Predicting Efficacies of Fractional Doses of Vaccines by Using Neutralizing Antibody Levels: Systematic Review and Meta-Analysis.

Background: With the emergence of SARS-CoV-2 variants that have eluded immunity from vaccines and prior infections, vaccine shortages and vaccine effectiveness pose unprecedented challenges for governments in expanding booster vaccination programs. The fractionation of vaccine doses might be an effective strategy for helping society to face these challenges, as fractional doses may have efficacies comparable with those of the standard doses. Objective: This study aims to investigate the relationship between vaccine immunogenicity and protection and to project efficacies of fractional doses of vaccines for COVID-19 by using neutralizing antibody levels. Methods: In this study, we analyzed the relationship between in vitro neutralization levels and the observed efficacies against both asymptomatic infection and symptomatic infection, using data from 13 studies of 10 COVID-19 vaccines and from convalescent cohorts. We further projected efficacies for fractional doses, using neutralization as an intermediate variable, based on immunogenicity data from 51 studies included in our systematic review. Results: In comparisons with the convalescent level, vaccine efficacy against asymptomatic infection and symptomatic infection increased from 8.8% (95% CI 1.4%-16.1%) to 71.8% (95% CI 63%-80.7%) and from 33.6% (95% CI 23.6%-43.6%) to 98.6% (95% CI 97.6%-99.7%), respectively, as the mean neutralization level increased from 0.1 to 10 folds of the convalescent level. Additionally, mRNA vaccines provided the strongest protection, which decreased slowly for fractional dosing with dosages between 50% and 100% of the standard dose. We also observed that although vaccine efficacy increased with the mean neutralization level, the rate of this increase was slower for vaccine efficacy against asymptomatic infection than for vaccine efficacy against symptomatic infection. Conclusions: Our results are consistent with studies on immune protection from SARS-CoV-2 infection. Based on our study, we expect that fractional-dose vaccination could provide partial immunity against SARS-CoV-2 and its variants. Our findings provide a theoretical basis for the efficacy of fractional-dose vaccines, serving as reference evidence for implementing fractional dosing vaccine policies in areas facing vaccine shortages and thereby mitigating disease burden. Fractional-dose vaccination could be a viable vaccination strategy comparable to full-dose vaccination and deserves further exploration.

Bone matrix properties in adults with osteogenesis imperfecta are not adversely affected by Setrusumab - a sclerostin neutralizing antibody.

Osteogenesis imperfecta (OI) is a skeletal dysplasia characterized by low bone mass and frequent fractures. Children with OI are commonly treated with bisphosphonates to reduce fracture rate, but treatment options for adults are limited. In the Phase 2b ASTEROID trial, setrusumab (a sclerostin neutralizing antibody, SclAb) improved bone density and strength in adults with type I, III and IV OI. Here, we investigate bone matrix material properties in tetracycline-labeled trans-iliac biopsies from three groups: i) control: individuals with no metabolic bone disease, ii) OI: individuals with OI, iii) SclAb-OI: individuals with OI after six months of setrusumab treatment (as part of the ASTEROID trial). In addition to bone histomorphometry, bone mineral and matrix properties were evaluated with nanoindentation, Raman spectroscopy, second harmonic generation imaging, quantitative backscatter electron imaging, and small-angle x-ray scattering. Spatial locations of fluorochrome labels were identified to differentiate inter-label bone of the same tissue age and intra-cortical bone. No difference in collagen orientation was found between the groups. The bone mineral density distribution and analysis of Raman spectra indicate that OI groups have greater mean mineralization, greater relative mineral content, and lower crystallinity than the control group, which was not altered by SclAb treatment. Finally, a lower modulus and hardness were measured in the inter-label bone of the OI-SclAb group compared to the OI group. Previous studies suggest that even though bone from OI has a higher mineral content, the ECM has comparable mechanical properties. Therefore, fragility in OI may stem from contributions from other yet unexplored aspects of bone organization at higher length scales. We conclude that SclAb treatment leads to increased bone mass while not adversely affecting bone matrix properties in individuals with OI.

