Neutralization Assay Research Articles

The impact of beauvericin on rainbow trout intestinal epithelial cells at different temperatures and dosing methods.

Mycotoxins in aquatic feeds and their effects on fish are becoming more important in aquaculture, as fishmeal and fish oil in feeds are being replaced with more sustainable plant protein. Here, we investigated the potential of the mycotoxin, beauvericin (BEA), to impact the rainbow trout (RT) intestine by using cultures of the epithelial cell line, RTgutGC. BEA was dosed in different ways and exposed at temperatures ranging from 4 to 26°C before being evaluated for cell viability by the metabolic reduction of Alamar Blue, by the accumulation of Neutral Red (lysosomal activity), cytotoxicity (CellTox Green), and for wound healing. BEA induces cell death in RTgutGC cells. The lysosomes are the main target (Neutral Red assay is the most sensitive) while cytotoxicity and plasma membrane rupture (CellTox Green) occur at considerably higher concentrations. BEA caused a dose-dependent decline in Neutral Red reading at all tested temperatures but Alamar Blue readings did not decline at 4°C. Under these conditions, BEA appears to impair only lysosomal activity. Wound healing was reduced at 4, 10, and 26°C compared to 18°C. Also BEA treatment, at non-cytotoxic concentrations, reduced wound healing, but the temperature had little influence on this. Different carrier vehicles (methanol, DMSO) and exposure methods (passive or active dispersal) for BEA exposure were also studied. Here, methanol and passive dispersal gave comparable results to exposure with DMSO and active dispersal. In contrast, when DMSO was dosed with passive dispersal, immediate cytotoxicity in combination with BEA was induced.

Atezolizumab neutralizing assay development: a novel way of evaluating assay cutpoint after sample pretreatment

Atezolizumab, a biotherapeutic monoclonal antibody directed against PD-L1, has been shown to be efficacious in multiple oncology indications. In clinical trials, a subset of patients developed anti-atezolizumab antibodies, necessitating the development of a neutralizing antibody (NAb) assay. A bead-based sample pretreatment method was developed to reduce high levels of therapeutic in patient serum samples. Untreated sample variability is reduced by this sample pretreatment, based on this, a novel approach was taken for determining an appropriate assay decision threshold (cutpoint). The therapeutic was added to the samples to mimic typical patient samples. The resulting assay was successfully validated and applied to sample analysis from multiple clinical trials.

Development and Efficacy Evaluation of a Novel Nanoparticle-Based Hemagglutination Inhibition Assay for Serological Studies of Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea virus (PEDV) is a major pathogen that causes serious economic losses to the swine industry. To aid PEDV clinical diagnosis and vaccine development, sensitive and precise serological methods are demanded for rapid detection of (neutralizing) antibodies. Aiming for the development of a novel virus-free hemagglutination inhibition (HI) assay, the N-terminal region of the PEDV S1 subunit, encompassing the sialic acid-binding motif, was first expressed as an Fc-fusion protein with a C-terminal Spy Tag (S10A-Spy). The S10A-Spy protein was then presented on SpyCatcher-mi3 nanoparticles, forming virus-like particles designated S10A-NPs. Electron microscopy and dynamic light scattering analysis confirmed its topology, and the hemagglutination assay showed that S10A-NPs can efficiently agglutinate red blood cells. The HI assay based on S10A-NPs was then validated with PEDV-positive and -negative samples. The results showed that the HI assay had high specificity for the detection of PEDV antibodies. Next, a total of 253 clinical serum samples were subjected to the HI testing along with virus neutralization (VN) assay. The area under the receiver operating characteristic curve with VN was 0.959, and the kappa value was 0.759. Statistical analysis of the results indicated that the HI titers of the samples tested exhibited high consistency with the VN titers. Taken together, a novel virus-free HI assay based on the multivalent display of a chimeric PEDV spike protein upon self-assembling nanoparticles was established, providing a new approach for PEDV serological diagnosis.

Development and Immunogenicity Study of Subunit Vaccines Based on Spike Proteins of Porcine Epidemic Diarrhea Virus and Porcine Transmissible Gastroenteritis Virus

Porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) are responsible for significant economic losses in the swine industry. The S1 proteins of these viruses serve as key targets for vaccine development. In this study, prokaryotic expression vectors for pCZN1-PEDV S1, pCZN1-TGEV S1, and pCZN1-PEDV S1-TGEV S1 were constructed. The corresponding proteins were expressed, purified, and used to prepare monovalent, bivalent, and mixed (PEDV S1 + TGEV S1) vaccines. Kunming (KM) mice were immunized with subunit vaccines, with PBS as the negative control (NC) and a commercial inactivated vaccine as the positive control (PC). Immune responses, including specific antibody (IgG, IgG1, IgG2a) levels, virus neutralization, and IFN-γ production, were evaluated. All vaccines induced high levels of specific IgG, IgG1, and IgG2a antibodies. At weeks 2 and 8, the PEDV S1 + TGEV S1 vaccine induced significantly higher levels of specific IgG and IgG1 compared to the PC (p < 0.001). The PEDV S1 vaccine also induced significantly higher specific IgG2a levels than the PC at week 4 (p < 0.0001). Virus neutralization assays demonstrated that the subunit vaccines induced neutralizing antibody levels comparable to or exceeding those of the PC. Furthermore, IFN-γ levels were significantly elevated in all vaccinated groups compared to the NC (p < 0.0001), indicating a robust immune response. These results suggest that the subunit vaccines are promising candidates for the safe and effective control of both PEDV and TGEV infections.

87. Machine Learning-Guided Generation of a Potent Combination of Broadly Neutralizing Sarbecovirus Antibodies with a High Barrier to Resistance

Abstract Background The elderly and immunocompromised remain at high risk of severe COVID-19 due to suboptimal immune responses. Monoclonal antibodies to SARS-CoV-2 spike were highly effective for both prophylaxis and treatment, but quickly became ineffective due to viral escape. Using a machine learning-guided optimization approach, we identified two antibodies against highly conserved epitopes, one targeting the spike S2 stem helix (GB-0669) and the other against the class 4 RBD site on S1 (PRO-37587) with the goal of developing a combination for prophylaxis that would be less susceptible to viral escape. Methods GB-0669 and PRO-37587 were characterized for breadth through binding and neutralization assays against a panel of SARS-CoV-2 variants and related sarbecoviruses. We also assessed the barrier to resistance, both individually and in combination using SARS-CoV-2 Omicron BA.1 in live virus escape studies in Vero E6 cells. Passaged viruses were sequenced and assessed for viral replication. The prevalence of any observed mutations was analyzed for conservation in the SARS-CoV-2 lineages and across the sarbecovirus family. Results Both antibodies exhibited potent neutralization across SARS-CoV-2 variants including those that had not yet emerged during development of our antibodies, while displaying activity against non-SARS-CoV-2 viruses. During passage with BA.1, escape from GB-0669 was observed in passage 3 (H1156Y), while escape from PRO-37587 emerged in passage 10 through two mutations (P381S, D402G) that impaired viral replication. By the end of passage 10, neither complete nor partial escape were observed for the combination of GB-0669 and PRO-37587. These escape mutations were observed in fewer than 1% of circulating variants from the beginning of the pandemic, probably due to low selective pressure and/or their impact on viral replication. With the exception of P381S, these mutations were not observed across a range of sarbecoviruses. Conclusion We demonstrate that the combination of GB-0669 and PRO-37587 has potent and broad neutralization activity. The robust viral escape profile and conservation of these epitopes suggest that this combination is more likely to remain effective in the face of ongoing viral evolution. Disclosures Alex Ramos, PhD, Generate Biomedicines: Stocks/Bonds (Private Company) Alan Leung, PhD, Generate Biomedicines: Stocks/Bonds (Private Company) Brett T. Hannigan, PhD, Generate Biomedicines: Employee|Generate Biomedicines: Stocks/Bonds (Private Company) Adam Root, MS, MBA, Generate Biomedicines: Grant/Research Support|Generate Biomedicines: Stocks/Bonds (Private Company) Heather A. Van Epps, PhD, Generate Biomedicines: Stocks/Bonds (Private Company) Kristen Hopson, Ph.D., Generate Biomedicines: Stocks/Bonds (Private Company) Gevorg Grigoryan, PhD, Generate Biomedicines, Inc.: Employee and shareholder|Generate Biomedicines, Inc.: Stocks/Bonds (Private Company) Gavin CKW Koh, PhD, AstraZeneca: Stocks/Bonds (Public Company)|Generate:Biomedicines: Stocks/Bonds (Private Company)|GlaxoSmithKline: Stocks/Bonds (Public Company) Daria Hazuda, PhD, Generate Biomedicines: Stocks/Bonds (Private Company)

P-2066. Impact of XXB 1.5 Monovalent Booster Vaccination on Mucosal and Systemic Immune Responses in Healthy Adults

