An integrated technology for quantitative wide mutational scanning of human antibody Fab libraries.

Antibodies are engineerable quantities in medicine. Learning antibody molecular recognition would enable the in silico design of high affinity binders against nearly any proteinaceous surface. Yet, publicly available experiment antibody sequence-binding datasets may not contain the mutagenic, antigenic, or antibody sequence diversity necessary for deep learning approaches to capture molecular recognition. In part, this is because limited experimental platforms exist for assessing quantitative and simultaneous sequence-function relationships for multiple antibodies. Here we present MAGMA-seq, an integrated technology that combines multiple antigens and multiple antibodies and determines quantitative biophysical parameters using deep sequencing. We demonstrate MAGMA-seq on two pooled libraries comprising mutants of nine different human antibodies spanning light chain gene usage, CDR H3 length, and antigenic targets. We demonstrate the comprehensive mapping of potential antibody development pathways, sequence-binding relationships for multiple antibodies simultaneously, and identification of paratope sequence determinants for binding recognition for broadly neutralizing antibodies (bnAbs). MAGMA-seq enables rapid and scalable antibody engineering of multiple lead candidates because it can measure binding for mutants of many given parental antibodies in a single experiment.

Amplifying mRNA vaccines: potential versatile magicians for oncotherapy.

Cancer vaccines drive the activation and proliferation of tumor-reactive immune cells, thereby eliciting tumor-specific immunity that kills tumor cells. Accordingly, they possess immense potential in cancer treatment. However, such vaccines are also faced with challenges related to their design and considerable differences among individual tumors. The success of messenger RNA (mRNA) vaccines against coronavirus disease 2019 has prompted the application of mRNA vaccine technology platforms to the field of oncotherapy. These platforms include linear, circular, and amplifying mRNA vaccines. In particular, amplifying mRNA vaccines are characterized by high-level and prolonged antigen gene expression at low doses. They can also stimulate specific cellular immunity, making them highly promising in cancer vaccine research. In this review, we summarize the research progress in amplifying mRNA vaccines and provide an outlook of their prospects and future directions in oncotherapy.

Separate optogenetic manipulation of Nerve/glial antigen 2 (NG2) glia and mural cells using the NG2 promoter.

Nerve/glial antigen 2 (NG2) is a protein marker of NG2 glia and mural cells, and NG2 promoter activity is utilized to target these cells. However, the NG2 promoter cannot target NG2 glia and mural cells separately. This has been an obstacle for NG2 glia-specific manipulation. Here, we developed transgenic mice in which either cell type can be targeted using the NG2 promoter. We selected a tetracycline-controllable gene induction system for cell type-specific transgene expression, and generated NG2-tetracycline transactivator (tTA) transgenic lines. We crossed tTA lines with the tetO-ChR2 (channelrhodopsin-2)-EYFP line to characterize tTA-dependent transgene induction. We isolated two unique NG2-tTA mouse lines: one that induced ChR2-EYFP only in mural cells, likely due to the chromosomal position effect of NG2-tTA insertion, and the other that induced it in both cell types. We then applied a Cre-mediated set-subtraction strategy to the latter case and eliminated ChR2-EYFP from mural cells, resulting in NG2 glia-specific transgene induction. We further demonstrated that tTA-dependent ChR2 expression could manipulate cell function. Optogenetic mural cell activation decreased cerebral blood flow, as previously reported, indicating that tTA-mediated ChR2 expression was sufficient to impact cellular function. ChR2-mediated depolarization was observed in NG2 glia in acute hippocampal slices. In addition, ChR2-mediated depolarization of NG2 glia inhibited their proliferation but promoted their differentiation in juvenile mice. Since the tTA-tetO combination is expandable, the mural cell-specific NG2-tTA line and the NG2 glia-specific NG2-tTA line will permit us to conduct observational and manipulation studies to examine in vivo function of these cells separately.

Single-cell variations in the expression of codominant alleles A and B on RBC of AB blood group individuals.