Individuals with osteogenesis imperfecta (OI), also known as "brittle bone disease," have low bone mass and frequent fractures. Low bone mass occurs due to an imbalance between cells that remove bone and cells that form bone. Pharmaceutical treatments that block removal of bone lead to reduced fracture rates in children with OI. Effective treatment options for adults are limited. Setrusumab is a drug that leads to increased bone mass and strength in adults with OI. Here, we investigate whether Setrusumab alters the bone material in addition to improving bone mass. Three groups are compared: individuals with OI treated with Setrusumab, individuals with OI not treated with Setrusumab, and individuals without OI. A lower modulus and hardness were measured with nanoindentation in the Setrusumab-treated group. However, we did not find any changes in the bone's multi-scale structure. Fragility in OI may stem from other yet unexplored aspects of bone organization. We conclude that Setrusumab treatment leads to increased bone mass while not adversely affecting bone material properties in individuals with OI.

A ferritin-based nanoparticle displaying a neutralizing epitope for foot-and-mouth disease virus (FMDV) confers partial protection in guinea pigs.

BACKGROUND: Foot-and-mouth disease (FMD) is a devastating disease affecting cloven-hoofed animals, that leads to significant economic losses in affected countries and regions. Currently, there is an evident inclination towards the utilization of nanoparticles as powerful platforms for innovative vaccine development. Therefore, this study developed a ferritin-based nanoparticle (FNP) vaccine that displays a neutralizing epitope of foot-and-mouth disease virus (FMDV) VP1 (aa 140-158) on the surface of FNP, and evaluated the immunogenicity and protective efficacy of these FNPs in mouse and guinea pig models to provide a strategy for developing potential FMD vaccines. RESULTS: This study expressed the recombinant proteins Hpf, HPF-NE and HPF-T34E via an E. coli expression system. The results showed that the recombinant proteins Hpf, Hpf-NE and Hpf-T34E could be effectively assembled into nanoparticles. Subsequently, we evaluated the immunogenicity of the Hpf, Hpf-NE and Hpf-T34E proteins in mice, as well as the immunogenicity and protectiveness of the Hpf-T34E protein in guinea pigs. The results of the mouse experiment showed that the immune efficacy in the Hpf-T34E group was greater than the Hpf-NE group. The results from guinea pigs immunized with Hpf-T34E showed that the immune efficacy was largely consistent with the immunogenicity of the FMD inactivated vaccine (IV) and could confer partial protection against FMDV challenge in guinea pigs. CONCLUSIONS: The Hpf-T34E nanoparticles stand out as a superior choice for a subunit vaccine candidate against FMD, offering effective protection in FMDV-infected model animals. FNP-based vaccines exhibit excellent safety and immunogenicity, thus representing a promising strategy for the continued development of highly efficient and safe FMD vaccines.

Identifying a Level of Neutralizing Antibody That Correlates With Protection From Clinical Mumps Disease During a 2017 Mumps Outbreak Among Military Service Members.

Background: In 2017, a mumps outbreak occurred in a US military barracks. Serum collected at service entry was used to compare pre-exposure with presumptive vaccine-induced antibody levels from persons who developed mumps (cases) and potentially exposed persons who did not develop mumps (non-cases). Sufficient information to determine levels of exposure during the outbreak was not available. Methods: Pre-outbreak serum samples from the Department of Defense Serum Repository were available from 254 potentially exposed service members. Twelve developed clinical symptoms and had post-outbreak serum collected. All sera were tested with a mumps-specific enzyme immunoassay for immunoglobulin M, immunoglobulin G (IgG), and IgG avidity. The neutralizing antibodies to vaccine strain (Jeryl Lynn [JL], genotype A) and wildtype virus (genotype G) was assessed by a plaque reduction neutralization test. A Fisher exact test and receiver operator characteristic curve were used to analyze the antibody response for non-cases and mumps cases. Results: Eight mumps cases were laboratory confirmed. Pre-outbreak neutralizing antibody titers to JL and genotype G mumps virus and pre-outbreak IgG index values were proportionately lower for most cases as compared with exposed non-cases. When compared with potentially exposed non-cases, cases with clinical symptoms had greater odds of having a pre-outbreak JL titer <41 and a genotype G titer <16. Conclusions: We identified potential correlates of protection for mumps neutralizing antibody titers against JL and genotype G mumps viruses.

