Knob Domain Research Articles

The Knob Domain of the Fiber-1 Protein Affects the Replication of Fowl Adenovirus Serotype 4

Fowl adenovirus serotype 4 (FAdV-4) outbreaks have caused significant economic losses in the Chinese poultry industry since 2015. The relationships among viral structural proteins in infected hosts are relatively unknown. To explore the role of different parts of the fiber-1 protein in FAdV-4-infected hosts, we truncated fiber-1 into fiber-1-Δ1 (73–205 aa) and fiber-1-Δ2 (211–412 aa), constructed pEF1α-HA-fiber-1-Δ1 and pEF1α-HA-fiber-1-Δ2 and then transfected them into leghorn male hepatocyte (LMH) cells. After FAdV-4 infection, the roles of fiber-1-Δ1 and fiber-1-Δ2 in the replication of FAdV-4 were investigated, and transcriptome sequencing was performed. The results showed that the fiber-1-Δ1 and fiber-1-Δ2 proteins were the shaft and knob domains, respectively, of fiber-1, with molecular weights of 21.4 kDa and 29.6 kDa, respectively. The fiber-1-Δ1 and fiber-1-Δ2 proteins were mainly localized in the cytoplasm of LMH cells. Fiber-1-Δ2 has a greater ability to inhibit FAdV-4 replication than fiber-1-Δ1, and 933 differentially expressed genes (DEGs) were detected between the fiber-1-Δ1 and fiber-1-Δ2 groups. Functional analysis revealed these DEGs in a variety of biological functions and pathways, such as the phosphoinositide 3-kinase–protein kinase b (PI3K–Akt) signaling pathway, the mitogen-activated protein kinase (MAPK) signaling pathway, cytokine–cytokine receptor interactions, Toll-like receptors (TLRs), the Janus tyrosine kinase–signal transducer and activator of transcription (Jak–STAT) signaling pathway, the nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) signaling pathway, and other innate immune pathways. The mRNA expression levels of type I interferons (IFN-α and INF-β) and proinflammatory cytokines (IL-1β, IL-6 and IL-8) were significantly increased in cells overexpressing the fiber-1-Δ2 protein. These results demonstrate the role of the knob domain of the fiber-1 (fiber-1-Δ2) protein in FAdV-4 infection and provide a theoretical basis for analyzing the function of the fiber-1 protein of FAdV-4.

Precise location of three novel linear epitopes using the generated monoclonal antibodies against the Knob domain of FAdV-4 surface structural protein, fiber1.

Fowl adenovirus serotype 4 (FAdV-4) is the main pathogen of hepatitis-hydropericardium syndrome (HHS), which brings huge economic losses to the poultry industry worldwide. Fiber-1 protein plays an important role in viral infection and pathogenesis by binding directly to cellular receptors of FAdV-4. In particular, the knob domain of fiber-1 protein has been reported to induce the production of neutralizing antibodies and arouse protection against the lethal challenge of chickens with FAdV-4. The fiber-1 knob (F1K) protein was expressed in a prokaryotic expression system and purified using Ni-NTA affinity chromatography. Monoclonal antibodies (mAbs) against FAdV-4 were generated by immunizing BALB/c mice with the purified F1K protein and screened using a series of immunoassays. Potential B cell epitopes on the knob domain of fiber-1 protein were mapped using enzyme-linked immunosorbent assay (ELISA) and dot-blot. Precious location and crucial amino acids of the identified epitopes were determined using peptide array scanning, truncations and alanine-scanning mutagenesis. The epitopes were analyzed and visualized on the knob trimer of FAdV-4 fiber-1 protein using the PyMOL software. Water-soluble recombinant fiber-1 knob (F1K) protein was obtained with the assistance of chaperone. Four monoclonal antibodies (5C10, 6F8, 8D8, and 8E8) against FAdV-4 were generated and characterized using indirect ELISA, Western blot, dot-blot, and immunological fluorescence assay (IFA). The mAbs were demonstrated to be from different hybridoma cell lines based on the sequences of the variable regions. Meanwhile, three distinct novel linear B-cell epitopes (319SDVGYLGLPPH329, 328PHTRDNWYV336, and 407VTTGPIPFSYQ417) on the knob domain of fiber-1 protein were identified and the key amino acid residues in the epitopes were determined. Structural analysis showed that the two adjacent epitopes 319SDVGYLGLPPH329 and 328PHTRDNWYV336 were exposed on the surface of the fiber-1 knob trimer, whereas the epitope 407VTTGPIPFSYQ417 was located inside of the spatial structure. This was the first identification of B-cell epitopes on the knob domain of fiber-1 protein and these findings provided a sound basis for the development of subunit vaccines, therapeutics, and diagnostic methods to control FAdV infections.