Abstract Background The FDA recently approved the first monovalent XBB1.5 booster vaccine. However, the mechanism by which this vaccine stimulates mucosal and systemic immune responses has been understudied.Figure 1.Impact of XBB 1.5 vaccination on viral neutralization titers using pseudo virus assay (left panel) and ACE2 inhibition assay (right panel).Each dot (white) represents a single serum sample tested in parallel with a post-vaccination sample (colored dot) for each of the 28 participants. The mean fold increase in neutralization titers pre- and post-vaccination are shown for Wuhan, XBB 1.5, and BA5 variants. All increases in neutralization titers are significant to P<0.0001 by employing Wilcoxon matched-pairs signed rank test. Methods We examined 28 participants' serum and saliva one week before and two to three weeks after the XBB1.5 mRNA vaccination for neutralizing and binding antibodies to Wuhan and XBB1.5 S protein.Figure 2:Inhibition of ACE2 binding to different S variants by saliva samples pre- and post-vaccination with XBB1.5 monovalent booster.Paired samples represent the percent ACE2 binding inhibition in participants before and after vaccination. Vaccination induced a 1.7-fold increase in ACE2 neutralization in saliva to XBB1.5 (p<0.0001) and 1.2-fold increase in ACE2 neutralization to Wuhan (p=0.03). Significance tested between pre and post-vaccination by Wilcoxon matched-paired sign rank test. *** p<0.001, **** p<0.0001. Results We observed a 2.9 to 2.6-fold and 5.0 to 7.8-fold increase in Wuhan and XBB1.5 serum neutralization titers following mRNA vaccination using pseudovirus and ACE2 neutralization assays respectively, that closely correlated with a rise in binding antibodies to S protein. Vaccination induced a 1.7-fold increase in ACE2 neutralization in saliva to XBB1.5 (p< 0.0001) and 1.2-fold increase in ACE2 neutralization to Wuhan (p=0.03). This increase in neutralization Ab levels in saliva failed to correlate with the number and time of prior COVID-19 infections, vaccination status, type of vaccine, or concomitant rise in S-specific IgA or IgG in saliva or serum. Depletion of IgA or IgG in saliva following vaccination demonstrated that IgA was primarily responsible for the increase in ACE2 blocking activity (mean reduction in blocking activity with IgA depletion=68%, range, 58-81%) compared to IgG (mean reduction=27%, range 0-71%).Figure 3.Role of IgA on percent reduction in ACE2 inhibition in saliva. Depletion of IgA or IgG in saliva following vaccination demonstrated that IgA was primarily responsible for the increase in ACE2 blocking activity (mean reduction in blocking activity with IgA depletion=68%, range, 58-81%) compared to IgG (mean reduction=27%, range 0-71%). Statistical analysis done using unpaired t test. Conclusion The XBB1.5 monovalent vaccine boosts systemic and mucosal viral neutralization antibody levels, primarily to the XBB1.5 variant. Most people have hybrid immunity to SARS-CoV2 now, so mRNA vaccines boost mucosal immune response better than observed earlier in the pandemic. Increased oral mucosal immunity may reduce the risk of COVID-19 progressing to pneumonia and more severe disease. Disclosures All Authors: No reported disclosures

P-2023. A ‘Variant-Proof’ Cocktail Of Two Heavy Chain-Only Antibodies Targets 3 Epitopes In The Spike Protein S1 And S2 Subunits And Broadly Neutralizes SARS-Cov-2 With Exceptional Potency

