Generation Of Antibodies Research Articles

Development of highly bioactive long-acting recombinant porcine FSH for batch production management of sows

To improve the economic benefits, exogenous hormones are used to control follicular development and synchronize ovulation in the batch flow management of gilts and weaned sows. Equine chorionic gonadotropin (eCG) is widely used to stimulate follicular development in both experimental and farm animals. Despite its effectiveness, several side effects have been found, including the occurrence of follicular cysts, follicular premature luteinization, and increased follicular atresia. As eCG is a heterologous protein, the generation of antibodies has been found in the superovulation of cattle. Moreover, the extraction of eCG from pregnant mare serum raises concerns regarding animal welfare, as well as potential risks for disease transmission. Follicle stimulating hormone (FSH) controls the follicular growth and maturation in the porcine ovary under physiological conditions. In the current study, we developed a novel long-acting recombinant porcine FSH (rpFSH-pFc) consisting of porcine FSH and porcine fragment crystallizable (Fc) via (G4S)3 linker by using the available protein fusion technology to control the follicular development and maturation. The results of pharmacokinetic studies indicated that rpFSH-pFc exhibited a prolonged half-life in both rats and sows. The efficacy of rpFSH-pFc was confirmed by cAMP level evaluation and germinal vesicle breakdown (GVBD) analysis in vitro. Through ovarian weight gain, superovulation and fertility testing assays, our results revealed that a single rpFSH-pFc treatment could effectively promote follicular development and maturation in vivo. Meanwhile, the mRNA expression levels of the target genes associated with follicular development, maturation and ovulation were significantly up-regulated after rpFSH-pFc treatment. Taken together, our results revealed that rpFSH-pFc could bind to the FSH receptor, stimulate follicular growth and development in female mice, and possess a prolonged half-life in both rats and sows. These characteristics suggest that rpFSH-pFc may be an ideal candidate for promoting follicular growth and development in livestock production.

Complete primer set for amplification and expression of full-length recombinant human monoclonal antibodies from single human B cells.

Human monoclonal antibodies (mAbs) are an important segment in precision therapeutics. Various methodologies are available for generating them. Recombinant human mAbs expression from sorted single B cells is preferred for its rapid expression using mammalian vectors while maintaining in vivo immunoglobulin (Ig) pairing. The success rate of generating recombinant mAbs from single sorted human B cells directly relies on Ig heavy (IgH) and light (IgL) gene coverage of the PCR primers. Existing primer sets fail to cover all functional human Ig gene rearrangements, exhibit high degeneracy leading to non-specific amplifications and mutations arising from primer mismatch/degeneracy, and require high amplification cycles. Some existing primer sets have high coverage but are not designed for expression as recombinant mAbs. Here, we have designed a primer set to amplify all functional V(D)J transcripts in human B cell repertoire using a nested RT-PCR approach. The resultant amplicons can be cloned into mammalian vectors for expression of recombinant mAb. Non-specific amplifications were minimized using isotype-specific primers for cDNA synthesis and limiting primer degeneracy. We validated the designed primers on single sorted B cells, bulk sorted B cells and peripheral blood mononuclear cells. We were successfully able to amplify paired heavy and light chain transcripts in 38.46 % (80/208) from naive, memory and B1 B cell subsets sorted as single B cells. Paired Ig transcripts from five single B cells were cloned into expression vectors and purified from mammalian cells as recombinant mAbs. Thus, our new primer set offers significant advantages over existing primers as it allows amplification of all functional V(D)J rearrangements, facilitating rapid generation of antigen-specific recombinant antibodies from diverse human B cell repertoires following vaccinations and infections previously inaccessible due to primer limitations.

Antibody-secreting cell repertoires hold high-affinity anti-rocuronium specificities that can induce anaphylaxis in vivo.