A novel subunit vaccine based on Fiber1/2 knob domain provides full protection against fowl adenovirus serotype 4 and induces stronger immune responses than a Fiber2 subunit vaccine

Outbreaks of hepatitis-hydropericardium syndrome (HHS) caused by fowl adenovirus serotype 4 (FAdV-4) have resulted in huge economic losses to the poultry industry in China since 2015. However, commercially available vaccines against the FAdV-4 infection remain scarce. In our study, subunit vaccine candidates derived from the bacterially expressed recombinant Fiber1 knob domain and Fiber2 knob domain fusion protein (termed as Fiber1/2 knob subunit vaccine) and Fiber2 protein (termed as Fiber2 subunit vaccine) of the FAdV-4 SDSX strain were developed. Immunogenicity evaluation showed that the Fiber1/2 knob subunit vaccine induced the production of antibodies at 7 d postvaccination (dpv), earlier than the Fiber2 subunit vaccine. Moreover, the neutralizing antibody level of the Fiber1/2 subunit vaccine group was higher than the Fiber2 subunit vaccine group, showing significant differences at 14, 21, and 28 dpv. Immune protection test results revealed that both Fiber1/2 knob subunit and Fiber2 subunit vaccines could protect chickens from death against FAdV-4 challenge, although the weight of chickens in the Fiber1/2 knob subunit vaccine group decreased less. Furthermore, analysis of plasma Glutamic oxaloacetic transaminase (AST) and blood glutamic pyruvic transaminase (ALT) levels suggested that the Fiber1/2 subunit vaccine can significantly inhibit liver damage caused by FAdV-4 infection and is more effective in blocking the pathogenicity of FAdV-4 in target organs. In addition, the Fiber1/2 knob subunit vaccine further reduced the viral load in different tissues and virus shedding in chickens than the Fiber2 subunit vaccine. Overall, the Fiber1/2 knob subunit vaccine was more effective than the Fiber2 subunit vaccine. These findings lay the foundation for the development of more effective FAdV-4 subunit vaccines.

Modular design of bi- and multi-specific knob domain fusions.

The therapeutic potential of bispecific antibodies is becoming widely recognised, with over a hundred formats already described. For many applications, enhanced tissue penetration is sought, so bispecifics with low molecular weight may offer a route to enhanced potency. Here we report the design of bi- and tri-specific antibody-based constructs with molecular weights as low as 14.5 and 22 kDa respectively. Autonomous bovine ultra-long CDR H3 (knob domain peptide) modules have been engineered with artificial coiled-coil stalks derived from Sin Nombre orthohantavirus nucleocapsid protein and human Beclin-1, and joined in series to produce bi- and tri-specific antibody-based constructs with exceptionally low molecular weights. Knob domain peptides with coiled-coil stalks retain high, independent antigen binding affinity, exhibit exceptional levels of thermal stability, and can be readily joined head-to-tail yielding the smallest described multi-specific antibody format. The resulting constructs are able to bind simultaneously to all their targets with no interference. Compared to existing bispecific formats, the reduced molecular weight of the knob domain fusions may enable enhanced tissue penetration and facilitate binding to cryptic epitopes that are inaccessible to conventional antibodies. Furthermore, they can be easily produced at high yield as recombinant products and are free from the heavy-light chain mispairing issue. Taken together, our approach offers an efficient route to modular construction of minimalistic bi- and multi-specifics, thereby further broadening the therapeutic scope for knob domain peptides.

