and significant change in immune and inflammatory responses. Conclusions: Use of short-term systemic E2/P4 is associated with decrease in biomarkers of respiratory and GI disease inflammation and change in biomarkers of immune and inflammatory response. Presentation: 6/3/2024

Transcutaneous Immunization of 1D Rod-Like Tobacco-Mosaic-Virus-Based Peptide Vaccine via Tip-Loaded Dissolving Microneedles.

Peptide vaccines induce specific neutralizing antibodies and are effective in disease prevention and treatment. However, peptide antigens have a low immunogenicity and are unstable, requiring efficient vaccine carriers to enhance their immunogenicity. Here, we develop a tobacco mosaic virus (TMV)-based peptide vaccine for transdermal immunization using a tip-loaded dissolving microneedle (MN) patch. TMV is decorated with the model peptide antigen PEP3. The prepared TMV-PEP3 promotes dendritic cell maturation and induces dendritic cells to overexpress MHC II, costimulatory factors, and pro-inflammatory factors. By encapsulation of TMV-PEP3 in the tips of a trehalose MN, TMV-PEP3 can be delivered by MN and significantly promote local immune cell infiltration. In vivo studies show that both subcutaneous injection and MN administration of TMV-PEP3 increase the production of anti-PEP3 IgG antibodies and the harvested serum can induce complement-dependent cytotoxicity. This work provides a promising strategy for constructing efficient and health-care-friendly peptide vaccines.

Mechanism of modified mRNA structure in COVID-19 vaccines for inducing neutralizing antibodies

The development of SARS-CoV-2 mRNA vaccines is closely linked to advancements in mRNA manufacturing technology. Structural modifications, such as replacing uridine with 1-methylpseudouridine (1mψ), enhance translation efficiency and help the mRNA evade immune detection. Lipid nanoparticles (LNPs) serve as an effective delivery system. Vaccines like BNT162b2 and mRNA-1273 target the receptor-binding domain (RBD) of the spike (S) protein, prompting B cells to produce neutralizing antibodies that block the RBD from binding to the Angiotensin-Converting Enzyme 2 (ACE2) receptor, preventing infection. These vaccines also stimulate adaptive immune responses by activating CD4+ and CD8+ T cells, with mRNA functioning as an endogenous antigen. Antigen-presenting cells (APCs) present the vaccine antigens via major histocompatibility complex (MHC) class I and II pathways, with CD8+ T cells recognizing MHC class I and destroying infected cells, while CD4+ T cells recognize MHC class II and assist in B cell maturation and antibody production. While mRNA vaccines have proven effective in neutralizing SARS-CoV-2, challenges remain, including the decline in neutralizing antibody titers over time and the emergence of new viral variants.

Prophylaxis of respiratory syncytial virus infection: current status and prospects for vaccine development

INTRODUCTION. Respiratory syncytial virus (RSV) is one of the most widespread pathogens that typically cause acute upper and lower respiratory tract infections in children and adults. Monoclonal antibody products have long been used for passive immunoprophylaxis in premature infants with bronchopulmonary dysplasia. However, vaccines have recently been licensed for the prophylactic immunisation of older adults with various risk factors for severe RSV infection (primarily, chronic cardiovascular and respiratory diseases and diabetes mellitus).AIM. This study aimed to review the current status of the development of vaccines for active immunisation against RSV infection, including the epidemiological rationale for their development, clinical trial results for licensed vaccines, recommendations for vaccination, and promising pipeline vaccines for RSV prevention.DISCUSSION. Initially hindered by the unique immunopathogenesis of RSV infection and the genetic hypervariability of RSV for a long time, attempts to develop an RSV vaccine succeeded when international researchers managed to identify a conservative neutralising antibody target capable of inducing specific immunity against RSV. This target, the RSV capsid protein stabilised in its prefusion (pre-F) conformation, can mediate viral entry into the cell following a conformational change. Several subunit vaccines based on recombinant pre-F proteins have successfully passed clinical trials and have been approved for the immunisation of older adults. In addition, one of these vaccines has been recommended for use during pregnancy to prevent RSV infection in newborns and infants. Currently, programmes are being implemented to develop novel RSV vaccines based on messenger RNA (mRNA) and non-replicating attenuated influenza vectors. This article examines and summarises the efficacy and safety parameters of two RSV vaccines approved in multiple countries around the world. Both vaccines have satisfactory safety profiles and comparable prophylactic efficacy parameters.CONCLUSIONS. Prelicensure clinical trials of novel RSV vaccines, including those being developed in the Russian Federation, should include prophylactic efficacy assessments in target patient populations. For vaccines approved by leading international regulators, clinical trials required for approval in the Russian Federation will be limited to bridging studies confirming the immunogenicity and safety of the vaccines.

