Search Results (1,898)

Extensive research on homologous-recombination-deficient (HRD) tumors has led to advancements in targeted therapies, such as PARP inhibitors (PARPis). Around 50% of high-grade serous ovarian cancer (HGSOC) cases exhibit HR deficiency, but understanding the remaining half, referred to as homologous-recombination-proficient (HRP) tumors, is limited. This review explores existing knowledge regarding HGSOC patients with HRP tumors and offers insights into potential targets for innovative treatments. Patients with HRP tumors do not experience the same benefits from PARPi and have poorer survival outcomes compared to those with HRD tumors. CCNE1 amplification is a common, well-established molecular feature in HGSOC HRP tumors, occurring in about 20% of cases. Targeting CCNE1 amplification and/or overexpression shows promise with emerging therapies like CDK2 or Wee1 inhibitors. Additionally, approaches using immunotherapy and antibody–drug conjugates could represent promising targets for HRP patients. This review also covers lesser-known molecular features in HRP tumors, such as fold-back inversions and CARM1 amplification and/or overexpression, as well as HRD tumors that acquire HR proficiency (BRCA1/2 reversion mutations, demethylation of BRCA1 and RAD51C). We also discuss controversial topics regarding HRP tumors and limitations of HRD detection. Addressing this need is critical to reduce toxicity and improve disease management. Full article

Influenza virus is a leading cause of global morbidity and mortality due to acute lower respiratory infection, even with the widespread use of multiple licensed influenza vaccines. However, antigenic drift during influenza replication can cause vaccine-induced antibodies to poorly neutralize influenza virus, thereby reducing vaccine effectiveness. To help overcome this problem, we leveraged a hydrogel platform with influenza hemagglutinin (HA) protein to induce prolonged antigen exposure. The hydrogel platform, Vaccine Self-Assembling Immune Matrix (VacSIM^®), in combination with recombinant influenza H1 or H3 HA protein antigens, increased antigen-specific antibody titers in vaccinated mice, which led to decreased disease severity after H1N1 infection for H1 HA-vaccinated mice and decreased lung viral titers after H3N2 challenge for H3 HA-vaccinated mice. Sera collected from mice immunized with VacSIM and HA also showed broader HAI activity, increasing by 1–3 log against a panel of influenza viruses. These results were consistent with the use of cocktail immunization, containing both an H1 and H3 HA, where mice immunized with VacSIM had an increase in antigen-specific antibody titers and decreased disease severity and lung viral titers against H1N1 and H3N2 influenza challenges, respectively. Finally, it was determined that a single immunization with VacSIM and H1 HA could provide protection against lethal H1N1 challenge compared to a group without VacSIM. In summary, we demonstrate that use of the slow-release platform VacSIM can improve the host immune response to vaccination and increase protection against influenza infection. Full article

Background: Type 1 diabetes (T1D) is an autoimmune disorder characterized by destruction of insulin-producing β-cells. While conventional insulin therapy manages hyperglycemia, it fails to halt autoimmunity. Oral immunotherapy targeting autoantigens like GAD65 offers potential for antigen-specific tolerance; however, its efficacy is limited by gastrointestinal degradation and poor mucosal uptake. Lactococcus lactis (L. lactis), a food-grade delivery vector, enables sustained antigen release and intestinal tract immune modulation, yet the differential transcriptomic mechanisms underlying mucosal versus systemic immune responses remain uncharacterized. Methods: Non-obese diabetic (NOD) mice were randomized into control and GAD65 groups, receiving oral PBS or the GAD65 recombinant L. lactis vaccine, respectively. Fasting blood glucose was monitored weekly. GAD65-specific IgA and IgG, along with immune tolerance-related factors, were quantified using ELISA. Lymphocyte subsets were analyzed by flow cytometry, alongside RNA sequencing and transcriptional profiling. Results: The study demonstrated that the orally administered GAD65-L. lactis vaccine could significantly induce GAD65-specific IgA antibody and TGF-β cytokine and alleviate hyperglycemia and diabetes symptoms in NOD mice. Our study facilitated the induction of GAD65-specific regulatory T cells within both intestinal lamina propria lymphocytes (LPLs) and splenic lymphocytes. Notably, antigen-specific tolerance was mainly observed in intestinal LPLs. Crucially, the immune responses elicited by the vaccine demonstrated significant disparities between intestinal LPLs and splenic lymphocytes, with intestinal LPLs exhibiting unique local immune tolerance transcriptomic profiles. Conclusions: Our findings have enhanced the comprehension of the mechanisms by which oral vaccines influence the interplay between mucosal and systemic immune responses, thereby establishing a foundational framework for the design of oral vaccines. This understanding is instrumental in advancing antigen-specific immune tolerance strategies for autoimmune diseases such as Type 1 Diabetes (T1D). Full article