Abstract Background More than 7 million people have died from COVID-19 to date and SARS-CoV-2 continues to cause substantial disease around the world. High-risk patients like immunocompromised and elderly are often not able to elicit adequate immune responses to vaccination and strongly benefit from an additional layer of protection in the form of complementary antibody therapy. Monoclonal antibodies act immediately and can provide immune support for months. However, all previously authorized antibodies lost their neutralization potency against the currently circulating and constantly mutating SARS-CoV-2 variants, leaving a vast unmet medical need. The combination XVR012 consists of the molecules XVR013m and XVR014. Methods The combination XVR012 consists of the molecules XVR013m and XVR014 (Figure 1). XVR013m is a mono-specific VHH-Fc antibody that targets a unique epitope in the S2 subunit, XVR014 is a bi-specific bivalent VHHx-Fc-VHHy antibody construct, targeting 2 non-competing epitopes in the Receptor-Binding Domain of the spike (Figure 2). Both antibodies contain a human IgG1 Fc with LS mutations for half-life extension. Structural analysis of the XVR013m epitope Left panel: Structural analysis of the VHH R3DC23 (green) bound to the HR2 domain of the S2 unit of the SARS-CoV-2 spike protein, between the terminal N1194 glycan (orange) and the viral membrane. Right panel: One R3DC23 (green) binds the interface of two HR2 coils and each HR2 coil is bound by two R3DC23 VHHs. Results XVR012 delivers a triple mode of action in preventing infection of host cells, i.e., (1) sterical hinderance of ACE2 receptor binding, (2) induction of S1 subunit shedding thereby preventing viral attachment and (3) inhibition of the fusion process between the viral and host cell membrane. XVR012 broadly neutralizes SARS-CoV-1 and SARS-CoV-2 viruses in vitro and demonstrates an IC50 ranging from 4.8 to 8,7 ng/mL against all SARS-CoV-2 variants tested so far in a pseudovirus neutralization assay, including the currently circulating BA.2.86.1, HK.3, EG.5.1 and HV.1 (Table 1). In vivo, efficacy was demonstrated in the Syrian Golden Hamster model in a therapeutic setting, showing complete reduction of lung viremia for all XVR012 dose levels tested, even with a very low dose of 0,5 mg/kg for the XVR013m component. (Figure 3). PK parameters have been determined in a Tg32 SCID mouse model and support an envisioned duration of protection up to 6 months. Neutralization potency of VSV pseudo-typed with spike proteins of recent SARS-CoV-2 variants. Data are presented as mean IC50 based on 3 independent experiments, except for (*) 1 independent experiment. Conclusion In summary, this second-generation cocktail targeting three unique and highly conserved epitopes in the S1 and S2 subunits of the spike is ready to proceed to clinical testing and may provide a long-term solution to the populations at highest risk. Lung infectious viral titers in Syrian golden hamster post-infection challenge model (Wuhan strain) on day 4 post infection. Mean values + standard error of the mean (SEM) are reported. * Two animals in 2 mg/kg group of XVR013m and one animal in the 20 mg/kg group of XVR014 were experimentally confirmed in PK assays to not have been exposed to the drug. Dotted lines represent the lower limit of detection (LLOD) range. Disclosures Florence M. Herschke, PhD, ExeVir Bio: Patent inventor|ExeVir Bio: Employee Bert Schepens, PhD, Exevir: Bert Schepens is Inventor on patents that describe the nanobodies that are mentioned in the abstract and presentation Loes van Schie, PhD, Exevir: Grant/Research Support|Exevir: I am a co-inventor on the patents protecting the biologicals described in the abstract and presentation Xavier Saelens, PhD, ExeVir Bio: I am an inventor on patent applications WO2022/167666 A1 and WO2023/222825 A1 which incorporate discoveries and inventions described here.|ExeVir Bio: I am a scientific co-founder of ExeVir Bio and in receipt of ExeVir Bio share options

Long-Term Immune Consequences of Initial SARS-CoV-2 A.23.1 Exposure: A Longitudinal Study of Antibody Responses and Cross-Neutralization in a Ugandan Cohort

Background: This study assessed the long-term dynamics of neutralizing antibodies in a Ugandan cohort primarily exposed to the A.23.1 SARS-CoV-2 variant, examining how this shaped immune breadth and potency against diverse strains following infection and prototype-based vaccination. Methods: We conducted a 427-day retrospective analysis of 41 participants across multiple SARS-CoV-2 waves, assessing binding and neutralizing antibody responses using in-house ELISA and pseudotyped virus neutralization assays. We quantified immune responses to key SARS-CoV-2 variants, A.23.1, D614G, Delta, and BA.4, capturing evolving immunity across the pandemic. Results: Neutralizing antibody titers against A.23.1 remained significantly higher than those against D614G, Delta, and BA.4, highlighting the solid immune memory following A.23.1 infection. Consistently lower titers were observed for BA.4 across all time points, aligning with its strong immune-evasion capability. Correlations between neutralizing titers and spike-directed IgG (S-IgG) concentrations were significantly stronger for A.23.1 than for D614G, with no correlation for BA.4. ChAdOx1-S vaccination substantially elevated the neutralizing titers across all variants, most notably BA.4, highlighting the essential role of vaccination in boosting immunity, even in individuals with initially low titers. Conclusions: Initial exposure to the A.23.1 variant triggered potent immune responses, shaping neutralizing antibody dynamics during subsequent exposures. These findings highlight the importance of accounting for early viral exposures in vaccine development and public health planning. The distinctly lower immune response to BA.4 highlights the need for continuous antigenic monitoring and timely vaccine updates for protection against emerging variants. Vaccination remains essential for reinforcing and sustaining immunity against evolving variants.

Cross-Reactive Fc-Mediated Antibody Responses to Influenza HA Stem Region in Human Sera Following Seasonal Vaccination

Background: Current influenza A vaccines primarily induce neutralizing antibodies targeting the variable hemagglutinin (HA) head domain, limiting their effectiveness against diverse or emerging influenza A virus (IAV) subtypes. The conserved HA stem domain, particularly the long α-helix (LAH) epitope, is a focus of universal vaccine research due to its cross-protective potential. Additionally, Fc-mediated functions such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) are recognized as important protective immune mechanisms. This study evaluated IgG responses to the HA head, stem, and LAH regions and assessed cross-reactive potential through neutralization, ADCC, and ADCP assays. Methods: IgG responses to the HA head, stem, and LAH regions were measured in vaccinated individuals. Functional assays were conducted for neutralization, ADCC, and ADCP to evaluate the association between antibody levels and immune function. Results: The results showed that HA head-specific IgG increased significantly after vaccination in 50 individuals, whereas stem-specific IgG increased by 72% and LAH-specific IgG by 12–14%. Among the induced antibody subclasses, IgG1 was predominantly increased. Neutralization titers were detected in viruses of the same strain as the vaccine strain, but not in classical or pandemic strains (H5N1, H7N9). HA stem-specific IgG1 antibody titers showed a significant correlation with ADCC/ADCP activity breadth, but no correlation was observed with neutralization breadth. Conclusion: These findings suggest that although current influenza vaccines can induce HA stem-targeted cross-reactive antibodies, their quantity may be insufficient for broad cross-protection, underscoring the need for improved vaccine strategies.