Complete combinatorial mutational enumeration of a protein functional site enables sequence-landscape mapping and identifies highly-mutated variants that retain activity.

Understanding how proteins evolve under selective pressure is a longstanding challenge. The immensity of the search space has limited efforts to systematically evaluate the impact of multiple simultaneous mutations, so mutations have typically been assessed individually. However, epistasis, or the way in which mutations interact, prevents accurate prediction of combinatorial mutations based on measurements of individual mutations. Here, we use artificial intelligence to define the entire functional sequence landscape of a protein binding site in silico, and we call this approach Complete Combinatorial Mutational Enumeration (CCME). By leveraging CCME, we are able to construct a comprehensive map of the evolutionary connectivity within this functional sequence landscape. As a proof of concept, we applied CCME to the ACE2 binding site of the SARS-CoV-2 spike protein receptor binding domain. We selected representative variants from across the functional sequence landscape for testing in the laboratory. We identified variants that retained functionality to bind ACE2 despite changing over 40% of evaluated residue positions, and the variants now escape binding and neutralization by monoclonal antibodies. This work represents a crucial initial stride toward achieving precise predictions of pathogen evolution, opening avenues for proactive mitigation.

Neutralizing Antibody Sample Testing and Report Harmonization.

Immunogenicity testing and characterization is an important part of understanding the immune response to administration of a protein therapeutic. Neutralizing antibody (NAb) assays are used to characterize a positive anti-drug antibody (ADA) response. Harmonization of reporting of NAb assay performance and results enables efficient communication and expedient review by industry and health authorities. Herein, a cross-industry group of NAb assay experts have harmonized NAb assay reporting recommendations and provided a bioanalytical report (BAR) submission editable template developed to facilitate agency filings. This document addresses key bioanalytical reporting gaps and provides a report structure for documenting clinical NAb assay performance and results. This publication focuses on the content and presentation of the NAb sample analysis report including essential elements such as the method, critical reagents and equipment, data analysis, study samples, and results. The interpretation of immunogenicity data, including the evaluation of the impact of NAb on safety, exposure, and efficacy, is out of scope of this publication.

Immunogenicity of 4-dose Essen intramuscular regimen for rabies post-exposure prophylaxis: A multi-center cross-sectional study in China.

BACKGROUND: The 4-dose Essen intramuscular (IM) regimen for rabies post-exposure prophylaxis (PEP) has been recommended by Advisory Committee on Immunization Practices (ACIP) and World Health Organization (WHO), but the large-sample clinical evidence is still limited. METHOD: Rabies virus neutralizing antibodies of 11,752 patients were detected from 409 rabies prevention clinics in 27 provinces in China. Patients with serum collected before or no later than 1 h after injection on the day of the fifth dose (day 28) of 5-dose Essen regimen were included in Group A to observe the immune efficacy of 4-dose Essen IM regimen, and patients with serum collected 14-28 days after injection of the fifth dose were included in Group B to observe the immune efficacy of 5-dose Essen IM regimen. RESULTS: Finally, 2351 cases met the inclusion and exclusion criteria, including 2244 cases in Group A and 107 cases in Group B. The antibody titer of Group A was higher than that of Group B [12.21 (4.15, 32.10) IU/ml vs. 9.41 (3.87, 27.38) IU/ml] (P = 0.002). In Group A, the median antibody titers were 4.01IU/ml, 11.63IU/ml and 29.46IU/ml in patients vaccinated with purified hamster kidney cell vaccine (PHKCV), purified Vero cell vaccine (PVRV), and human diploid cell rabies vaccine (HDCV), respectively, with statistical significance (P < 0.001). CONCLUSIONS: The 4-dose Essen IM regimen could provide satisfactory immune effect, and HDCV induced higher antibody titer than PHKCV or PVRV.