Background/Objectives: Avian influenza represents a major threat to both animal and public health. Our group has tracked avian influenza viruses circulating in wild birds in Peru during the last 20 years. While most of these viruses are low-pathogenic avian influenza strains, some exhibit genetic changes that significantly diverge from common circulating viruses. We selected a highly divergent hemagglutinin H2 gene from a genetically characterized avian influenza virus to develop a recombinant protein using a baculovirus system. Methods: We administered 5 µg and 20 µg doses of the recombinant H2 protein (rH2) into 3-week-old chickens using an abdominal cavity inoculation model to evaluate the activation of innate immune responses. Chickens were euthanized at 24 and 72 h post inoculation and an abdominal lavage was performed to harvest the abdominal cavity content. Results: Infiltrating cells were counted and their cell viability was measured using an Annexin V/PI staining. At 24 h, a large proportion of infiltrating leukocytes were identified as heterophils, monocyte/macrophages and lymphocytes. These proportions changed at 72 h, with a decrease in heterophils and increase in monocyte and lymphocyte pools. We observed strong cellular activity in abdominal leukocytes at 24 h, with a decline in activation levels at 72 h. Cytokine expression suggested a tightly regulated immune response during the 72 h period, while a more sustained response was observed at the 20 µg dose. Antibody levels demonstrated the capacity of the rH2 protein to induce long-term responses. Conclusions: These results revealed that the baculovirus-expressed rH2 protein induces a controlled immune activation, a long-term immune response, holding promise as a potential vaccine candidate for animal health. Full article

MICA/B molecules (MICs) are stress-induced molecules expressed by infected and tumor cells. Their expression also characterizes trophoblast cells. Cytotoxic lymphocytes, including natural killer (NK) cells, express the NKG2D receptor, aiding them in the recognition and destruction of target cells that present MICs. To evade destruction, target cells employ various defense mechanisms, including the secretion of soluble forms of MICs. Choriocarcinoma JEG-3 cells and NK-92 cells were used to assess the expression of MICs and NKG2D. The cytotoxicity of NK-92 cells against JEG-3 cells in the presence of trichostatin A (TSA), anti-MICA/B antibodies (anti-MICA/B), and recombinant MIC proteins (rMICA/B) was evaluated. JEG-3 cells and NK-92 cells express MICs. Additionally, NK-92 cells exhibit high levels of NKG2D receptor expression. TSA treatment reduced the surface expression of MICs on choriocarcinoma cells, and was also associated with the release of soluble MICB. However, the TSA-induced decrease in MIC expression by choriocarcinoma cells did not protect them from the cytotoxic effects of NK cells. Only the activation of NK cells by IL-12 resulted in a decline in susceptibility of TSA-treated choriocarcinoma cells to NK cell-mediated cytotoxicity. Thus, NK cells activated by IL-12 lose their ability to effectively kill TSA-treated choriocarcinoma cells through the MIC-mediated mechanisms. Full article