Development of a Mammalian Cell Line for Stable Production of Anti-PD-1.

Background/Objectives: Immune checkpoint blockade, particularly targeting the programmed cell death 1 (PD-1) receptor, is a promising strategy in cancer immunotherapy. The interaction between PD-1 and its ligands, PD-L1 and PD-L2, is crucial in immune evasion by tumors. Blocking this interaction with monoclonal antibodies like Nivolumab can restore anti-tumor immunity. This study aims to develop a stable expression system for Nivolumab-based anti-PD-1 in the Chinese Hamster Ovary (CHO) DG44 cell line using two different expression vector systems with various signal sequences. Methods: The heavy chain (HC) and light chain (LC) of Nivolumab were cloned into two expression vectors, pOptiVEC and pcDNA3.3. Each vector was engineered with two distinct signal sequences, resulting in the creation of eight recombinant plasmids. These plasmids were co-transfected into CHO DG44 cells in different combinations, allowing for the assessment of stable antibody production. Results: Both pOptiVEC and pcDNA3.3 vectors were successful in stably integrating and expressing the Nivolumab-based anti-PD-1 antibody in CHO DG44 cells. This study found that the choice of signal sequence significantly influenced the quantity of antibodies produced. The optimization of production conditions further enhanced antibody yield, indicating the potential for large-scale production. Conclusions: This study demonstrates that both pOptiVEC and pcDNA3.3 expression systems are effective for the stable production of Nivolumab-based anti-PD-1 in CHO DG44 cells. Signal sequences play a critical role in determining the expression levels, and optimizing production conditions can further increase antibody yield, supporting future applications in cancer immunotherapy.

A-235 Systemic Inflammation is associated with worse outcomes from SARS-CoV-2 infection but not neutralizing antibody

Abstract Background Numerous studies have demonstrated an association between systemic inflammation and mortality in patients with COVID-19. However, it is unclear how neutralizing antibody to severe acute respiratory syndrome related coronavirus 2 (SARS-CoV-2) associates with outcomes. Further, how systemic inflammation impacts neutralizing antibody production is relatively poorly understood. The aim of this study was to determine the association between the neutralizing antibody response to SARS-CoV-2, inflammatory markers, and clinical outcomes in a cohort of hospitalized patients without prior exposure/vaccination. Methods This prospective observational study approved by the Washington University IRB included adults admitted through the ED at Barnes Jewish Hospital between 10/2020 and 10/2021 with symptomatic RT-PCR confirmed SARS-CoV-2 infection. A total of 395 serial samples were assessed from 208 patients without previous vaccination or documented exposure to SARS-CoV-2. Neutralization of SARS-CoV-2 was assessed using the Architect ACE2 binding inhibition assay (Abbott, Research Use only) which measures the % inhibition of interactions between antibodies and the SARS-COV-2 viral Spike Receptor Binding Domain. While 12.5% inhibition of ACE2 was considered positive >80% neutralization is the expected standard. CRP was assessed using the Architect Multigent CRP assay with 9.6mg/dL as the categorical cutoff for risk and interleukin 6 (IL-6) was assessed using the Alinity IL-6 assay (Abbott, Research Use Only) using 30 pg/mL as the threshold for risk in categorical analyses. The electronic medical record (EPIC) was interrogated for comorbidities, age, gender, 30-day mortality, 10-day intubation, and pharmacological interventions. Correlation between biomarkers was assessed using Spearman r. Kaplan-Meier curves and Cox proportional hazards models were constructed for 30-day mortality and 10-day intubation. Results 37 of 208 (18%) of patients died within thirty days of ED presentation and 59 (28%) required intubation within 10-days. There was a significant correlation between IL-6 and CRP (r=0.34) but not with ACE-2 binding (Spearman r <0.06 for both IL-6 and CRP). There was significantly higher CRP in those that died (median = 14 mg/dL, IQR = 8-21) than those that survived (5 mg/dL, 2-11). IL-6 was higher in patients that died (344 pg/mL, 0-870) than those that survived (65 pg/mL, 28-140). Median ACE-2 inhibition trended higher in those that survived (18%, 0-65%) than those that died (3%, 0-48%). Survival curves demonstrated that patients with IL-6 > 30 pg/mL, CRP > 9.6 mg/dL, and ACE2 binding <12.5% at baseline were more likely to die within 30-days. Multivariate modeling found that only IL-6 (Hazard Ratio, 1.28, 95%CI 1.08-1.52 for each doubling) and age (1.04, 1.01-1.08) were significantly associated with 30-day mortality. Conclusions Systemic inflammation measured by IL-6 and CRP upon admission is highly associated with 30-day mortality from SARS-CoV-2 infection. While ACE-2 inhibition was lower in those that died from COVID-19 at admission, it was not independently associated with mortality or correlated with inflammatory markers. This implies an importance of other aspects of the immune response for mediating inflammation and reducing risk of mortality from SARS-CoV-2.