Non-clinical and clinical characterization of MAU868, a novel human-derived monoclonal neutralizing antibody targeting BK polyomavirus VP1.

Reactivation of BK polyomavirus (BKPyV) can cause significant kidney and bladder disease in immunocompromised patients. There are currently no effective, BKPyV-specific therapies. MAU868 is a novel, human IgG1 monoclonal antibody that binds the major capsid protein VP1 of BKPyV with picomolar affinity, neutralizes infection by the four major BKPyV genotypes (EC50 ranging from 0.009 to 0.093 µg/ml; EC90 ranging from 0.102 to 4.160 µg/ml), and has comparable activity against variants with highly prevalent VP1 polymorphisms. No resistance-associated variants were identified in long-term selection studies, indicating a high in vitro barrier-to-resistance. The high-resolution crystal structure of MAU868 in complex with VP1 pentamer identified three key contact residues in VP1 (Y169, R170, K172). A first-in-human study was conducted to assess the safety, tolerability, and pharmacokinetics of MAU868 following intravenous and subcutaneous administration to healthy adults in a randomized, placebo-controlled, double-blinded, single ascending dose design. MAU868 was safe and well-tolerated. All adverse events were Grade 1 and resolved. The pharmacokinetics of MAU868 was typical of a human IgG, with dose-proportional systemic exposure and an elimination half-life ranging between 23 and 30 days. These results demonstrate the potential of MAU868 as a first-in-class therapeutic agent for the treatment or prevention of BKPyV disease.

A significant outbreak of respiratory human adenovirus infections among children aged 3-6 years in Hokkaido, Japan, in 2023.

Human adenovirus (HAdV) infections present diverse clinical manifestations upon infecting individuals, with respiratory infections predominating in children. We surveyed pediatric hospitalizations due to respiratory HAdV infections across 18 hospitals in Hokkaido Prefecture, Japan, from July 2019 to March 2024, recording 473 admissions. While hospitalizations remained below five cases per week from July 2019 to September 2023, a notable surge occurred in late October 2023, with weekly admissions peaking at 15-20 cases from November to December. There were dramatic shifts in the age distribution of hospitalized patients: during 2019-2021, 1-year-old infants and children aged 3-6 years represented 51.4%-54.8% and 4.1%-13.3%, respectively; however, in 2023-2024, while 1-year-old infants represented 19.0%-20.1%, the proportion of children aged 3-6 years increased to 46.2%-50.0%. Understanding the emergence of significant outbreaks of respiratory HAdV infections and the substantial changes in the age distribution of hospitalized cases necessitates further investigation into the circulating types of HAdV in Hokkaido Prefecture and changes in children's neutralizing antibody titers against HAdV.

Efficacy, immunogenicity, and safety of a monovalent mRNA vaccine, ABO1020, in adults: A randomized, double-blind, placebo-controlled, phase 3 trial.

BACKGROUND: ABO1020 is a monovalent COVID-19 mRNA vaccine. Results from a phase 1 trial showed ABO1020 was safe and well tolerated, and phase 3 trials to evaluate the efficacy, immunogenicity, and safety of ABO1020 in healthy adults are urgently needed. METHODS: We conducted a multinational, randomized, placebo-controlled, double-blind, phase 3 trial among healthy adults (ClinicalTrials.gov: NCT05636319). Participants were randomly assigned (1:1) to receive either 2 doses of ABO1020 (15 µg per dose) or placebo, administered 28 days apart. The primary endpoint was the vaccine efficacy in preventing symptomatic COVID-19 cases that occurred at least 14 days post-full vaccination. The second endpoint included the neutralizing antibody titers against Omicron BA.5 and XBB and safety assessments. FINDINGS: A total of 14,138 participants were randomly assigned to receive either vaccine or placebo (7,069 participants in each group). A total of 366 symptomatic COVID-19 cases were confirmed 14 days after the second dose among 93 participants in the ABO1020 group and 273 participants in the placebo group, yielding a vaccine efficacy of 66.18% (95% confidence interval: 57.21-73.27, p < 0.0001). A single dose or two doses of ABO1020 elicited potent neutralizing antibodies against both BA.5 and XBB.1.5. The safety profile of ABO1020 was characterized by transient, mild-to-moderate fever, pain at the injection site, and headache. CONCLUSION: ABO1020 was well tolerated and conferred 66.18% protection against symptomatic COVID-19 in adults. FUNDING: National Key Research and Development Project of China, Innovation Fund for Medical Sciences from the CAMS, National Natural Science Foundation of China.