Mixed lipopeptide-based mucosal vaccine candidate induces cross-variant immunity and protects against SARS-CoV-2 infection in hamsters.

The global dissemination of SARS-CoV-2 led to a worldwide pandemic in March 2020. Even after the official downgrading of the COVID-19 pandemic, infection with SARS-CoV-2 variants continues. The rapid development and deployment of SARS-CoV-2 vaccines helped to mitigate the pandemic to a great extent. However, the current vaccines are suboptimal; they elicit incomplete and short-lived protection and are ineffective against evolving virus variants. Updating the spike antigen according to the prevailing variant and repeated boosters is not the long-term solution. We have designed a lipopeptide-based, mucosal, pan-coronavirus vaccine candidate, derived from highly conserved and/or functional regions of the SARS-CoV-2 spike, nucleocapsid, and membrane proteins. Our studies demonstrate that the designed lipopeptides (LPMix) induced both cellular and humoral (mucosal and systemic) immune responses upon intranasal immunization in mice. Furthermore, the antibodies bound to the wild-type and mutated S proteins of SARS-CoV-2 variants of concern, including Alpha, Beta, Delta and Omicron, and also led to efficient neutralization in a surrogate viral neutralization assay. Our sequence alignment and 3-dimensional molecular modeling studies demonstrated that spike-derived epitopes, P1 and P2, are sequentially and/or structurally conserved among the SARS-CoV-2 variants. The addition of a novel mucosal adjuvant, heat-killed Caulobacter crescentus, to the lipopeptide vaccine significantly bolstered mucosal antibody responses. Finally, the lipopeptide-based intranasal vaccine demonstrated significant improvement in lung pathologies in a hamster model of SARS-CoV-2 infection. These studies are fundamentally important and open new avenues in the investigation of an innovative, broadly protective intranasal vaccine platform for SARS-CoV-2 and its variants.

Citrinin-Induced Cellular Damage: Insights from SH-SY5Y Cell Line Studies

Citrinin (CIT), a mycotoxin commonly found in cereals, is produced by fungi from the Aspergillus, Penicillium, and Monascus genera. While its nephrotoxic effects are well studied, its impact on neurons is less understood. This study investigates CIT-induced toxicity in human neuroblastoma cells (SH-SY5Y). The IC50 values for cells treated with CIT were 77.1 μM at 24 h and 74.7 μM at 48 h using MTT assay, and 101.0 μM at 24 h and 54.7 μM at 48 h using neutral red assay. CIT exposure caused G2/M phase arrest, with cells in this phase increasing from 11.83% (control) to 33.10% at 50 μM CIT. At 50 μM, the percentage of cells in the S phase also increased, which may suggest that cellular stress pathways were activated. Moreover, an increase in late apoptosis process was noted in cells exposed to CIT for 24 h, particularly at the highest concentrations (38.75 and 50 µM). Western blot analysis confirmed a rapid change in the anti-apoptotic protein Bcl-2, but no significant changes in Bax. In conclusion, CIT induces apoptosis and cell cycle arrest in SH-SY5Y cells. However, further transcriptomic studies in specific proteins involved in different pathways described in this work are needed to gain a comprehensive understanding of the specific mechanisms underlying CIT’s toxicity in SH-SY5Y cells.

Tousled-like kinase loss confers PARP inhibitor resistance in BRCA1-mutated cancers by impeding non-homologous end joining repair