Background: The vast majority of GBMs recur within 2 years following standard treatment, including radiotherapy. Seizures and epilepsy are common in GBM patients, suggesting tumor-cell-induced neuron toxicity. Additionally, the tumor cells and neurons interact during tumor development; however, the effects of tumor cells on the neurons remain unclear. Methods: Orthotopic xenografts initiated from GSCs expressing GFP implanted into the right striatum of nude mice were irradiated (10 Gy) 35 days after implantation, followed by immunohistochemistry (IHC) to investigate the tumor cell–neuron interactions. Moreover, we established a direct coculture of human GSCs and neurons differentiated from human iPSC-derived neural progenitor cells (NPCs) to investigate the impact of the tumor cells on the neurons. Neuronal cell counts were monitored to assess neurotoxicity. Culture CM were analyzed through cytokine profiling. Results: In untreated mice, tumors invaded across the right hemisphere (RH), with increased cell contact with the mouse neurons. In irradiated mice, the tumor regrowth was less invasive and had fewer neurons. In vitro, the GSCs induced neuronal death in the direct coculture. Similarly, the CM from the direct cocultures caused significant neuronal death. The cytokine analysis revealed that the cocultures uniquely secreted IL-8 into the CM. Furthermore, treatment with recombinant (r) human IL-8 caused significant neuron death, while IL-8 blocking antibodies prevented this neurotoxicity in the coculture. Conclusions: This study demonstrates that GBM tumors regrown after radiation lack neurons, and direct interaction between GSCs and the neurons is necessary for GSC-mediated neurotoxicity, likely involving IL-8 in neuronal death. Full article

Background/Objectives: Single-domain immunoglobulins are small protein modules with specific affinities. Among them, the variable domains of heavy chains of heavy-chain-only antibodies (VHH) as the antigen-binding fragment of heavy-chain-only antibodies (also termed nanobodies) have been widely investigated for their applicability, e.g., therapeutics and immunodiagnostics. However, despite their advantageous biochemical and biophysical characteristics, protein aggregation throughout recombinant synthesis is a serious drawback in the development of nanobodies with application perspectives. Therefore, we aimed to develop a computational method to predict the aggregation propensity of VHH antibodies for the selection of promising candidates in early discovery. Methods: We employed a deep learning-based structure prediction for VHHs and derived from it likely biophysical and biochemical properties of the framework region 2 with relevance for aggregation. A total of 106 nanobody variants were produced by recombinant expression and characterized for their aggregation behavior using size exclusion chromatography (SEC). Results: Quantitative characteristics of framework region 2 patches were combined into a function that defines an aggregation score (AS) predicting the aggregation propensities of VHH variants. AS was evaluated for its capability to forecast recombinant VHH aggregation by experimentally studying VHH Fc-fusion proteins for their aggregation. We observed a clear correlation between the calculated aggregation score and the actual aggregation propensities of biochemically characterized VHHs Fc-fusion proteins. Moreover, we implemented an easily accessible pipeline of software modules to design nanobodies with desired solubility properties. Conclusions: AI-based prediction of VHH structures, followed by analysis of framework region 2 properties, can be used to predict the aggregation propensities of VHHs, providing a convenient and efficient tool for selecting stable recombinant nanobodies. Full article

Schmallenberg virus (SBV) is a negative-sense RNA virus transmitted by insect vectors, causing arthrogryposis-hydranencephaly syndrome in newborn ruminants. Since its discovery in Germany and the Netherlands in 2011, SBV has rapidly spread across multiple European countries, resulting in significant economic losses in the livestock industry. With the increasing global animal trade and the expanded range of insect transmission, the risk of SBV introduction into non-endemic regions is also rising. As the gold standard for serological testing, the virus neutralization test (VNT) is crucial for tracking the spread of SBV and evaluating the efficacy of vaccines. However, in non-endemic regions, the lack of local viral strains and the biosafety risks associated with introducing foreign strains pose challenges to the implementation of VNT. In this study, we employed reverse genetics techniques using vesicular stomatitis virus (VSV) to substitute the VSV G protein with the envelope glycoproteins of SBV, thereby successfully generating and rescuing the recombinant virus rVSVΔG-eGFP-SBVGPC. The recombinant virus was then thoroughly characterized in terms of SBV Gc protein expression, viral morphology, and growth kinetics. Importantly, rVSVΔG-eGFP-SBVGPC exhibited SBV-specific cell tropism and was capable of reacting with SBV-positive serum, enabling the measurement of neutralizing antibody titers. The results suggest that this recombinant virus can serve as a feasible alternative for SBV neutralization tests, with promising potential for application in serological screening and vaccine evaluation. Full article