Identification of neutralizing antibodies against monkeypox virus using high-throughput sequencing of A35+H3L+B cells from patients with convalescent monkeypox.

The global monkeypox virus (MPXV) outbreak in 2022 emphasizes the urgent need for effective and accessible new-generation vaccines and neutralizing antibodies. Herein, we identified MPXV-neutralizing antibodies using high-throughput single-cell RNA and V(D)J sequencing of antigen-sorted B cells from patients with convalescent monkeypox. IgG1-expressing B cells were obtained from 34 paired heavy- and light-chain B cell receptor sequences. Subsequently, three potent neutralizing antibodies, MV127, MV128, and MV129, were identified and reacted with the MPXV A35 protein. Among these, MV129, which has a half-maximal inhibitory concentration of 2.68µg/mL against authentic MPXV, was considered to be the putative candidates for MPXV neutralization in response to monkeypox disease.

Generation of novel respiratory syncytial virus vaccine candidate antigens that can induce high levels of prefusion-specific antibodies.

Respiratory syncytial virus (RSV) infection is an acute respiratory infection caused by RSV. It occurs worldwide, and for over 50 years, several attempts have been made to research and develop vaccines to prevent RSV infection; effective preventive vaccines are eagerly awaited. The RSV fusion (F) protein, which has gained attention as a vaccine antigen, causes a dynamic structural change from the preF to postF state. Therefore, the structural changes in proteins must be regulated to produce a vaccine antigen that can efficiently induce antibodies with high virus-neutralizing activity. We successfully discovered several mutations that stabilized the antigen site Ø in the preF state, trimerized it, and improved the level of protein expression through observation and computational analysis of the RSV-F protein structure and amino acid mutation analysis of RSV strains. The four RSV-F protein mutants that resulted from the combination of these effective mutations stably conserved a wide range of preF- and trimeric preF-specific epitopes with high virus-neutralizing activity. Absorption assay using human serum revealed that mutants constructed bound to antibodies with virus-neutralizing activity that were induced by natural RSV infection, whereas they hardly bound to anti-postF antibodies without virus-neutralizing activity. Furthermore, mouse immunization demonstrated that our constructed mutants induced a high percentage of antibodies that bind to the preF-specific antigen site. These characteristics suggest that the mutants constructed can be superior vaccine antigens from the viewpoint of RSV infection prevention effect and safety.

Comparable antibody levels in heterologous and homologous mRNA COVID-19 vaccination, with superior neutralizing and IgA antibody responses in mRNA homologous boosting.

BACKGROUND: Priming with two doses of AZD1222 (Oxford-AstraZeneca; ChAd) followed by a third mRNA vaccine boosting is considered in several countries, yet comparisons between heterologous and homologous booster efficacy remain unexplored. AIM: To evaluate and contrast the immunogenicity of homologous and heterologous boosting regimens. METHOD: The study examined antibody responses in 1113 subjects, comprising 895 vaccine-naïve individuals across different vaccination strategies (partial, primary series, heterologous booster, homologous booster) and 218 unvaccinated, naturally infected individuals. Assessments included neutralizing total antibodies (NTAbs), total antibodies (TAbs), anti-S-RBD IgG, and anti-S1 IgA levels. RESULTS: The study found mRNA vaccines to exhibit superior immunogenicity in primary series vaccination compared to ChAd, with mRNA-1273 significantly enhancing NTAbs, TAbs, anti-S-RBD IgG, and anti-S1 IgA levels (p < 0.001). Both booster types improved antibody levels beyond primary outcomes, with no significant difference in TAbs and anti-S-RBD IgG levels between regimens. However, homologous mRNA boosters significantly outperformed heterologous boosters in enhancing NTAbs and anti-S1 IgA levels, with the BNT/BNT/BNT regimen yielding particularly higher enhancements (p < 0.05). CONCLUSION: The study concludes that although TAbs and anti-S-RBD IgG antibody levels are similar for both regimens, homologous mRNA boosting outperform heterologous regimen by enhancing anti-S1 IgA and neutralizing antibody levels.

