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(Group A Streptococcus, Strep A) is a Gram-positive bacterium and one of the most significant global bacterial pathogens, with disproportionate disease burden affecting low and middle-income countries. Despite substantial mortality and immune sequelae caused by Strep A, no licensed vaccines exist, and correlates of protection remain unestablished. Advancing Strep A vaccine development requires standardized and qualified assays to accurately measure vaccine-induced immune responses to support vaccine development and licensure/post-licensure monitoring. This study reports a direct comparison of two qualified multiplex immunoassays designed to detect human IgG antibodies targeting four distinct Strep A vaccine antigens. The MSD assay platform employs positional multiplexing with electrochemiluminescent detection, while the Luminex system uses bead-based multiplexing with fluorescent detection. Both assays used standardized intravenous immunoglobulin dilutions as reference standards. Assay precision was evaluated by analyzing eight samples from human volunteers selected to span the dynamic range for the four antigens Streptolysin O, cell envelope protein, adhesion and division protein, and group A carbohydrate. Both platforms demonstrated comparable intermediate precision and repeatability profiles. Increased variance was observed in samples with very low antibody concentrations. Neither operator variability nor day-to-day factors significantly impacted assay results. Additionally, inter-assay coefficients of variation for standards remained below 15% within the pre-established quantitative ranges for both platforms. The findings demonstrate that both multiplex immunoassays show equivalent precision and repeatability for quantifying human IgG to four Strep A vaccine antigens, making both platforms suitable for supporting early clinical trials and seroepidemiological studies.

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 emerged in China in 2019 and has since travelled the world infecting millions. SARS-CoV-2 causes Corona Virus Disease (COVID-19), that has to date taken over 4 million lives. The Kingdom of Bahrain’s vaccine roll-out has consisted of Sinopharm’s BBIBP-CorV (Sinopharm) and Pfizer/BioNtech’s BNT162b2 (Pfizer/BioNtech). Testing for SARS-CoV-2 anti-Spike (S) antibodies is a useful technique in estimating an individual’s immune protection against the infection. In this study we evaluated S antibody levels by electro-chemiluminescence immunoassay in 379 individuals double vaccinated with Sinopharm and 15 of whom were given a booster with the Pfizer/BioNtech vaccine. Among our double vaccinated cohort, we found a spectrum of S antibody levels. Indeed, we found that a significant proportion of individuals with low S antibody levels had clinical conditions, which were mainly immune-related disorders. Furthermore, a significant proportion of individuals with low S antibody levels were above 50 years of age. Finally, we observed a significant increase in S antibody levels after the Pfizer/BioNtech booster was administered. These findings reveal that while a large proportion of Sinopharm vaccinated individuals did not develop high levels of antibodies against the S protein, a booster dose of the Pfizer/BioNtech vaccine significantly enhances S antibody levels, revealing this “triple dose” vaccination strategy as a useful method of ensuring protective immunity against SARS-CoV-2.

Metastasis-initiating cells are key players in progression, resistance, and relapse of colorectal cancer (CRC), by leveraging the regulatory relationship between Transforming Growth Factor-beta (TGF-β) signaling and anti-L1 cell adhesion molecule (L1CAM). This study introduces a novel strategy for CRC targeted therapy and imaging based on the use of a hybrid nanosystem made of gold nanoparticles-covered porous biosilica further modified with the (L1CAM) antibody. The nanosystem intracellularly delivers galunisertib (LY), a TGF-β inhibitor, aiming to inhibit epithelial-mesenchymal transition (EMT), a process pivotal for metastasis. Anti-L1CAM antibody-functionalized nanoparticles (NPs) target tumor-initiating cells expressing L1CAM, inhibiting cancer growth. The number of antibody molecules conjugated to the single NP is precisely quantified, revealing a high surface coverage that facilitates the tumor targeting. The therapeutic efficacy of the nanosystem is investigated in organoid-like cultures of CRC cells and in vivo mouse models, showing a significant reduction in tumor growth. The spatial distribution of NPs within CRC tumors from mice is investigated using a label-free optical approach based on Raman micro-spectroscopy. This research highlights the multifunctional capabilities of engineered biosilica NPs, which offer new insights in targeted CRC therapy and imaging, improving patient outcomes and paving the way for personalized therapies.