A key question in biology is whether all cells of a given 'cell type' within an individual have more or less the same phenotype, especially in relation to nonimprinted autosomal loci. Some studies have shown differential allelic expression of autosomal genes to confer phenotypic variability at the individual cell level. Here, we report the amount of A and B histo-blood group antigens, products of classic examples of codominant alleles, in individual red blood cells (RBCs). Using immunofluorescence with Cy3-tagged and FITC-tagged antibodies, we quantified the levels of these antigens in 2512 RBCs from 24 individuals in the AB blood group. When these data were fit to a normal distribution, we could detect four groups: showing normal distribution for both antigens, either antigen, and neither antigen. Surprisingly, very few samples showed a significant positive correlation between the amounts of A and B antigens on individual RBC; in fact, the ratio of antigen A to antigen B in the entire set of samples spanned over five orders of magnitude. This variability in the amount of antigens A and/or B, combined with a lack of correlation between the amounts of these two antigens, resulted in unique staining patterns of RBC, generating widespread mosaicism in the RBC population of AB blood group individuals.

Physical and chemical advances of synthetic delivery vehicles to enhance mRNA vaccine efficacy.

The successful translation of mRNA vaccines slows down the spread of viral infectious diseases, which may be accomplished by developing novel chemically modified nucleotides (or nucleosides) and highly efficient, safe mRNA delivery vehicles. Delivery vehicles protect vulnerable antigen mRNA and increase the uptake of mRNA into antigen-presenting cells in the peripheral tissue or lymph nodes. This review introduces essential characteristics of mRNA vaccines (e.g., particle sizes, colloidal stability, surface charges/endosomal escape ability, and ligand conjugation) that may be used to generate high immune responses against foreign antigens. The significance and mechanism of each characteristic are described based on the results obtained from in vitro and in vivo studies. We also discuss the development of next generation delivery vehicles for future mRNA vaccines.

Easily Established and Multifunctional Synthetic Nanobody Libraries as Research Tools.

Nanobodies, or VHHs, refer to the antigen-binding domain of heavy-chain antibodies (HCAbs) from camelids. They have been widely used as research tools for protein purification and structure determination due to their small size, high specificity, and high stability, overcoming limitations with conventional antibody fragments. However, animal immunization and subsequent retrieval of antigen-specific nanobodies are expensive and complicated. Construction of synthetic nanobody libraries using DNA oligonucleotides is a cost-effective alternative for immunization libraries and shows great potential in identifying antigen-specific or even conformation-specific nanobodies. This review summarizes and analyses synthetic nanobody libraries in the current literature, including library design and biopanning methods, and further discusses applications of antigen-specific nanobodies obtained from synthetic libraries to research.

Lack of evidence of significant homology of SARS-CoV-2 spike sequences to myocarditis-associated antigens.

BACKGROUND: COVID-19 mRNA vaccines have proven to be highly safe and effective. Myocarditis is an adverse event associated with mRNA vaccination, especially in young male subjects. These events are rare and, in the majority of cases, resolve quickly. As myocarditis can be driven by autoimmune responses, we wanted to determine if the SARS-CoV-2 spike protein antigen encoded in the mRNA COVID vaccines had potential cross-reactivity with auto-antigens previously associated with myocarditis. METHODS: We performed a sequence identity comparison between SARS-CoV-2 spike protein-derived peptides and myocarditis-associated antigens. We also performed a structural analysis of these antigens and the SARS-CoV-2 spike protein to identify potential discontinuous 3-D epitope similarities. FINDINGS: We found no significant enrichment in the frequency of spike-derived peptides similar to myocarditis-associated antigens as compared to several controls. INTERPRETATION: Our results do not support the notion that increased occurrence of myocarditis after SARS-CoV-2-spike vaccination is mediated by a cross-reactive adaptive immune response.

Screening for the "Achilles Heel" of Hyalomma anatolicum Ticks by RNA Interference Technology and an Update on Anti-Tick Vaccine Design.

Over the years, RNA interference (RNAi) has evolved as a valuable tool to study the tick gene function, screening and preliminary characterization of tick-protective antigens in a relatively short time, with a minimal use of laboratory animals before conducting expensive vaccine trials for the development of improved vaccine composition. In this process, a double-stranded RNA (dsRNA) of gene of interest is introduced into the tick system which specifically suppresses expression of a target gene. The results of RNAi-based gene silencing were interpreted by reduction in targeted gene transcript, changes in phenotypic data and anatomical/ biochemical changes in ticks; thereby, providing a clue to the probable role played by the gene in the tick biological system. Across the globe, various tick research groups applied RNAi technique for characterization and identification of new anti-tick vaccine targets. Herein, we used the RNAi tool in Hyalomma anatolicum ticks for identification and characterization of vaccine candidates.

Sperm Cell Capture Based on ABH Antigen Differences to Separate Two Men in Mixed Seminal Stains.