The presently used vaccines do not offer solid immunity/protection against the currently circulating strains of the H5N1 viruses. We aim to design a pan-H5N1 vaccine that protects birds against the presently circulating clade 2.3.4.4b in chickens. We used AI tools, including epitope mapping, molecular docking, and immune simulation, to design a multiepitope DNA vaccine including the top-ranked B and T cell epitopes within four major proteins (HA, NA, NP, and M2) of H5N1 clade 2.3.4.4b. We selected the top-ranked 12 epitopes and linked them together using linkers. The designed vaccine is linked to IL-18 as an adjuvant. The molecular docking results showed a high binding affinity of those predicted epitopes from the MHC I and MHC II classes of molecules with chicken alleles. The immune simulation results showed that the designed vaccine has the potential to stimulate the host immune response, including antibody and cell-mediated immunity in chickens and other birds. We believe this vaccine is going to be a universal vaccine that offers good protection against HPAI-H5N1 clade 2.3.4.4b. We are reporting the successful molecular cloning of a recombinant multiepitope-based vaccine spanning some key epitopes within some key proteins of the currently circulating H5N1 clade 2.3.4.4b. These designed vaccines could be a great positive impact on the protection of birds and various species of animals, as well as humans, against the HP-H5N1 influenza virus. Further studies are required to validate this vaccine candidate in chickens. Full article

Background: Monoclonal antibodies play an important role in therapeutic and analytical applications. For recombinant expression, the coding sequences of the variable regions of the heavy and light chains are required. In addition, cloning antibody sequences, including constant regions, reduces the impact of hybridoma cell loss and ensures preservation of the naturally occurring full antibody sequence. Method: We combined amplification of IgG antibody variable regions from hybridoma mRNA with an advanced method for full-length cloning of monoclonal antibodies in a simple two-step workflow. Following Sanger sequencing and evaluation of consensus sequences, the best matching variable, diversity, and joining (V-(D-)J) gene segments were identified according to identity scores from IgBLAST reference sequences. Simultaneously, the mouse IgG subclass was determined at the DNA level based on isotype-specific sequence patterns in the C_H1 domain. Knowing the DNA sequence of V-(D-)J recombination responsible for the complementary determining region 3 (CDR 3), variable region-specific primers were designed and used to amplify the corresponding antibody constant regions. Results: To verify the approach, we applied it to the hybridoma clone BAM-CCMV-29-81 and obtained identical full-length antibody sequences as with RNA Illumina sequencing. Further validation at the protein level using an established MALDI-TOF MS-fingerprinting protocol showed that five out of six genetically encoded CDR domains of the monoclonal antibody BAM-CCMV-29-81 could be efficiently correlated. Conclusion: This simple, streamlined method enables the cost-effective determination of the full-length sequence of monoclonal antibodies from hybridoma cell lines, with the added benefit of obtaining the DNA sequence of the antibody ready for recombinant expression. Full article

Fruit bats in the genus Pteropus are the natural reservoirs for zoonotic paramyxoviruses, notably henipaviruses and pararubulaviruses, which are found across Southeast Asia and Oceania. The genetic and antigenic diversity of viruses in both genera, and region specificity, are ill-defined, limiting health security measures aimed at minimizing spillover. For example, Nipah virus has been isolated from bats in the Battambang province of western Cambodia, and surveys suggest bat foraging behaviors occur in close proximity to human settlements. However, there have been no historical cases of Nipah virus in Cambodia. Here, we use a multiplex microsphere immunoassay to identify cryptic human exposure to selected henipaviruses and pararubulaviruses in Cambodia. Convalescent human sera from persons presenting with acute respiratory illness were screened to detect the presence or absence of antibodies reactive with attachment glycoprotein antigens from Nipah virus, Hendra virus, Cedar virus, and Ghana virus, and a hemagglutinin-neuraminidase antigen from Menangle virus. In this sero-survey, we detected antibodies that were specifically reactive with Cedar virus and Menangle virus, including one serum sample that neutralized a recombinant Cedar virus. Additionally, we detected a pattern of cross-reactivity with Hendra virus, Cedar virus, and Ghana virus, suggesting previous infection by an antigenically-related henipavirus. We did not detect high antibody reactivity with the NiV glycoprotein. Future studies should expand serological surveillance for these transboundary pathogens, including genetic surveillance to aid in henipavirus discovery, and focused biosurveillance where interfaces with livestock and humans occur. Full article