Trimerized S expressed by modified vaccinia virus Ankara (MVA) confers superior protection against lethal SARS-CoV-2 challenge in mice.

The reoccurrence of successive waves of SARS-CoV-2 variants suggests the exploration of more vaccine alternatives is imperative. Modified vaccinia virus Ankara (MVA) is a virus vector exhibiting excellent safety as well as efficacy for vaccine development. Here, a series of recombinant MVAs (rMVAs) expressing monomerized or trimerized S proteins from different SARS-CoV-2 variants are engineered. Trimerized S expressed from rMVAs is found predominantly as trimers on the surface of infected cells. Remarkably, immunization of mice with rMVAs demonstrates that S expressed in trimer elicits higher levels of binding IgG and IgA, as well as neutralizing antibodies for matched and mismatched S proteins than S in the monomer. In addition, trimerized S expressed by rMVA induces enhanced cytotoxic T-cell responses than S in the monomer. Importantly, the rMVA vaccines expressing trimerized S exhibit superior protection against a lethal SARS-CoV-2 challenge as the immunized animals all survive without displaying any pathological conditions. This study suggests that opting for trimerized S may represent a more effective approach and highlights that the MVA platform serves as an ideal foundation to continuously advance SARS-CoV-2 vaccine development. IMPORTANCE: MVA is a promising vaccine vector and has been approved as a vaccine for smallpox and mpox. Our analyses suggested that recombinant MVA expressing S in trimer (rMVA-ST) elicited robust cellular and humoral immunity and was more effective than MVA-S-monomer. Importantly, the rMVA-ST vaccine was able to stimulate decent cross-reactive neutralization against pseudoviruses packaged using S from different sublineages, including Wuhan, Delta, and Omicron. Remarkably, mice immunized with rMVA-ST were completely protected from a lethal challenge of SARS-CoV-2 without displaying any pathological conditions. Our results demonstrated that an MVA vectored vaccine expressing trimerized S is a promising vaccine candidate for SARS-CoV-2 and the strategy might be adapted for future vaccine development for coronaviruses.

Anti-idiotype isolation of a broad and potent influenza A virus-neutralizing human antibody.

The VH6-1 class of antibodies includes some of the broadest and most potent antibodies that neutralize influenza A virus. Here, we elicit and isolate anti-idiotype antibodies against germline versions of VH6-1 antibodies, use these to sort human leukocytes, and isolate a new VH6-1-class member, antibody L5A7, which potently neutralized diverse group 1 and group 2 influenza A strains. While its heavy chain derived from the canonical IGHV6-1 heavy chain gene used by the class, L5A7 utilized a light chain gene, IGKV1-9, which had not been previously observed in other VH6-1-class antibodies. The cryo-EM structure of L5A7 in complex with Indonesia 2005 hemagglutinin revealed a nearly identical binding mode to other VH6-1-class members. The structure of L5A7 bound to the isolating anti-idiotype antibody, 28H6E11, revealed a shared surface for binding anti-idiotype and hemagglutinin that included two critical L5A7 regions: an FG motif in the third heavy chain-complementary determining region (CDR H3) and the CDR L1 loop. Surprisingly, the chemistries of L5A7 interactions with hemagglutinin and with anti-idiotype were substantially different. Overall, we demonstrate anti-idiotype-based isolation of a broad and potent influenza A virus-neutralizing antibody, revealing that anti-idiotypic selection of antibodies can involve features other than chemical mimicry of the target antigen.