B-086 Evaluation of Long-Term Adaptive Immune Responses Specific to SARS-CoV-2: Effect of Various Vaccination and Omicron Exposure

Abstract Background The immune response to SARS-CoV-2 becomes increasingly complex as individuals receive different combinations of vaccines and encounter breakthrough infections. Our study aims to describe the immunogenicity observed over a two-year period in healthy individuals who completed two-dose series and then experienced booster and/or Omicron infection. Methods In June 2023, we recruited 78 healthcare workers who had previously participated in clinical research initiated in March 2021 at a single medical center. At 1, 5, 11, and 25 months after second dose, we assessed SARS-CoV-2-specific humoral and cellular immune responses using commercially available immunoassays and surrogate virus neutralization tests, along with interferon gamma (IFN-γ)-based enzyme-linked immunosorbent spot (ELIspot) assay using SARS-CoV-2 spike peptide pools. Results As the humoral immune response was monitored longitudinally, robust antibody production persisted after the third antigen exposure, in contrast to the rapid decline observed between the second and third dose injections. All immune metrics tended to increase as the number of vaccine doses increased. We also found a trend toward increased humoral immune response in the homologous mRNA-based vaccine group and enhanced cellular immune response in the vector-based primary series with mRNA booster heterologous combination group. Interestingly, no significant difference was found between Omicron exposure through breakthrough infection and bivalent vaccination. Omicron exposure significantly increased the breadth of neutralizing activity, while it did not significantly alter the cross-reactivity in the cellular immune response. Conclusions We identified individuals with diminished neutralization, aiding decisions for future vaccination prioritization. In addition, our findings suggest the role of cellular immunity as an important correlate of long-term protection through cross-reactivity to emerging variants.

CXCL13/CXCR5 axis facilitates TFH expansion and correlates with disease severity in adults with immune thrombocytopenia

BackgroundImmune thrombocytopenia (ITP) is an autoimmune bleeding disorder defined by a diminished platelet count. ITP pathogenesis involves intricate changes to both cellular and humoral immunity. The pivotal roles of follicular helper T (TFH) cells in the maturations of B cells and the production of antibodies are well-established. However, the specific role of TFH to the immunopathogenesis of ITP remain incompletely understood. This study aimed to clarify the association of CXCL13/CXCR5 axis with TFH in adults with ITP. MethodsA total of 97 ITP patients and 41 healthy controls were enrolled. CD4+CXCR5+ TFH, CD4+CXCR5+PD-1+ TFH, CD4+CXCR5+Foxp3+ follicular regulatory T cells (TFR), and desialylated platelets in peripheral blood were measured by flow cytometry. Plasma cytokines were assessed by enzyme-linked immunosorbent assay. CD4+ T cells cocultured with chemokine CXCL13 in vitro was performed for the measurement of TFH proliferation. Intracellular production of reactive oxygen species (ROS) was examined by dichlorodihydrofluorescein diacetate (DCFH-DA) probe staining. ResultsWe observed a significant increase in circulating TFH and a marked decrease in circulating TFR in the entire ITP cohort. The ratio of TFH/TFR was elevated, accompanied by heightened levels of platelet desialylation, cytokines BAFF, HMGB1, and IL-21, while levels of IL-10 were downregulated in adults with ITP. Notably, patients with ITP exhibiting platelet count below 50 × 109/L had dramatically elevated levels in both chemokine CXCL13 and its receptor CXCR5+ TFH compared to those with platelet count above 100 × 109/L. High frequencies of TFH correlated with poor therapeutic response. Furthermore, in vitro CD4+ T cell proliferation assay demonstrated a CXCL13 dose-dependent increase in the frequencies in both CD4+CXCR5+ TFH and CD4+CXCR5+PD-1+ TFH from ITP patients. Intriguingly, DCFH-DA assay illustrated a significant enhancement in intracellular ROS generation in CXCR5+ T cell subsets, especially in CD4+CXCR5+PD-1+ TFH from 4 patients with ITP. ConclusionsThese results underscore the pivotal role of CXCL13/CXCR5 axis-drived TFH expansion in the pathogenesis of ITP, providing a potential disease severity biomarker.