Neuromuscular blocking agents (NMBA) are muscle relaxants used to assist mechanical ventilation but lead in 1/10,000 anesthesia to severe acute hypersensitivity reactions i.e., anaphylaxis. Incidences vary between types of NMBAs. Rocuronium, a widely used non-depolarizing aminosteroid NMBA, induces among the highest anaphylaxis rates. Rocuronium-induced anaphylaxis is proposed to rely on pre-existing rocuronium-binding antibodies, but no such antibodies have ever been identified. Identify rocuronium-specific antibody repertoires from plasma cells or plasmablasts of rocuronium-immunized mice, and determine the affinities, structures and anaphylactogenic potential of these antibodies for rocuronium. Herein, we engrafted rocuronium onto carrier proteins allowing immunization of mice against rocuronium, screening for rocuronium-specific antibody responses, and sorting of rocuronium-specific plasma cells using droplet microfluidics coupled to single-cell antibody gene (VH and VL) sequencing. The two different repertoires of >500 VH-VL pairs were oligoclonal, comprised of three major clonal families, and displayed convergence. Expressed as human IgG1, these antibodies demonstrated subnanomolar affinities for rocuronium with families either monospecific for rocuronium or cross-reactive only for closely-related NMBAs. Expressed as human IgE, they triggered human mast cell and basophil activation, and severe passive systemic anaphylaxis in mice humanized for the IgE receptor FcεRI. Co-crystal structures between rocuronium and antibody representatives of three different VH-VL families revealed distinct interaction modes with, however, the ammonium group involved systematically in the binding interface. This work identifies the epitopes of antibody reactivity to rocuronium, demonstrates anaphylactogenic potential of anti-rocuronium IgE and establishes the first mouse model of NMBA anaphylaxis.

γδ T Cells’ Role in Donor-Specific Antibody Generation: Insights From Transplant Recipients and Experimental Models

The generation of donor-specific antibodies (DSA) requires that alloreactive B cells receive help from follicular helper T (TFH) cells. Recent works have suggested that γδ T cells could contribute to T cell-dependent humoral responses, leading us to investigate their role in DSA generation. Analysis of a cohort of 331 kidney transplant recipients found no relation between the number of circulating γδ T cells and the risk to develop DSA. Coculture models demonstrated that activated γδ T cells were unable to promote the differentiation of B cells into plasma cells, ruling out that they can be “surrogate” TFH. In line with this, γδ T cells preferentially localized outside the B cell follicles, in the T cell area of lymph nodes, suggesting that they could instead act as “antigen-presenting cell” (APC) to prime αβ TFH. This hypothesis was proven wrong since γδ T cells failed to acquire APC functions in vitro. These findings were validated in vivo by the demonstration that following transplantation with an allogeneic Balb/c (H2d) heart, wild-type and TCRδKO C57BL/6 (H2b) mice developed similar DSA responses, whereas TCRαKO recipients did not develop DSA. We concluded that the generation of DSA is unfazed by the absence of γδ T cells.

P-1967. Characteristics of the first immunocompromised patients to receive Sipavibart as an early access treatment for COVID-19 pre-exposure prophylaxis in France

Abstract Background Immunocompromised patients account for a large proportion of COVID-19 hospitalizations and deaths, even in the most recent periods, despite having received several COVID-19 vaccine doses. Monoclonal antibodies effectively prevented COVID-19 in the general population in randomized control trials and the immunocompromised population in real-world studies. Unfortunately, the emergence of new variants precluded their use. Sipavibart (AZD3152) is an investigational long-acting monoclonal antibody designed to provide broad coverage across Omicron and ancestral viral variants currently being studied in the SUPERNOVA prevention trial. France was the first country to grant Sipavibart as a COVID-19 pre-exposure prophylaxis treatment in immunocompromised individuals with an early access authorization in December 2023. Methods We performed a retrospective multicentric study in France, including the first consecutive patients to receive one intramuscular dose of Sipavibart in January and February 2024, to collect their characteristics and COVID-19-related medical history. Results Overall, we included 47 patients with a median age of 61 years (IQR:64—69) and 64% male. The three main immunosuppressive conditions were Stem cell transplants (38%), solid organ transplants (32%), and immune-based therapies (mostly Rituximab) (19%) for chronic inflammatory diseases or haematologic malignancies. Nearly half had hypogammaglobulinemia and 71% had at least another key comorbidity (Table 1). 15% and 11% had a history of severe and persistent COVID-19, respectively. The median number of COVID-19 doses was 4 (IQR 3-6), and nearly one-third had previously received previous generation of monoclonal antibodies for COVID-19 prophylaxis (Casirivimab/imdevimab or Tixagevimab/Cilgavimab). SARS-CoV-2 serology results before Sipavibart administration were available in 11 patients (without Immunoglobulins substitution). The median anti-Spike IgG titers was 30 UI/L (IQR 0-179). Conclusion The first patients to receive Sipavibart in France had different profiles, but they were all highly immunocompromised with frequently associated hypogammaglobulinemia and other chronic conditions. Disclosures Paul LOUBET, MD, PhD, Astrazeneca: Advisor/Consultant|Gilead: Advisor/Consultant|Moderna: Advisor/Consultant|Pfizer: Advisor/Consultant Iliès Benotmane, n/a, Astrazeneca: Advisor/Consultant|Astrazeneca: Board Member