Construction and immunogenicity of SARS-CoV-2 virus-like particle expressed by recombinant baculovirus BacMam.

The pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve to give rise to variants of concern that can escape vaccine-induced immunity. As such, more effective vaccines are urgently needed. In this study, we evaluated virus-like particle (VLP) as a vaccine platform for SARS-CoV-2. The spike, envelope, and membrane proteins of the SARS-CoV-2 Wuhan strain were expressed by a single recombinant baculovirus BacMam and assembled into VLPs in cell culture. The morphology and size of the SARS-CoV-2 VLP as shown by transmission electron microscopy were similar to the authentic SARS-CoV-2 virus particle. In a mouse trial, two intramuscular immunizations of the VLP BacMam with no adjuvant elicited spike-specific binding antibodies in both sera and bronchoalveolar lavage fluids. Importantly, BacMam VLP-vaccinated mouse sera showed neutralization activity against SARS-CoV-2 spike pseudotyped lentivirus. Our results indicated that the SARS-CoV-2 VLP BacMam stimulated spike-specific immune responses with neutralization activity. IMPORTANCE: Although existing vaccines have significantly mitigated the impact of the COVID-19 pandemic, none of the vaccines can induce sterilizing immunity. The spike protein is the main component of all approved vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) due primarily to its ability to induce neutralizing antibodies. The conformation of the spike protein in the vaccine formulation should be critical for the efficacy of a vaccine. By way of closely resembling the authentic virions, virus-like particles (VLPs) should render the spike protein in its natural conformation. To this end, we utilized the baculovirus vector, BacMam, to express virus-like particles consisting of the spike, membrane, and envelope proteins of SARS-CoV-2. We demonstrated the immunogenicity of our VLP vaccine with neutralizing activity. Our data warrant further evaluation of the virus-like particles as a vaccine candidate in protecting against virus challenges.

The kinetics and durability of antibody and T-cell responses to SARS-CoV-2 in children.

BACKGROUND: The kinetics and durability of T-cell responses to SARS-CoV-2 in children are not well-characterized. We studied a cohort of children aged 6 months to 20 years with COVID-19 in whom peripheral blood mononuclear cells (PBMC) and sera were archived at approximately 1, 6, and 12 months post-symptom onset. METHODS: We compared antibody (N = 85) and T-cell responses (N = 26) to nucleocapsid (N) and spike (S) glycoprotein over time across four age strata: 6 months to 5 years, 5-9, 10-14, and 15-20 years. RESULTS: N-specific antibody responses declined over time, becoming undetectable in 26/32 (81%) children by approximately one year post-infection. Functional breadth of anti-N CD4+ T-cell responses also declined over time and were positively correlated with N-antibody responses (Pearson's r = 0.31, p = 0.008). CD4+ T-cell responses to S displayed greater functional breadth than N in unvaccinated children, and, along with neutralization titers, were stable over time and similar across age strata. Functional profiles of CD4+ T-cell responses against S were not significantly modulated by vaccination. CONCLUSIONS: Our data reveal durable, age-independent T-cell immunity to SARS-CoV-2 structural proteins in children over time following COVID-19 infection as well as S-Ab responses overall, in comparison to declining antibody responses to N.

Low pre-infection levels of neutralizing antibody in breakthrough infections after bivalent BA.4-5 vaccine and practical application of dried blood spots.

The level of neutralizing antibodies required to confer protection against COVID-19 breakthrough infections (BIs) is unclear, and the ability to know the immune status of individuals against the rapidly changing endemic variants is limited. We assessed longitudinal serum anti-RBD antibody levels and neutralizing activities (NTs) against Omicron BA.5 and XBB.1.5 in healthcare workers following the fourth monovalent and fifth bivalent BA.4-5 vaccines. The occurrence of BIs was also followed, and pre-infection antibody levels were compared between patients who developed BI and those who did not. In addition, we collected whole blood samples on the same day as the sera and stored them on filter papers (nos. 545, 590, and 424) for up to two months, then measured their NTs using dried blood spots (DBS) eluates, and compared them with the NTs in paired sera. Pre-infection levels of NTs were lower in patients who developed BI than those who did not, but the anti-RBD antibody levels were not different between them. The NTs below 50 % using 200-fold diluted sera might be one of the indicators of high risk for COVID-19 BI. However, the NTs against XBB.1.5 at 6 months after the fifth dose of bivalent BA.4-5 vaccine were lower than this threshold in almost half of infection-naïve participants. NTs measured using DBS eluates were strongly correlated with those measured using paired sera, but the time and temperature stability varied with the type of filter paper; no. 545 filter paper was found to most suitable for NT evaluation.