Virus-specific antibodies are crucial for protective immunity against SARS-CoV-2. Assessing functional antibodies through conventional or pseudotyped virus neutralisation tests (pVNT) requires high biosafety levels. Alternatively, the virus-free surrogate virus neutralisation test (sVNT) quantifies antibodies interfering with spike binding to angiotensin-converting enzyme 2. We evaluated secreted nanoluciferase-tagged spike protein fragments as diagnostic antigens in the sVNT in a vaccination cohort. Initially, spike fragments were tested in a capture enzyme immunoassay (EIA), identifying the receptor binding domain (RBD) as the optimal diagnostic antigen. The sensitivity of the in-house sVNT applying the nanoluciferase-labelled RBD equalled or surpassed that of a commercial sVNT (cPass, GenScript Diagnostics) and an in-house pVNT four weeks after the first vaccination (98% vs. 94% and 72%, respectively), reaching 100% in all assays four weeks after the second and third vaccinations. When testing serum reactivity with Omicron BA.1 spike, the sVNT and pVNT displayed superior discrimination between wild-type- and variant-specific serum reactivity compared to a capture EIA. This was most pronounced after the first and second vaccinations, with the third vaccination resulting in robust, cross-reactive BA.1 construct detection. In conclusion, utilising nanoluciferase-labelled antigens permits the quantification of SARS-CoV-2-specific inhibitory antibodies. Designed as flexible modular systems, the assays can be readily adjusted for monitoring vaccine efficacy.

Hyperspectral imaging (HSI) is a non-invasive, contrast-free optical-based tool that has recently been applied in medical and basic research fields. The opportunity to use HSI to identify exogenous tumor markers in a large field of view (LFOV) could increase precision in oncological diagnosis and surgical treatment. In this study, the anti-high mobility group B1 (HMGB1) labeled with Alexa fluorophore (647 nm) was used as the target molecule. This is the proof-of-concept of HSI’s ability to quantify antibodies via an in vitro setting. A first test was performed to understand whether the relative absorbance provided by the HSI camera was dependent on volume at a 1:1 concentration. A serial dilution of 1:1, 10, 100, 1000, and 10,000 with phosphatase-buffered saline (PBS) was then used to test the sensitivity of the camera at the minimum and maximum volumes. For the analysis, images at 640 nm were extracted from the hypercubes according to peak signals matching the specificities of the antibody manufacturer. The results showed a positive correlation between relative absorbance and volume (r = 0.9709, p = 0.0013). The correlation between concentration and relative absorbance at min (1 µL) and max (20 µL) volume showed r = 0.9925, p < 0.0001, and r = 0.9992, p < 0.0001, respectively. These results demonstrate the HSI potential in quantifying HMGB1, hence deserving further studies in ex vivo and in vivo settings.

We studied the quantification of an intact therapeutic monoclonal antibody (mAb), rituximab (RTX), using (reverse-phase) high-performance liquid chromatography with diode array detection ((RP)HPLC/DAD). To this end, we developed a chromatographic method and validated it as stability-indicating in accordance with the International Conference on Harmonization guidelines (ICH). A 300-Å C8 column (250 mm × 4.6 mm, 5 μm) was used to perform the analysis, and the temperature was maintained at 70 °C. Although only one mAb was analyzed, it was necessary to apply a gradient to elute it with a complex organic mixture. Chromatograms were registered at several wavelengths, with λ  = 214 nm employed for quantification purposes. The method was developed to quantify marketed RTX under typical hospital administration conditions. Further dilution was avoided in order to prevent additional mAb modification, and in this way the method was shown to be linear from 60 to 5000 mg/L. The precision of the method (repeatability and intermediate precision, estimated as the relative standard deviation, RSD %), was less than 1.0 %. Accuracy, specificity, robustness, and system suitability were also evaluated as specified in the ICH guidelines. We conducted a comprehensive chromatographic analysis by submitting RTX to several informative stress conditions. These forced degradation studies were conducted for two reasons: to estimate the specificity of the method, and to evaluate the robustness of the mAb formulation against external stress factors when handling it in preparation for administration. Thus, we investigated the effects of acid, base, oxidation, ionic strength, temperature, and UV light. Although a slight modification to the intact mAb could not be distinguished chromatographically in the stress studies we conducted, the procedure proposed here to evaluate peak purity enabled us to detect it with a satisfactory level of confidence. The proposed method could therefore be considered stability-indicating for quantyfying the intact mAb since it is qualified to detect its degradation/modification. Finally, the method was used to evaluate RTX in a long-term stability study performed under hospital conditions of use.