P0326 Environmental factors, genetic predisposition and subclinical inflammation in the development of perinuclear-antineutrophil cytoplasmic antibody (P-ANCA)-positive ulcerative colitis: A European twin study

Abstract Background Perinuclear-antineutrophil cytoplasmic antibodies (P-ANCAs) have been identified in familial ulcerative colitis (UC), but the precise mechanism underlying their expression remains elusive. We assessed the role of genetic predisposition, environmental factors and systemic subclinical inflammation in the development of P-ANCA in a twin cohort with UC. Methods A total of 48 twin pairs (Leuven, Belgium n=4, Maastricht, The Netherlands n=6 and Örebro, Sweden n=38) with UC were included. Among these, 18 were monozygotic (3 concordant and 15 discordant for UC) and 30 were dizygotic (1 concordant and 29 discordant for UC). P-ANCA was detected through standardised ELISA, an indirect immunofluorescence assay and DNase treatment. In addition to high sensitivity C-reactive protein (hs-CRP), 92 inflammatory protein markers were measured in serum by proximity extension assay (Olink proteomics). Results P-ANCA was present in 16/52 (31%) of UC twins vs. 4/44 (9%) healthy twin siblings (p=0.01). No agreement in the presence of P-ANCA or their levels was observed between twin siblings in monozygotic pairs discordant for UC [intraclass correlation coefficient (ICC)=0.09] or dizygotic pairs (ICC=-0.20). Female sex was associated with an increased likelihood of P-ANCA (odds ratio, OR 5.49; 95% confidence interval, CI 1.47-20.57) and higher ANCA levels (ratio of geometric means 1.80; 95% CI 1.12-2.88). Active smoking was associated with lower concentrations of ANCA (ratio of geometric means 0.33; 95% CI 0.15-0.75) and potentially reduced the likelihood of P-ANCA (OR 0.23; 95% CI 0.02-2.21) in twins with UC but not in their healthy siblings. In twin siblings without UC, significant correlations between ANCA levels and hs-CRP, CDCP1, IL17A, CXCL9 and IL5 (correlation coefficients 0.36-0.41, p-values <0.05) were observed. Conclusion Female sex and tobacco smoking outweighed genetics regarding the generation and levels of P-ANCA and ANCA antibodies. The correlations between ANCA levels and inflammatory markers in healthy twin siblings suggest that P-ANCA may result from subclinical inflammation in these healthy siblings.

Acquisition of innate B cell properties and generation of autoreactive IgA antibodies by follicular B cells during homeostatic proliferation.

The physiologic process of homeostatic proliferation serves to restore the pool of peripheral lymphocytes in response to lymphopenia. However, functional changes in B cell responses during homeostatic proliferation are still only insufficiently characterized. Mature peripheral B cells consist of functionally distinct B cell subsets, such as adaptive follicular B cells (FoBs) and the innate B cell subsets, marginal zone B cells (MZBs) and B1a B cells. During homeostatic proliferation, B cells undergo antigen-independent clonal expansion and differentiation into antibody-producing plasma cells (PCs). However, it is still largely unknown which B cell lineages are involved in the formation of antibodies in response to lymphopenia and what functional properties these antibodies have. Employing adoptive transfer of different mature B cell subsets into lymphopenic Rag2-/- hosts, we here show that not only innate B cells - MZBs and B1a cells - but also adaptive FoBs were capable to differentiate into PCs and to produce IgM and class-switched IgA serum antibodies in a T cell-independent fashion during homeostatic proliferation. In light of the poor reactivity of FoBs to innate stimulation in vitro, the observed high expansion capacity of FoBs, their sustained repopulation of lymphoid and intestinal organs and their particularly prominent ability to induce class-switched auto-/polyreactive IgA antibodies in this antigen- and T cell-independent system was rather unexpected. These properties, which are more typical for innate B cells, were associated with a striking plasticity of FoBs that transdifferentiate into MZB-like cells under lymphopenic conditions. Together, our study indicates that the reconstitution of antibodies in response to lymphopenia-induced homeostatic B cell proliferation is mainly elicited by innate MZB-like B cell responses via antigen- and T cell-independent pathways resulting in the selection of autoreactive IgA antibodies. In addition, our data point to the pathogenic potential of the conversion of conventional adaptive B cells, which are the most common population of mature B cells, into innate-like B cells and the production of autoreactive IgA antibodies during homeostatic proliferation. This process could also manifest as clinical complication of therapy-induced lymphopenia in the context of transplantation and cancer in human patients.