IL-17A enhances the inflammatory response of glaucoma through Act1/TRAF6/NF-κB pathway.

OBJECTIVES: To investigate the possible roles of Interleukin 17A (IL-17A) and IL-17A neutralizing antibodies (IL-17Ab) in glaucoma and the potential mechanisms. METHODS: The two glaucoma animal models, chronic ocular hypertension (COH) and N-methyl-D-aspartate (NMDA)-induced retinal ganglion cell (RGC) damage, were established and treated with intravitreal injection of IL-17A or IL-17Ab. Intraocular pressure (IOP) was measured by a rebound tonometer. The retina and RGC injury were evaluated by HE staining, TUNLE assay and Brn3a immunofluorescence staining. The frequency of IL-17A+CD4+T cells in peripheral blood was detected by flow cytometry. The expression of glial fibrillary acidic protein (GFAP) was detected by immunofluorescence staining, Western Blot and qPCR in retina. The RNA and protein expression of Act1/TRAF6/NF-κB were detected by Western Blot and qPCR in retina. RESULTS: The expression of IL-17A increased in glaucoma models. After intravitreal injection of IL-17A, in the retina, the number of RGCs decreased, the apoptosis of RGCs increased, the Müller cell gliosis was more obvious. In addition, peripheral inflammation aggravated. Whereas the intravitreal injection of IL-17Ab alleviated the relevant manifestations and peripheral inflammation, reduced the gliosis of Müller cells. In the COH model, IOP increased after the injection of IL-17A, while the intravitreal injection of IL-17Ab led to a decrease in IOP. Furthermore, IL-17A promotes the apoptosis of RGCs by binding to IL-17A receptor, activating Act1/TRAF6/NF-κB pathways. CONCLUSION: IL-17A plays a role in and aggravates RGC damage in glaucoma. IL-17Ab can neutralize the pro-inflammatory effect of IL-17A and have a protective function in glaucoma. These findings reveal the importance of IL-17A in the pathogenesis of glaucoma, which will shed light on a novel direction for the prevention and treatment of glaucoma, and also provide a reference for further research on other retinal diseases.

Unique challenges required reassessment and alterations to critical reagents to rescue a neutralizing antibody assay.

Aim: To redevelop a neutralizing antibody (NAb) assay to be much more drug tolerant, have a large dynamic range and have high inhibition when using high levels of positive control (PC). Materials & methods: Early assay data suggested that typical biotin labeling of the capture reagent (Drug 1, produced in a human cell line) was blocking it from binding with the PC or the detection target, and that the detection target was out competing the PC. Methodical biotin labeling experiments were performed at several challenge ratios and an Fc linker was added to the detection target. Results & conclusion: A larger dynamic range, high inhibition and higher drug tolerance were achieved by adding an acid dissociation step to the assay, performing atypical biotin labeling of Drug 1 and switching to a detection target that contained an Fc linker to increase steric hinderance and decrease its binding affinity to Drug 1.

Many of the drugs available today are produced by a living organism and these are called biologics. Biologics are larger than chemical drugs and the human body can detect them as foreign and create antibodies against them. This is called immunogenicity. When the antibodies created against the biologic blocks the drug's ability to work correctly, they are called neutralizing antibodies (NAbs). Testing for NAbs is one of the requirements of regulatory agencies for biologics. Here we describe challenges encountered developing an assay to test for NAbs against a biologic.