Monovalent Omicron XBB.1.5 mRNA vaccines were newly developed and approved by the FDA in Autumn 2023 for preventing COVID‐19. However, clinical efficacy for these vaccines is currently lacking. We previously established the quantification of antigen‐specific antibody sequence (QASAS) method to assess the response to severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) vaccination at the mRNA level using B‐cell receptor (BCR) repertoire assay and the coronavirus antibody database (CoV‐AbDab). Here, we used this method to evaluate the immunogenicity of monovalent XBB.1.5 vaccines. We analyzed repeated blood samples of healthy volunteers before and after monovalent XBB.1.5 vaccination (BNT162b2 XBB.1.5 or mRNA‐1273.815) for the BCR repertoire to assess BCR/antibody sequences that matched SARS‐CoV‐2‐specific sequences in the database. The number of matched unique sequences and their total reads quickly increased 1 week after vaccination. Matched sequences included those bound to the Omicron strain and Omicron XBB sublineage. The antibody sequences that can bind to the Omicron strain and XBB sublineage revealed that the monovalent XBB.1.5 vaccines showed a stronger response than previous vaccines or SARS‐CoV‐2 infection before the emergence of XBB sublineage. The QASAS method was able to demonstrate the immunogenic effect of monovalent XBB.1.5 vaccines for the 2023–2024 COVID‐19 vaccination campaign.

A monovalent Omicron XBB.1.5 mRNA RBD analogue vaccine, MAFB-7256a (DS-5670d), was newly developed and approved in Japan in the Spring of 2024 for the prevention of COVID-19. However, clinical efficacy data for this vaccine are currently lacking. We previously established the Quantification of Antigen-specific Antibody Sequence (QASAS) method to assess the response to SARS-CoV-2 vaccination at the mRNA level using B-cell receptor (BCR) repertoire assays and the Coronavirus Antibody Database (CoV-AbDab). Here, we used this method to evaluate the immunogenicity of MAFB-7256a. We analyzed repeated blood samples using the QASAS method from three healthy volunteers before and after MAFB-7256a vaccination. BCR response increased rapidly one week post-vaccination and then decreased, as with conventional vaccine. Notably, the matched sequences after MAFB-7256a vaccination specifically bound to the receptor-binding domain (RBD), with no sequences binding to other epitopes. These results validate that MAFB-7256a is an effective vaccine that exclusively induces antibodies specific for the RBD, demonstrating its targeted immunogenic effect.

To support large-scale efficacy trials, especially where efficacy trials are not feasible, the ability to compare immune response data across candidate Shigella vaccines can be very valuable for identifying the most promising vaccine platform and immunobridging to other populations, vaccine formulations, or additional platforms in the future. However, international standards for antibody assays are not yet available for Shigella vaccines currently in clinical development. Lack of standardization of Shigella immunoassays means that the results of antibody measurement in clinical samples from different vaccine trials or those from seroepidemiology studies cannot be easily compared. The results from this study will facilitate the comparison of immunological titers obtained across different Shigella vaccine studies as an interim measure until such time that immunoassays can be better harmonized through the use of an International Standard Serum.