Background: Respiratory syncytial virus (RSV) poses a significant global health threat, particularly to children and the elderly. While progress has been made in RSV vaccine development, gaps remain, especially in protecting the elderly population. BARS13, a recombinant non-glycosylated G protein-based RSV vaccine, has shown promise in preclinical and Phase 1 studies. This phase II trial sought to determine whether escalating doses of BARS13 could enhance immune responses while maintaining safety and tolerability in healthy older adults aged 60–80 years. Methods: This study employed a rigorous randomized, double-blind, placebo-controlled design involving 125 participants across two Australian centers. Participants were randomized in a 3:1 (vaccine–placebo) ratio for Cohorts 1–2 (30 active, 10 placebo each) and a 2:1 ratio for Cohort 3 (30 active, 15 placebo). Cohort 1 (low dose) received 10 µg rRSV-G + 10 µg CsA in one arm + a placebo in the other (Days 1 and 29); Cohort 2 (high dose) received 10 µg rRSV-G + 10 µg CsA in both arms (20 µg total per dose, Days 1 and 29); Cohort 3 (multi-dose) received the same dose as that of Cohort 2 but with a third dose on Day 57. The placebo groups received IM injections in both arms at matching timepoints. The primary endpoints included safety and tolerability assessments, while the secondary endpoints evaluated the RSV G protein-specific IgG antibody concentrations using enzyme-linked immunosorbent assays (ELISAs). Statistical analysis was performed on both the safety and immunogenicity populations. Results: BARS13 was well-tolerated across all cohorts, with no serious adverse events (SAEs) related to the vaccine. The most common adverse events were mild local reactions (pain and tenderness) and systemic reactions (headache and fatigue), which resolved within 24–48 h. Immunogenicity analysis demonstrated a dose-dependent increase in the RSV G protein-specific IgG geometric mean concentrations (GMCs). Cohort 3, which received multiple high-repeat dose administrations, showed the highest immune response, with the IgG GMC rising from 1195.4 IU/mL on Day 1 to 1681.5 IU/mL on Day 113. Response rates were also the highest in Cohort 3, with 86.2% of participants showing an increase in antibody levels by Day 29. Conclusions: BARS13 demonstrated a favorable safety profile and strong immunogenicity in elderly participants, with a clear dose-dependent antibody response. These results support further development of BARS13 as a potential RSV vaccine candidate for the elderly. Further studies are needed to evaluate the long-term efficacy and optimal dosing schedule. Full article

Urine-based immunoassay is a non-invasive method with demonstrated utility in detecting anti-SARS-CoV-2 antibodies in unvaccinated patients with COVID-19. To evaluate urine’s potential for serological surveys in a real-world setting, SARS-CoV-2 serology was performed on urine samples from vaccinated individuals, both with and without prior confirmed COVID-19. (1) Methods: An in-house indirect ELISA was used to measure antibodies against recombinant spike (S) and nucleocapsid (N) proteins of SARS-CoV-2 in urine and paired serum from 149 individuals vaccinated with Janssen AD26.COV2.S, an S protein-based COVID-19 vaccine. (2) Results: Anti-S and anti-N levels were higher in the urine and serum of participants with confirmed prior COVID-19 compared to those without prior infection. Urinary anti-S effectively distinguished vaccinated individuals with (AUC = 0.96) and without (AUC = 0.88) prior infection from negative controls (non-vaccinated, non-previously infected individuals) (p < 0.0001). Among vaccinated participants, urinary anti-S and anti-N identified prior infection, with AUC values of 0.73 (p < 0.0001) and 0.60 (p = 0.03), respectively, being recorded. (3) Conclusions: Findings indicate that urinary anti-SARS-CoV-2 antibodies reflect AD26.COV2.S vaccination and previous COVID-19. To further advance the methodology, studies with larger sample sizes and a greater diversity of COVID-19 vaccines are required. Full article