Short-term oral exposure to nanoplastics does not significantly impact the antiviral immune response of the mouse.

The increasing prevalence of nanoplastics (NPs) in the environment, particularly polystyrene (PS) nanoparticles, raises concerns regarding their potential impact on human and animal health. Given their small size, NPs can cross biological barriers and accumulate in organs, including those critical for immune functions. This study investigates the effects of short-term oral exposure to 100 and 500 nm PS NPs on the adaptive immune responses during viral infections in vivo, using vesicular stomatitis virus (VSV) and lymphocytic choriomeningitis virus (LCMV) as models. Male and female C57BL/6 mice were orally exposed to PS NP for a period of 28 days, during which they were infected with either VSV or LCMV to study the humoral and cellular responses, respectively. The humoral responses were assessed by measuring total and VSV-specific antibody levels, and splenic immune populations. T cell phenotypes, activation, exhaustion and functionality towards LCMV epitopes were studied as readouts of the cellular responses. Our results demonstrate that short-term NP exposure does not significantly affect the generation or neutralizing capacity of antibodies against VSV, nor the cellular responses directed against LCMV. These findings indicate that, under these conditions, PS NP exposure does not significantly compromise the adaptive immune responses during viral infections, underscoring the value of in vivo models.

Anti-immune complex antibodies are elicited during repeated immunization with HIV Env immunogens.

Vaccination strategies against HIV-1 aim to elicit broadly neutralizing antibodies (bnAbs) using prime-boost regimens with HIV envelope (Env) immunogens. Epitope mapping has shown that early antibody responses are directed to easily accessible nonneutralizing epitopes on Env instead of bnAb epitopes. Autologously neutralizing antibody responses appear upon boosting, once immunodominant epitopes are saturated. Here, we use electron microscopy-based polyclonal epitope mapping (EMPEM) to elucidate how repeated immunization with HIV Env SOSIP immunogens results in the generation of Ab2α anti-idiotypic antibodies in rabbits and rhesus macaques. We present the structures of six anti-immune complex antibodies and find that they target idiotopes composed of framework regions of antibodies bound to Env. Examination of cryo-electron microscopy density enabled prediction of sequences for an anti-immune complex antibody, the paratope of which is enriched with aromatic amino acids. This work sheds light on current vaccine development efforts for HIV, as well as for other pathogens in which repeated exposure to antigen is required.

Profiling the broad antibody diversity of lymphatic filariasis immune antibody repertoire by deep sequencing.

Lymphatic filariasis is caused by infections of thread-like filarial worms, namely Wuchereria bancrofti, Brugia Malayi and Brugia timori. However, in-depth analysis of the antibody repertoire against Lymphatic filariasis is lacking. Using high-throughput sequencing of antibody repertoires, immunome analysis of IgG (LG) and IgM (LM) repertoires were studied. Despite significant differences between LG and LM in V(D)J gene usage, IGHV4-34, IGHV6-1, IGHD3-10 and IGHJ4 were preferred in both repertoires. The CDR3 in the LG repertoire showed a longer length than LM. Higher SHM level were observed in LG sequences and presence of oligoclonal sequences indicates the extent of clonal expansion. The prevalence of rare clonotypes in LM repertoire depicts the high clonal diversity when compared to LG repertoire. Monoclonal antibodies against closely related parasitic infections were present within the LG repertoire suggesting that immune repertoires may not be as exclusive and biased against the target infection as initially thought. The characterization of the immune repertoire can provide critical insight into the antibody response patterns in disease state, antibody generation process during infections and future antibody designs.