BackgroundDouble-strand breaks (DSBs) are primarily repaired through non-homologous end joining (NHEJ) and homologous recombination (HR). Given that DSBs are highly cytotoxic, PARP inhibitors (PARPi), a prominent class of anticancer drugs, are designed to target tumors with HR deficiency (HRD), such as those harboring BRCA mutations. However, many tumor cells acquire resistance to PARPi, often by restoring HR in HRD cells through the inactivation of NHEJ. Therefore, identifying novel regulators of NHEJ could provide valuable insights into the mechanisms underlying PARPi resistance.MethodsCellular DSBs were assessed using neutral comet assays and phospho-H2AX immunoblotting. Fluorescence-based reporter assays quantified repair via NHEJ or HR. The recruitment of proteins that promote NHEJ and HR to DSBs was analyzed using immunostaining, live-cell imaging following laser-induced microirradiation, and FokI-inducible single DSB generation. Loss-of-function experiments were performed in multiple human cancer cell lines using siRNA-mediated knockdown or CRISPR-Cas9 gene knockout. Cell viability assays were conducted to evaluate resistance to PARP inhibitors. Additionally, bioinformatic analyses of public databases were performed to investigate the association between TLK expression and BRCA1 status.ResultsWe demonstrate that human tousled-like kinase (TLK) orthologs are essential for NHEJ-mediated repair of DSBs and for PARPi sensitivity in cells with BRCA1 mutation. TLK1 and TLK2 exhibit redundant roles in promoting NHEJ, and their deficiency results in a significant accumulation of DSBs. TLKs are required for the proper localization of 53BP1, a key factor in promoting the NHEJ pathway. Consequently, TLK deficiency induces PARPi resistance in triple-negative breast cancer (TNBC) and ovarian cancer (OVCA) cell lines with BRCA1 deficiency, as TLK deficiency in BRCA1-depleted cells, impairs 53BP1 recruitment to DSBs and reduces NHEJ efficiency, while restoring HR.ConclusionsWe have identified TLK proteins as novel regulators of NHEJ repair and PARPi sensitivity in BRCA1-depleted cells, suggesting that TLK repression may represent a previously unrecognized mechanism by which BRCA1 mutant cancers acquire PARPi resistance.

SARS-CoV-2 IgG antibodies in COVID-19 convalescent plasma and conventional plasma units.

The Association for the Advancement of Blood and Biotherapies guidelines recommend the use of high-titer COVID-19 convalescent plasma (CCP) for patients with SARS-CoV-2 at high risk of disease progression, including those who are immunocompromised. We hypothesized that conventional plasma units have comparable neutralizing antibody levels to CCP. Conventional plasma and CCP units were obtained from blood suppliers. Quantitatively measured antibodies to SARS-CoV-2 were assessed using the MesoScale Discovery multiplex electrochemiluminescence immunoassay. Binding antibody distributions were compared with Wilcoxon rank-sum tests. SARS-CoV-2 neutralizing antibodies were analyzed using the GeneScript ELISA-based neutralization assay. The proportion of conventional and CCP units with a percent signal inhibition of ≥80% (as defined by the United States Food and Drug Administration for CCP in 2021) and exact binomial confidence intervals (CIs) were calculated. Among 218 conventional plasma units and 74 CCP units collected between September 2023 and July 2024, the distribution of total antibody binding levels largely overlapped between conventional plasma and CCP, though statistically significant differences in median nucleocapsid and spike Omicron variant concentrations were observed. Median percent signal neutralization was 97.5% (range 3.4%-98.6%) among conventional plasma units and 97.7% (range 95.4%-98.6%) among CCP units. For conventional plasma, 95.0% (95% CI = 91.2%-97.5%) met the neutralization antibody threshold for high-titer CCP. For CCP, 100% (95% CI = 95.1%-100.0%) met the neutralization threshold for high-titer CCP. Conventional plasma units demonstrate similar median antibody concentration to CCP units. In countries or regions where licensed CCP is unavailable and titers are unknown, transfusion of multiple conventional plasma units may be of clinical utility.

Innovative production of highly potent equine neutralizing antibody against Hemiscorpius lepturus scorpion venom using recombinant mPLD1 protein.

Scorpion envenomation, especially from Hemiscorpius lepturus, poses a significant health risk, leading to considerable morbidity and mortality. The venom's major toxin, which includes phospholipase D (PLD), is responsible for various systemic complications. In prior studies, we identified a native phospholipase D (PLD) toxin as a key lethal factor in the venom of H. lepturus. A recombinant PLD that retained its toxicity was developed and designated as PLD1. Additionally, a non-toxic and devoid of lethal effects mutant form of the recombinant PLD1 protein, was produced and named as mPLD1. Building on this knowledge, we aimed to produce a novel antivenom using recombinant mPLD1-based immunogen and commercial antisera were included for comparison. Two horses were immunized separately with either recombinant or mutant PLD1, resulting in high titer antisera with no significant difference between the two immunogens. Purified F(ab')2 fragments derived from horse antisera demonstrated a markedly enhanced specificity in the detection of PLD1 and crude venom when compared to commercial alternatives. Furthermore, in vivo neutralization assays revealed that the antisera generated from mPLD1 protein was 89 and 36 times more potent than those of commercial ones. Horses produced highly neutralizing antibodies against PLD1 than the two local commercial antisera. These findings underscore the promise of the developed anti-mPLD1 as a highly effective therapeutic molecule for H. lepturus envenomation. Given that the production process for the recombinant immunogen is straightforward and utilizes cost-effective technologies, focusing on the manufacture of this highly efficient antisera could lead to significant advancements in horse antisera production platforms.