Personal identification of two individuals in mixed semen samples in forensic DNA testing in general usually involves analysis using autosomal and Y chromosome short tandem repeats (STRs). Results may exclude unrelated donors but cannot identify individuals. In this study, sperm cell capture based on ABH antigen differences was used to obtain the cells with the single ABO blood type. Immunohistochemical staining using labeled anti-A, anti-B, and anti-H antibodies and the laser microdissection system can be used to enrich sperm with different ABO types in mixed seminal stains from two individuals. Then, PCR amplification and capillary electrophoresis were performed to genotype the STR loci. To some extent, after sperm cell capture based on ABH antigen differences, autosomal STR typing using enriched single blood group cells can be utilized to partially identify different individuals in a mixed seminal stain sample from two individuals.

Protein phosphatase 2A inactivation induces microsatellite instability, neoantigen production and immune response.

Microsatellite-instable (MSI), a predictive biomarker for immune checkpoint blockade (ICB) response, is caused by mismatch repair deficiency (MMRd) that occurs through genetic or epigenetic silencing of MMR genes. Here, we report a mechanism of MMRd and demonstrate that protein phosphatase 2A (PP2A) deletion or inactivation converts cold microsatellite-stable (MSS) into MSI tumours through two orthogonal pathways: (i) by increasing retinoblastoma protein phosphorylation that leads to E2F and DNMT3A/3B expression with subsequent DNA methylation, and (ii) by increasing histone deacetylase (HDAC)2 phosphorylation that subsequently decreases H3K9ac levels and histone acetylation, which induces epigenetic silencing of MLH1. In mouse models of MSS and MSI colorectal cancers, triple-negative breast cancer and pancreatic cancer, PP2A inhibition triggers neoantigen production, cytotoxic T cell infiltration and ICB sensitization. Human cancer cell lines and tissue array effectively confirm these signaling pathways. These data indicate the dual involvement of PP2A inactivation in silencing MLH1 and inducing MSI.

Engineered Attenuated Salmonella typhimurium Expressing Neoantigen Has Anticancer Effects.

Neoantigen vaccines are an immunotherapy strategy for treating cancer. The vaccine degrades quickly, so the strategy must include protection and precise targeting for immune cell stimulation. In this study, we engineered attenuated Salmonella typhimurium, which is highly infiltrative to tumors, to act as a carrier for Neoantigen peptide vaccine. Our system used a constitutive promoter vector, so that a single injection of Salmonella expressing Neoantigen could be used without requiring additional induction injections. In vivo experiments on bacteria-treated mice showed that Neoantigen expressed by the engineered carrier infiltrated tumors and resulted in suppressed tumor growth, higher survival rates and longer survival times, a relative increase of CD4 and CD8 T cells, and cytokine release. These results indicate that engineered Salmonella can be used as a carrier for Neoantigen immunotherapy.

Protein-coding repeat polymorphisms strongly shape diverse human phenotypes.

Many human proteins contain domains that vary in size or copy number because of variable numbers of tandem repeats (VNTRs) in protein-coding exons. However, the relationships of VNTRs to most phenotypes are unknown because of difficulties in measuring such repetitive elements. We developed methods to estimate VNTR lengths from whole-exome sequencing data and impute VNTR alleles into single-nucleotide polymorphism haplotypes. Analyzing 118 protein-altering VNTRs in 415,280 UK Biobank participants for association with 786 phenotypes identified some of the strongest associations of common variants with human phenotypes, including height, hair morphology, and biomarkers of health. Accounting for large-effect VNTRs further enabled fine-mapping of associations to many more protein-coding mutations in the same genes. These results point to cryptic effects of highly polymorphic common structural variants that have eluded molecular analyses to date.

Identification of anti-horn fly vaccine antigen candidates using a reverse vaccinology approach.