Background/Objectives: Effective prophylaxis for Mycobacterium tuberculosis (Mtb) requires greater understanding of immune correlates of protection. With renewed interest in BCG as an Mtb vaccine, particularly via the intravenous (IV) route, our objective was to characterize both innate and adaptive immune correlates of vaccine-induced pulmonary immunity as potential biomarkers for protective efficacy in a murine model of Mtb infection. Methods: Mice were given BCG via different routes and some boosted with recombinant virus constructs encoding Mtb Ag85B. Responding innate lymphoid cell (ILC) populations, T cells and B cells were analyzed by fluorescence activated cell sorting (FACS) for surface markers and by intracellular cytokine staining or antibody ELISPOT. Some immunized mice were challenged with aerosolized Mtb and monitored for bacterial growth in the lungs and spleen. Results: BCG given IV, but not intranasally or subcutaneously, resulted in marked increases in IFNγ expression at 72 h by pulmonary CD49⁺ NK cells, CD69⁺ ILC1, and two ILC3 populations, NCR-ILC3 and LTi cells, the latter also producing IL-22. Pulmonary ILC2 populations in these mice had significantly increased IL-13 expression at 24 h compared to the other routes. Interestingly, high levels of NK cells and ILC1 expressing IFNγ and/or TNFα were sustained at 8 wk, with sustained expression of IL-17A by pulmonary NCR-ILC3 and pronounced tissue-resident and effector memory CD4⁺ and CD8⁺ T cell responses. Intranasal boosting with Ad-Ag85B enhanced these T cell responses and generated Mtb-specific pulmonary IgA and IgG B cells, correlating with significantly reduced bacterial loads following Mtb challenge. Conclusions: BCG given IV primed for both early and persistent pulmonary ILC1/ILC3 responses of a predominantly Th1/Th17-type profile along with local Mtb-specific memory T cell and B cell populations, correlating with enhanced protective efficacy. These are worthy of further study as compartmentalized biomarkers for effective vaccine-induced local immunity against Mtb. Full article

Background: T-cell receptor excision circles (TRECs) and kappa-deleting recombination excision circles (KRECs) are markers of recent thymic and bone marrow output, respectively. As they have previously been associated with immunosenescence, we aimed to investigate their association with anti-spike SARS-CoV-2 (S1RBD) IgG antibody response after COVID-19 vaccination in nursing home residents (NHRs) and staff (NHS). Methods: We measured TREC and KREC levels and S1RBD IgG antibody levels from dried blood spots (DBSs) using in-house qPCRs and a commercial ELISA kit, respectively, in 200 participants (50 NHRs and 150 NHS). DBSs were collected in April 2021, approximately two months after primary course COVID-19 vaccination (BNT162b2). We assessed the association between TREC and KREC as dependent variables and age, sex, infection-priming status, and post-vaccination S1RBD-specific IgG concentrations as independent variables by simple and multiple linear regression. Results: TREC and KREC levels were significantly lower in NHRs compared with NHS and were negatively correlated with age (p < 0.001). Neither TREC nor KREC levels were significantly associated with SARS-CoV-2 antibody concentrations (p > 0.05). Conclusions: In our study population, TREC and KREC levels decreased with age and were statistically significantly lower in NHRs than NHS. They were, however, not associated with the antibody response after COVID-19 vaccination. Yet, additional research is warranted to explore their potential relevance in cellular immune responses or in combination with other biomarkers of immune function. Full article