Generation of antagonistic biparatopic anti-CD30 antibody from an agonistic antibody by precise epitope determination and utilization of structural characteristics of CD30 molecule

Abstract Background CD30 is a member of the tumor necrosis factor receptor superfamily. Recently, blocking CD30-dependent intracellular signaling has emerged as potential strategy for immunological regulation. Development of antibody-based CD30 antagonists is therefore of significant interest. However, a key challenge is that the bivalent form of natural antibody can crosslink CD30 molecules, leading to signal transduction even in the absence of specific ligand, CD153. Biparatopic antibodies (BpAbs) offer a solution, using two different variable fragments (Fvs) to bind distinct epitopes on a single antigen molecule. BpAbs format is an attractive alternative of natural antibody by potentially avoiding unwanted crosslinking and signaling induction. Methods We systematically characterized 36 BpAbs, each designed with pairs of Fvs binding to nine distinct epitopes across the CD30 extracellular domain. We first identified the precise epitope sites of the nine antibodies by assessing the binding to multiple orthologous CD30 proteins and mutants. We then produced the 36 BpAbs and analyzed their biological activities and binding modes. Results Among 36 BpAbs, we identified both potent ligand-independent agonists and ligand-blocking antagonists, with many displayed reduced signal activation, including 1:1-binding antagonists derived from AC10, a strong agonist developed for lymphoma therapy. Epitope dependency in reduced signaling activity was observed and associated with the flexible nature of CD30 protein. Conclusions We successfully developed antagonistic BpAbs against CD30 by controlling the stoichiometry of antibody–antigen binding mode. This study elucidated the mechanism of signaling induction, informing the design strategies of the development of biparatopic antibodies. Statement of Significance Thirty-six bivalent biparatopic antibodies targeting pairs of nine distinct topographical epitopes distributed over CD30 were developed. These BpAbs exhibited a range of signaling activities from agonism to antagonism, with many displaying reduced signal activation. Notably, 1:1-binding antagonists were developed with no signal induction. The mechanism was elucidated by epitope information.

Evaluation of an F Protein-Based Recombinant Protein for Immunization Against Respiratory Syncytial Virus.

Respiratory syncytial virus (RSV) is one of the most important etiologies of acute respiratory infections that cause bronchiolitis in children under 5 years of age. Treatments are expensive, no vaccine is available, and this is an important cause of hospitalization. Costimulatory molecules have been reported to be good inducers of antiviral type 1 immune response. This study aimed to generate a recombinant vaccine with the RSV F protein fused to 4-1BBL to evaluate the activation of an antiviral response in vitro and the production of neutralizing antibodies invivo. The codon-optimized F gene was subcloned into an expression vector as follows: streptavidin core, gene F, and costimulatorytumor necrosis factor receptor superfamily member 9 -TNFRS9- ligand (4-1BBL). After the induction of expression in Escherichia coli C43, the recombinant protein (SA-F3x-4-1BBL, denominated SF4) was purified and verified by western blotting. Cultured RAW264.7 macrophages were stimulated with SF4 protein, then tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2), p38, and nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) were analyzed by western blot, and mRNA cytokines were analyzed by RT-qPCR. Finally, male C57BL/6 mice were inoculated with SF4, and the generation of anti-RSV neutralizing antibodies and serum cytokines was examined. SF4 had a size of 84.4 kDa with a 5.6% yield. SA-F-4-1BBL upregulated TRAF2, TNF-α, and interferon (IFN)-γ expression levels and activated p38 mitogen-activated protein kinase and NF-κΒ pathways in RAW264.7 cells. Importantly, antibodies capable of neutralizing RSV infection and producing type 1 cytokines were detected in the sera of immunized animals. These results suggest that the fusion protein SF4 activates the 4-1BBL signaling pathway, resulting in an effective antiviral response mediated by neutralizing antibodies and antiviral cytokines.

A comprehensive review of probiotics and human health-current prospective and applications.