BACKGROUND: The horn fly, Haematobia irritans irritans, causes significant production losses to the cattle industry. Horn fly control relies on insecticides; however, alternative control methods such as vaccines are needed due to the fly's capacity to quickly develop resistance to insecticides, and the pressure for eco-friendly options. METHODS: We used a reverse vaccinology approach comprising three vaccine prediction and 11 annotation tools to evaluate and rank 79,542 translated open reading frames (ORFs) from the horn fly's transcriptome, and selected 10 transcript ORFs as vaccine candidates for expression in Pichia pastoris. The expression of the 10 selected transcripts and the proteins that they encoded were investigated in adult flies by reverse transcription polymerase chain reaction (RT-PCR) and mass spectrometry, respectively. Then, we evaluated the immunogenicity of a vaccine candidate in an immunization trial and the antigen's effects on horn fly mortality and fecundity in an in vitro feeding assay. RESULTS: Six of the ten vaccine candidate antigens were successfully expressed in P. pastoris. RT-PCR confirmed the expression of all six ORFs in adult fly RNA. One of the vaccine candidate antigens, BI-HS009, was expressed in sufficient quantity for immunogenicity and efficacy trials. The IgG titers of animals vaccinated with BI-HS009 plus adjuvant were significantly higher than those of animals vaccinated with buffer plus adjuvant only from days 42 to 112, with a peak on day 56. Progeny of horn flies feeding upon blood from animals vaccinated with BI-HS009 plus adjuvant collected on day 56 had 63% lower pupariation rate and 57% lower adult emergence than the control group (ANOVA: F (1, 6) = 8.221, P = 0.028 and F (1, 6) = 8.299, P = 0.028, respectively). CONCLUSIONS: The reverse vaccinology approach streamlined the discovery process by prioritizing possible vaccine antigen candidates. Through a thoughtful process of selection and in vivo and in vitro evaluations, we were able to identify a promising antigen for an anti-horn fly vaccine.

Coding variants in the PCNT and CEP295 genes contribute to breast cancer risk in Chinese women.

BACKGROUND: Centrioles play pivotal roles in the assembly of centrosomes, their dysfunction is associated with multiple inherited diseases or cancers. To date, few studies have focused on the associations between coding single nucleotide polymorphisms (SNPs) in the centriole duplication cycle genes and the risk of breast cancer in Chinese women. METHODS: Twenty-one SNPs were selected from the coding regions of 10 critical centriole genes. The associations between the selected SNPs and breast cancer susceptibility were assessed in a case-control study of Chinese women, which included 1032 cases and 1063 controls. Potential biological functions in the influence of protein stability and the profile of expression quantitative trait loci (eQTL) of the identified SNPs were further evaluated using in silico databases. RESULTS: Multivariate logistic regression analyses revealed that a missense SNP rs7279204 in PCNT was significantly associated with an increased risk of breast cancer (additive model: adjusted OR=1.19, 95% CI: 1.02-1.38), while a missense SNP rs77922978 in CEP295 was significantly associated with a decreased risk of breast cancer (additive model: adjusted OR=0.74, 95% CI: 0.56-0.97). Stratification analyses suggested that rs7279204 and rs77922978 exhibited different effects among later first live birth, ER-negative and PR-negative women (P<0.05). Moreover, rs77922978 showed significant differences for ER and PR status strata (heterogeneity test P=0.028, P=0.046). In addition, bioinformatic analyses indicated that the two variants may possess potential functions of reducing the protein stability of their host genes. Further eQTL analysis showed that the rs7279204 was not only correlated with the expression of its host gene PCNT, but also correlated with the expression of its nearby genes, implying its potential roles in regulation of some cancer susceptibility genes. CONCLUSIONS: The SNPs rs7279204 and rs77922978 within the coding region of the PCNT and CEP295 genes may contribute to the susceptibility of breast cancer in Han Chinese population.

Thyroid hormone deficiency during zebrafish development impairs central nervous system myelination.

Thyroid hormones are messengers that bind to specific nuclear receptors and regulate a wide range of physiological processes in the early stages of vertebrate embryonic development, including neurodevelopment and myelogenesis. We here tested the effects of reduced T3 availability upon the myelination process by treating zebrafish embryos with low concentrations of iopanoic acid (IOP) to block T4 to T3 conversion. Black Gold II staining showed that T3 deficiency reduced the myelin density in the forebrain, midbrain, hindbrain and the spinal cord at 3 and 7 dpf. These observations were confirmed in 3 dpf mbp:egfp transgenic zebrafish, showing that the administration of IOP reduced the fluorescent signal in the brain. T3 rescue treatment restored brain myelination and reversed the changes in myelin-related gene expression induced by IOP exposure. NG2 immunostaining revealed that T3 deficiency reduced the amount of oligodendrocyte precursor cells in 3 dpf IOP-treated larvae. Altogether, the present results show that inhibition of T4 to T3 conversion results in hypomyelination, suggesting that THs are part of the key signaling molecules that control the timing of oligodendrocyte differentiation and myelin synthesis from very early stages of brain development.

Accurate determination of epitope for antibodies with unknown 3D structures.