Immunogenicity of HIV-1 Env mRNA and Env-Gag VLP mRNA Vaccines in Mice.

The development of a protective vaccine is critical for conclusively ending the human immunodeficiency virus (HIV) epidemic. We constructed nucleotide-modified mRNA vaccines expressing HIV-1 Env and Gag proteins. Env-gag virus-like particles (VLPs) were generated through co-transfection with env and gag mRNA vaccines. BALB/c mice were immunized with env mRNA, env-gag VLP mRNA, env plasmid DNA vaccine, or lipid nanoparticle (LNP) controls. HIV Env-specific binding and neutralizing antibodies in mouse sera were assessed via enzyme-linked immunosorbent assay (ELISA) and pseudovirus-based neutralization assays, respectively. Env-specific cellular immune responses in mouse splenocytes were evaluated using an Enzyme-linked immunosorbent assay (ELISpot) and in vivo cytotoxic T cell-killing assays. The Env-specific humoral and cellular immune responses elicited by HIV-1 env mRNA and env-gag VLP mRNA vaccine were stronger than those induced by the DNA vaccine. Specific immune responses induced by the env mRNA vaccine were significantly stronger in the high-dose group than in the low-dose group. Immunization with co-formulated env and gag mRNAs elicited superior cellular immune responses compared to env mRNA alone. These findings suggest that the env-gag VLP mRNA platform holds significant promise for HIV-1 vaccine development.

The Anti‐Inflammatory Activities of Sphagnum palustre L. Ethanol Extract to Control Inflammation in RAW264.7 Cells

ABSTRACTDiscomfort caused by inflammation leads to stress and anxiety in patients and seriously decreases the patients' quality of life. People prefer to use natural products instead of anti‐inflammatory drugs because of their low toxicity and side effects. Studies have shown that Sphagnum palustre L. (S. palustre) can be used as medicinal plant, but few studies have focused on its anti‐inflammatory effects. This study explored the mechanism of action of the ethanol extract of the peat moss plant S. palustre on lipopolysaccharide‐induced inflammation in macrophage RAW264.7 cells. Components in S. palustre ethanol extracts (SPE) were identified by HPLC‐MS, which mainly included 4‐methoxybenzaldehyde, 4‐methoxycinnamaldehyde and oleanolic acid. The effects of different concentrations (6.25–100 μg/mL) of SPE after 24 h administration were evaluated to establish a cellular inflammation model. Three biological replicates were performed based on each experiment, the MTT assay results showed that a low concentration of SPE promoted cell proliferation marked by Formazan. In a neutral red uptake assay, the SPE group was effectively inhibited the cell phagocytosis rate. With the increase of SPE concentration, intracellular ROS release decreased, which detected by DCFH‐DA. Immunofluorescence assay result showed that SPE inhibited the release of reactive oxygen species from macrophages with fluorescent markers and DAPI. SPE inhibited the release of nitric oxide from macrophages as well. What's more, SPE significantly decreased the protein expression of interleukin (IL)‐1, IL‐6, and nuclear factor (NF)‐κB according to enzyme‐linked immunosorbent and immunocytochemical assays. SPE reduces inflammation in macrophage RAW264.7 cells and thus is a promising natural anti‐inflammatory plant.

Monovalent Lectin Microvirin Utilizes Hydropathic Recognition of HIV-1 Env for Inhibition of Virus Cell Infection.

Microvirin is a lectin molecule known to have monovalent interaction with glycoprotein gp120. A previously reported high-resolution structural analysis defines the mannobiose-binding cavity of Microvirin. Nonetheless, structure does not directly define the energetics of binding contributions of protein contact residues. To better understand the nature of the MVN-Env glycan interaction, we used mutagenesis to evaluate the residue contributions to the mannobiose binding site of MVN that are important for Env gp120 glycan binding. MVN binding site amino acid residues were individually replaced by alanine, and the resulting purified recombinant MVN variants were examined for gp120 interaction using competition Enzyme-Linked Immunosorbent Assay (ELISA), biosensor surface plasmon resonance, calorimetry, and virus neutralization assays. Our findings highlight the role of both uncharged polar and non-polar residues in forming a hydropathic recognition site for the monovalent glycan engagement of Microvirin, in marked contrast to the charged residues utilized in the two Cyanovirin-N (CVN) glycan-binding sites.

Characterization of Vaccine-Enhanced Humoral Immune Responses Against Emergent SARS-CoV-2 Variants in a Convalescent Cohort.