The beneficial properties of probiotics have always been a point of interest. Probiotics play a major role in maintaining the health of Gastrointestinal Tract (GIT), a healthy digestive system is responsible for modulating all other functions of the body. The effectiveness of probiotics can be enhanced by formulating them with prebiotics the formulation thus formed is referred to as synbiotics. It not only improves the viability and stability of probiotic cells, but also inhibits the growth of pathogenic strains. Lactobacillus and Bifidobacterium spp. are most commonly used as probiotics. The other microbial spp. that can be used as probiotics are Bacillus, Streptococcus, Enterococcus, and Saccharomyces. Probiotics can be used for the treatment of diabetes, obesity, inflammatory, cardiovascular, respiratory, Central nervous system disease (CNS) and digestive disorders. It is also essential to encapsulate live microorganisms that promote intestinal health. Encapsulation of probiotics safeguards them against risks during production, storage, and gastrointestinal transit. Heat, pressure, and oxidation eradicate probiotics and their protective qualities. Encapsulation of probiotics prolongs their viability, facilitates regulated release, reduces processing losses, and enables application in functional food products. Probiotics as microspheres produced through spray drying or coacervation. This technique regulates the release of gut probiotics and provides stress resistance. Natural encapsulating materials including sodium alginate, calcium chloride, gel beads and polysaccharide promoting safeguards in probiotics during the digestive process. However, several methods including, spray drying where liquid is atomized within a heated air chamber to evaporate moisture and produce dry particles that improves the efficacy and stability of probiotics. Additionally, encapsulating probiotics with prebiotics or vitamins enhance their efficacy. Probiotics enhance immune system efficacy by augmenting the generation of antibodies and immunological cells. It combats illnesses and enhances immunity. Recent studies indicate that probiotics may assist in the regulation of weight and blood glucose levels and influence metabolism and insulin sensitivity. Emerging research indicates that the "gut-brain axis" connects mental and gastrointestinal health. Probiotics may alleviate anxiety and depression via influencing neurotransmitter synthesis and inflammation. Investigations are underway about the dermatological advantages of probiotics that forecasting the onsite delivery of probiotics, encapsulation is an effective technique and requires more consideration from researchers. This review focuses on the applications of probiotics, prebiotics and synbiotics in the prevention and treatment of human health.

Expression Cloning of Antibodies from Single Human B Cells.

The majority of lymphomas originate from B cells at the germinal center stage. Preferential selection of B-cell clones by a limited set of antigens has been suggested to drive lymphoma development. While recent studies in B-cell chronic lymphocytic leukemia (CLL) have shown that self-reactive B-cell receptors (BCR) can generate cell-autonomous signaling and proliferation, our knowledge about the role of BCRs for the development or survival of other lymphomas remains limited. Here, we describe a strategy to characterize the antibody reactivity of human B cells. The approach allows for unbiased characterization of the human antibody repertoire on single-cell level through the generation of recombinant monoclonal antibodies from primary human B cells of defined origin. This protocol offers a detailed description of the method starting from the flow cytometric isolation of single human B cells to the RT-PCR-based amplification of the expressed immunoglobulin (Ig) transcripts (IGH, IGK, and IGL) and their subsequent cloning into expression vectors for the in vitro production of recombinant monoclonal antibodies. The strategy may be used to obtain information about the clonal evolution of B-cell lymphomas by single-cell sequencing of Ig transcripts and on the antibody reactivity of human lymphoma B cells.

Methods for Detecting Phosphorylated Proteins and Observing Intracellular Localization During Mitosis.

This protocol describes the detection of phosphorylated proteins within cells and the identification of their intracellular localization, with a particular focus on mitotic cells. While the detection of phosphorylated proteins can be achieved using radioactive labeling with 32Pi, this method presents experimental challenges due to the requirement for radioisotopes. Alternatively, detection using phosphorylation-specific antibodies is a potent method; however, it necessitates the identification of phosphorylation sites and further requires the generation of antibodies targeting these sites, making it effective only for thoroughly analyzed phosphorylations. Here, we outline a convenient method for detecting intracellular phosphorylation using the Phos-tag technique. Additionally, we discuss the establishment of inducible stable cell lines using lentivirus, synchronization methods for cells in the mitotic phase using thymidine and nocodazole, as well as some tips for immunoprecipitation, Western blotting, and immunostaining from mitotic cells.

Safety and immunogenicity of an acellular pertussis vaccine containing genetically detoxified pertussis toxin administered to pregnant women living with and without HIV and their newborns (WoMANPOWER): a randomised controlled trial in Uganda.