MAbTope is a docking-based method for the determination of epitopes. It has been used to successfully determine the epitopes of antibodies with known 3D structures. However, during the antibody discovery process, this structural information is rarely available. Although we already have evidence that homology models of antibodies could be used instead of their 3D structure, the choice of the template, the methodology for homology modeling and the resulting performance still have to be clarified. Here, we show that MAbTope has the same performance when working with homology models of the antibodies as compared to crystallographic structures. Moreover, we show that even low-quality models can be used. We applied MAbTope to determine the epitope of dupilumab, an anti- interleukin 4 receptor alpha subunit therapeutic antibody of unknown 3D structure, that we validated experimentally. Finally, we show how the MAbTope-determined epitopes for a series of antibodies targeting the same protein can be used to predict competitions, and demonstrate the accuracy with an experimentally validated example.3D: three-dimensionalRMSD: root mean square deviationCDR: complementary-determining regionCPU: central processing unitsVH: heavy chain variable regionVL: light chain variable regionscFv: single-chain variable fragmentsVHH: single-chain antibody variable regionIL4Rα: Interleukin 4 receptor alpha chainSPR: surface plasmon resonancePDB: protein data bankHEK293: Human embryonic kidney 293 cellsEDTA: Ethylenediaminetetraacetic acidFBS: Fetal bovine serumANOVA: Analysis of varianceEGFR: Epidermal growth factor receptorPE: PhycoerythrinAPC: AllophycocyaninFSC: forward scatterSSC: side scatterWT: wild typeKeywords: MAbTope, Epitope Mapping, Molecular docking, Antibody modeling, Antibody-antigen docking.

An in vivo selection-derived d-peptide for engineering erythrocyte-binding antigens that promote immune tolerance.

When displayed on erythrocytes, peptides and proteins can drive antigen-specific immune tolerance. Here, we investigated a straightforward approach based on erythrocyte binding to promote antigen-specific tolerance to both peptides and proteins. We first identified a robust erythrocyte-binding ligand. A pool of one million fully d-chiral peptides was injected into mice, blood cells were isolated, and ligands enriched on these cells were identified using nano-liquid chromatography-tandem mass spectrometry. One round of selection yielded a murine erythrocyte-binding ligand with an 80 nM apparent dissociation constant, Kd We modified an 83-kDa bacterial protein and a peptide antigen derived from ovalbumin (OVA) with the identified erythrocyte-binding ligand. An administration of the engineered bacterial protein led to decreased protein-specific antibodies in mice. Similarly, mice given the engineered OVA-derived peptide had decreased inflammatory anti-OVA CD8+ T cell responses. These findings suggest that our tolerance-induction strategy is applicable to both peptide and protein antigens and that our in vivo selection strategy can be used for de novo discovery of robust erythrocyte-binding ligands.

Lipofection with Synthetic mRNA as a Simple Method for T-Cell Immunomonitoring.

The quantification of T-cell immune responses is crucial for the monitoring of natural and treatment-induced immunity, as well as for the validation of new immunotherapeutic approaches. The present study presents a simple method based on lipofection of synthetic mRNA in mononuclear cells as a method to determine in vitro T-cell responses. We compared several commercially available transfection reagents for their potential to transfect mRNA into human peripheral blood mononuclear cells and murine splenocytes. We also investigated the impact of RNA modifications in improving this method. Our results demonstrate that antigen-specific T-cell immunomonitoring can be easily and quickly performed by simple lipofection of antigen-coding mRNA in complex immune cell populations. Thus, our work discloses a convenient solution for the in vitro monitoring of natural or therapy-induced T-cell immune responses.

Triple deletion of TP53, PCNT, and CEP215 promotes centriole amplification in the M phase.

Supernumerary centrioles are frequently observed in diverse types of cancer cells. In this study, we investigated the mechanism underlying the generation of supernumerary centrioles during the M phase. We generated the TP53;PCNT;CEP215 triple knockout (KO) cells and determined the configurations of the centriole during the cell cycle. The triple KO cells exhibited a precocious separation of centrioles and unscheduled centriole assembly in the M phase. Supernumerary centrioles in the triple KO cells were present throughout the cell cycle; however, among all the centrioles, only two maintained an intact composition, including CEP135, CEP192, CEP295 and CEP152. Intact centrioles were formed during the S phase and the rest of the centrioles may be generated during the M phase. M-phase-assembled centrioles lacked the ability to organize microtubules in the interphase; however, a fraction of them may acquire pericentriolar material to organize microtubules during the M phase. Taken together, our work reveals the heterogeneity of the supernumerary centrioles in the triple KO cells. .