Vaccination of COVID-19-convalescent individuals may generate 'hybrid' immunity of enhanced magnitude, durability, and cross-reactive breadth. Our primary goal was to characterize hybrid antibody (Ab) responses in a patient cohort infected with ancestral Wuhan-Hu-1 virus and vaccinated between 6 and 10 months later with the Wuhan-Hu-1-based BNT162b2 mRNA vaccine. We were particularly interested in determining the efficacy of neutralizing Ab responses against subsequently emergent SARS-CoV-2 variants. Sera collected at 3-monthly intervals over a period of 12 months were analyzed by ELISA for SARS-CoV-2 RBD-specific Ab responses, and also for neutralizing Ab activity using pseudovirus-based neutralization assays. We found that convalescent RBD-reactive IgG and IgA Ab responses did not decline significantly through 9 months post-diagnosis. These responses improved significantly following vaccination and remained elevated through at least 12-months. SARS-CoV-2 neutralizing Ab activity was detected in convalescent sera through 9 months post-diagnosis, although it trended downwards from 3 months. Neutralizing Ab activity against the Wuhan-Hu-1 strain was significantly improved by vaccination, to levels that persisted through the end of the study. However, sera collected from vaccinated convalescent subjects also had significant neutralization activity against Delta B.1.617.2 and Omicron variants that persisted for at least 2-3 months, unlike sera from unvaccinated convalescent controls. Thus, vaccination of Wuhan-Hu-1-convalescent individuals with the BNT162b2 vaccine improved and sustained protective neutralizing Ab activity against SARS-CoV-2, including cross-reactive neutralizing activity against variants that emerged months later.

Multiple Gene Deletion Mutants of Equine Herpesvirus 1 Exhibit Strong Protective Efficacy Against Wild Virus Challenge in a Murine Model.

Equine herpesvirus type 1 (EHV1) is a ubiquitous viral pathogen infecting the equine population worldwide. EHV1 infection causes respiratory illness, abortion, neonatal foal mortality, and myeloencephalopathy. The currently available modified live EHV1 vaccines have safety and efficacy limitations. The two mutant EHV1 viruses (vToH-DMV (∆IR6/gE) and vToH-QMV (∆IR6/UL43/gE/UL56)), generated by the deletion of genes responsible for virulence (gE and IR6) and immunosuppression (uL43 and uL56), have been previously characterized by our group and found to generate good immune responses. The present study aimed to determine the safety and protective efficacy of the above mutants against a virulent EHV1 challenge in a murine model. BALB/c mice were intranasally immunized with a live vToH-QMV or vToH-DMV vaccine. Intranasal booster immunization was given at 14 days post-vaccination (dpv). Both mutants induced an optimal level of EHV1-specific humoral and cell-mediated immune responses, as determined by virus neutralization assay, ELISA, and immunophenotyping. At 35 dpv, the mice were intranasally challenged with wild-type EHV1 (vRaj strain). Amongst the two mutants, vToH-QMV induced a better immune response than the vToH-DMV vaccine. Furthermore, vToH-QMV provided good protection in mice against the virulent challenge. It specifically exhibited less severe clinical disease in terms of clinical signs, body weight reduction, and gross and histopathological lung lesions accompanied by early virus clearance. These studies are suggestive of vToH-QMV EHV1 being a potential vaccine candidate against EHV1 infection, which needs to be finally tested in the main host, i.e., horses.

Assessment of heat-killed E. coli expressing Chikungunya virus E2 protein as a candidate vaccine for dual protection against Chikungunya virus and E. coli.

The Chikungunya virus (CHIKV) is a mosquito-borne virus with a long history of recurring epidemics transmitted through Aedes mosquitoes. The rapid spread of CHIKV has intensified the need for potent vaccines. Escherichia coli (E.coli), a vital part of human gut microbiota, is utilized in recombinant DNA technology for cloning. However, its high adaptability can lead to severe infections in humans. This study aimed to develop the candidate dual vaccine against CHIKV and E. coli. For this, we expressed the CHIKV E2 protein in the E. coli Rosetta Bl21 cells and the protein expression was confirmed by western blotting. The IgG immune response of the candidate vaccine was determined against CHIKV and E. coli by ELISA. Further, the potential of antibodies to neutralize CHIKV was evaluated via Tissue Culture Infectious Dose 50 (TCID50). We observed that cells expressing E2 protein with alum immunized mice serum showed a five-fold higher IgG immune response against CHIKV, compared to control cells. The CHIKV neutralization assay results showed a two-fold decrease in CHIKV TCID50 value after 12 hours and a three-fold reduction after 120 hours. Similarly, the vaccine formulation also elicited a significantly higher IgG immune response against E. coli. The results suggested that expressing CHIKV E2 protein in E. coli is a potential approach for generating an IgG immune response against CHIKV and E. coli both. This study proposes a faster, safer, and cost-effective recombinant protein-based vaccine development.