Immunisation in pregnancy against pertussis can reduce severe disease in infancy. There are few data on the safety and immunogenicity of vaccines given to pregnant women living with HIV and their infants. We aimed to describe the safety and immunogenicity of a tetanus-diphtheria-acellular pertussis (TdaP) vaccine containing genetically detoxified pertussis toxin given to pregnant women living with HIV and the effect of the vaccine on infant whole-cell pertussis vaccine responses. We conducted an observer-blind, randomised, phase 2, multicentre, non-inferiority trial evaluating safety and immunogenicity of a vaccine containing genetically detoxified acellular pertussis in pregnant women living with HIV in Uganda. Women aged at least 18 years between 16 weeks and 26 weeks of gestation were randomly assigned to receive the tetanus-diphtheria (Td) vaccine or TdaP vaccine. Stratified block randomisation using blocks of four with a 1:1:1:1 ratio stratified by participant HIV status was used to distribute participants into equal groups (50 participants per group for a total of 200 participants). The intervention was a 0·5 mL single intramuscular dose of TdaP vaccine. Td or TdaP vaccination was randomly assigned to different clinic days using randomisation software. Primary immunogenicity endpoints were anti-pertussis toxin and anti-filamentous haemagglutinin IgG concentrations in infants at delivery and 18 weeks following three doses of a whole-cell pertussis containing vaccine. This study is registered at ClinicalTrials.gov, NCT04589312. Between Oct 28, 2020, and May 21, 2021, 438 pregnant women were screened and 181 were randomly assigned: 90 to TdaP vaccine (40 HIV-positive participants and 50 HIV-negative participants) and 91 to Td vaccine (41 HIV-positive participants and 50 HIV-negative participants). All participants received Td, and 4 weeks later, 177 received either Td or TdaP. 32 serious adverse events occurred, none related to the study vaccine. At delivery, anti-pertussis toxin IgG concentrations for TdaP versus Td were superior in infants who were HIV-exposed but uninfected (geometric mean ratio 9·61, 95% CI 5·21-17·74) and HIV-unexposed infants (21·6, 11·2-41·7). In infants at 18 weeks, anti-pertussis toxin IgG concentrations for TdaP versus Td-vaccinated mothers were significantly lower for both infants who were HIV-exposed but uninfected (0·19, 0·09-0·43) and infants who were not HIV-exposed (0·17, 0·08-0·33). Serum bactericidal antibody generation following whole-cell pertussis vaccination in infants was not affected. TdaP was safe and immunogenic in pregnant women living with HIV and their infants. TdaP provided superior anti-pertussis toxin IgG concentrations at delivery. Following routine vaccination with whole-cell pertussis vaccine, infants born to women receiving the TdaP vaccine had lower anti-pertussis toxin IgG concentrations than infants born to women receiving Td. In the absence of a correlate of protection against pertussis disease, the clinical significance of this finding is unclear. Medical Research Council Joint Clinical Trials, Canadian Institutes of Health Research, and British Columbia Children's Hospital Research Institute.

Overexpression and purification of YidRv gene from the hypervirulent Klebsiella pneumoniae, and the ability of the gene product in inducing a humoral response

The yidRv gene, isolated from the hypervirulent Klebsiella pneumoniae (hvKP), is a novel gene with an unknown function; however, it has exhibited high homology to the yidR, a gene recognized as potential vaccine candidate. The aim of this study was to clone the yidRv gene from the Indonesian hvKP and to investigate its overexpression in Escherichia coli. In the experiment, yidRv was cloned into pET21 to construct pYik23. Recombinant protein YidRv was produced by growing E. coli BL21 (DE3)/pYik23 in LB medium with ampicillin at 29 °C, inducing protein synthesis with 0.5 mM IPTG for 20 hours. Purification was performed using Ni‐NTA resin, and the purified protein (50 µg) was administered to BALB/c mice to test for the production of IgG, IgM and IgA on 2 days before and day 19th and 37th after the first vaccination. The results show a significant induction of IgG and IgM, but not of IgA antibodies. In conclusion, the yidRv gene was overexpressed in E. coli BL21 (DE3) at high levels in soluble form, with the recombinant protein able to be purified to 90% homogeneity. The recombinant YidRv demonstrated the ability to stimulate the generation of both IgM and IgG antibodies.

Enhanced Immunogenicity of Foot-and-Mouth Disease Virus-like Particles Using a Water-in-Oil-in-Water Adjuvant.