Phenotypic Characterization of B-Lymphocyte Subpopulations in Human Peripheral Blood: A Cost-Effective Seven-Color One-Tube Protocol.

B cells are crucial components of the immune system, responsible for producing specific antibodies in response to infections and vaccines. Despite their uniform appearance, B cells display diverse surface molecules and functional properties, which are not yet fully understood. Apart from antibody production, B cells also play roles in antigen presentation and cytokine secretion, essential for initiating T-cell immune responses. Their significance as disease biomarkers and therapeutic targets has led to increased research focus. However, the lack of standardized protocols for B-cell identification and the variability in defining B-lymphocyte subpopulations pose some challenges. This paper proposes a B-cell identification panel throughout the evaluation of previous cytometry panels and nomenclature heterogeneity for B-cell subpopulations. Major subpopulations recognized in human peripheral blood include transitional, naive, switched memory, unswitched memory, double negative, and plasmablasts, characterized based on their functional and phenotypic features. We present a standardized flow cytometry protocol utilizing surface phenotypic markers (CD3, CD19, IgD, CD27, CD38, and CD24) to differentiate and analyze B-cell subpopulations. This practical and cost-effective panel can be used in various research and laboratory settings. The challenges of standardizing names and markers for classifying B-lymphocyte subpopulations are discussed, along with protocols utilizing multiple markers and gating strategies, allied with the importance of considering viability markers. In summary, this standardized protocol and panel provide a comprehensive approach to identifying B-cell subpopulations to enhance the reproducibility and comparability of B-cell subpopulation studies.

Persistent Splenic-Derived IgM Preferentially Recognize Factor VIIIA2 and C2 Domain Epitopes but Do Not Alter Antibody Production

The most significant treatment complication for patients with hemophilia A is the development of neutralizing immunoglobin G (IgG), termed inhibitors, against factor VIII (FVIII) which prevent FVIII replacement therapy. Low titers of FVIII-specific immunoglobin M (IgM) have been identified in hemophilia A patients with and without inhibitors, as well as healthy individuals. However, the duration and influence of IgM on the immune response to FVIII remains unclear. To characterize the binding interactions of persistently secreted FVIII-specific IgM in hemophilia A mice and assess their effect on IgG antibody development. Splenic-derived monoclonal antibodies (MAbs) from immunized FVIII knockout mice were isolated and purified using hybridoma technology. Binding interactions were assessed utilizing a novel fluid-phase ELISA and computational modeling with HADDOCK to account for weak IgM binding. Sixteen porcine cross-reactive and non-inhibitory FVIII-specific IgM MAbs were identified. RNA sequencing of FVIII-specific IgM revealed 13 unique VDJ/VJ sequences indicating derivation from 13 unique B cell clones. IgM demonstrated polyclonal and polyreactive binding to FVIII in vitro and in silico. Molecular docking studies with reconstructed IgM VDJ/VJ regions identified frequent IgM interactions with amino acid residues K376, T381, K437, R2215 or K2249 within the FVIII A2 and C2 domains. Injections of individual IgM prior to FVIII exposure and co-injection of FVIII/IgM immune complexes did not affect de novo FVIII antibody production. Persistent FVIII-specific IgM are polyclonal but preferentially bind the A2 and C2 domains and FVIII/IgM immune complex formation do not significantly alter inhibitor development.