Foot-and-mouth disease (FMD) causes significant economic losses, prompting vaccination as a primary control strategy. Virus-like particles (VLPs) have emerged as promising candidates for FMD vaccines but require adjuvants to enhance their immunogenicity. In this study, we evaluated the immunogenicity of a VLP-based vaccine with a water-in-oil-in-water (W/O/W) emulsion adjuvant, named WT. The WT adjuvant was mixed with FMD VLPs to form the VLPs+WT vaccine. The size and stability of the vaccine were analyzed. BALB/c mice were immunized with the VLPs+WT vaccine, and immunological responses were assessed through antibody measurements, cytokine profiling, and gene expression analysis. In addition, splenic lymphocyte proliferation and signaling pathways were examined. The VLPs+WT vaccine exhibited a homogeneous size of 324.60 ± 2.30 nm and a viscosity of 8.76 mPa·s, indicating good stability. Immunized mice showed steady weight gain and no organ abnormalities. Compared to the VLPs group, the VLPs+WT group induced significantly higher levels of specific antibodies that persisted for 12 weeks, similar to the commercial VLPs+ISA201 vaccine. The VLPs+WT vaccine also enhanced the secretion of Th1-related (IgG2a, IFN-γ) and Th2-related (IgG1, IL-4) molecules. WT stimulated splenic lymphocyte proliferation and differentiation, primarily activating B-cell receptor signaling and phagosome pathways. It also upregulated genes associated with MHC and interferon stimulation while promoting the expression of MyD88, PI3K, AKT, p65, and p-p65 proteins. These findings suggest that WT is an effective adjuvant for FMD VLP-based vaccines, with potential for improving vaccine efficacy.

Binding mode–guided development of high-performance antibodies targeting site-specific posttranslational modifications

Posttranslational modifications (PTMs) of proteins play critical roles in regulating many cellular events. Antibodies targeting site-specific PTMs are essential tools for detecting and enriching PTMs at sites of interest. However, fundamental difficulties in molecular recognition of both PTM and surrounding peptide sequence have hindered the efficient generation of highly sequence-specific anti-PTM antibodies. Furthermore, the widespread use of potentially inconsistent, nonrenewable, and molecularly undefined antibodies presents experimental challenges thought to contribute to the reproducibility problem in biomedical research. In this study, we describe the binding mode-guided development of a platform that efficiently generates potent and selective recombinant antibodies to PTMs that are molecularly defined and renewable. Our platform is built on our previous discovery of an unconventional binding mode of anti-PTM antibodies, antigen clasping, where two antigen binding sites cooperatively sandwich a single antigen, creating extensive interactions with the antigen and leading to high selectivity and potency. We designed the platform that generates clasping antibodies with two distinct binding units, resulting in efficient generation of antibodies to a set of trimethylated histone H3 with high levels of specificity and affinity. Performance comparison in chromatin immunoprecipitation, a common application in epigenomics, revealed that a clasping antibody to trimethylated histone H3 at lysine 27 exhibited superior specificity to a widely used conventional antibody and captured symmetric and asymmetric nucleosomes in a less biased manner. We further generated clasping antibodies to phosphotyrosine antigens by using the same principle. These results suggest the broad applicability of our platform to generating high-performance clasping antibodies to diverse PTMs.

Single-chain antibody gene therapy strategy based on high-throughput screening triggers sustained antiviral activity in the body.

The occurrence of viral diseases poses a huge threat and impact on human public health safety and the development of the animal and fishery industry. Here, a strain of single-chain antibody fragment, scFv-1, was isolated from the phage antibody display library construct by immunizing New Zealand white rabbits with rhabdovirus. In vitro analysis showed that the single-chain antibody could inhibit the infection of the virus in multiple pathways, including adsorption, fusion, and release. In vivo analysis revealed scFv-1 had a preventive and protective effect against the infection of virus. In addition, we describe that transposon-based transport of neutralizing genes allows for long-term, continuous expression, avoiding the need for lifelong, repeated passive immunization for treatment. In sum, high-throughput screening of neutralization genes based on phage display technology and transposon vector-based gene transfer provides effective methods for treating and preventing diseases and avoiding repetitive passive immunotherapy. This study also provides a reference for the prevention and treatment of unknown pathogens.IMPORTANCELivestock and fisheries play an important role in economic development and food security. The frequent outbreaks of viral diseases have caused great losses to the livestock industry, while the increase in drug resistance caused by the use of antibiotics as well as the potential risks to human health have raised serious concerns. Here, we constructed a phage display antibody library by immunizing New Zealand white rabbits with purified rhabdovirus and selected a single-chain antibody, scFv-1, with good neutralizing activity, which was validated and found to be able to block multiple phases of the virus and thus play a neutralizing role. In addition, we describe that transposon-based transport of neutralizing genes allows for long-term, continuous expression, reducing the need for lifelong, repeated passive immunization for treatment. Our work not only provides methods for the prevention and treatment of viral diseases but also provides the body with long-lasting and even permanent protection against repeated passive immunization.