Abstract 5811: Ultralong CDR3 engineered interleukin-15 antibody fusion for T and NK cell expansion

Abstract Interleukin (IL)-15 is a promising immunotherapeutic cytokine for cancer treatment because it can stimulate the proliferation and cytotoxicity of CD8+ T lymphocytes and nature killer (NK) cells, similar to the action of IL-2, which has been used to treat metastatic melanoma and renal cancer. IL-15 may have improved pharmacologic properties over IL-2 because it doesn’t cause vascular leak syndrome or stimulate regulatory T cells. However, IL-15 is difficult to express as a stable soluble protein and has a short half-life in vitro and in vivo. To solve the problem, we designed an IL-15 ultralong CDR3 humanized bovine antibody fusion. Bovine ultralong CDR3 antibodies have a unique “stalk and knob” structure in which two antiparallel β-strands support a disulfide bonded knob protruding out of the antibody surface and forms a mini antigen binding domain. By replacing the knob domain with IL-15, we have created a chimeric CDR3-IL-15 IgG which functions as IL-15 in vitro and in vivo but can be easily produced in mammalian cells with increased stability. Thus, the CDR3 can stabilize IL-15 in the absence of its high affinity receptor α (Rα) chain. Furthermore, as IL-15 utilizes Rα for increased trans signaling to the receptor β and γ subunits, one variant candidate (CDR3-IL-15_Rα) was produced by co-expressing Rα sushi domain with the chimeric IgG. Both constructs have been demonstrated to be highly active in stimulating proliferation of NK cells and CD8+ T cells in vitro as well as in stimulating proliferation of NK cells and CD8+ T cells in rats and non-human primates (NHP). Although the CDR3-IL-15_Rα exhibited higher in vitro signaling activity than CDR3-IL-15, both were equally potent in stimulating the proliferation of NK cells and CD4+ and CD8+ T cells in NHP without any immunogenicity detected. Moreover, in vivo studies in NHP indicated that CDR3-IL-15 acted faster than CDR3-IL-15_Rα in stimulating the proliferation of these lymphocytes. CDR3-IL-15 is the first thermodynamically stabilized variant of IL-15 produced in the absence of its Rα chain, is highly active in vivo, and has multiple potential cancer applications as an T and NK cell stimulating agent. Citation Format: Ruiqi Huang, Duncan McGregor, Vaughn Smider. Ultralong CDR3 engineered interleukin-15 antibody fusion for T and NK cell expansion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5811.

Abstract 1860: The smallest antibody class: Cow ultralong CDR3 knobs as a new class of biomolecule for therapeutics and diagnostics

Abstract Engineered antibodies, including bispecifics and ADCs, are now a major drug class for oncology and other therapeutic areas. There are two fundamental antibody fragments: the Fv (heavy and light chain variable region fragments, derived from most species), and VHH (heavy chain ‘only’ V regions, derived from camelids and sharks). Cows produce antibodies with an unusually long VH CDR3 region, which can be up to seventy amino acids in length and is comprised of a β-ribbon “stalk” and disulfide-bonded “knob” minidomain which sits atop the stalk. The knob region is highly diverse and can bind cryptic epitopes with high affinity. We recently developed the technology to produce knob domains independently of the antibody, and at ~5 kDa are the smallest functional antibody fragment (https://doi.org/10.1073/pnas.2303455120). The high stability and small size could enable improved tissue penetration and development of novel therapeutics, including knob-drug conjugates or extremely small bispecific molecules. Citation Format: Vaughn Smider. The smallest antibody class: Cow ultralong CDR3 knobs as a new class of biomolecule for therapeutics and diagnostics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1860.

Variation in the affinity of three representative avian adenoviruses for the cellular coxsackievirus and adenovirus receptor.

According to previous studies, three representative avian adenoviral strains utilize coxsackievirus-adenovirus receptor (CAR) as a receptor and seem to exhibit diverse binding affinities and modes. Thus, further revealing the exact molecular mechanism underlying the interaction between different FAdVs and the attachment receptor CAR is necessary. In this study, we successfully solved the crystal structure of the FAdV-4 fiber1 knob at 1.6 Å resolution. The interaction between the fibre knob and different domains of CAR was verified by confocal microscopy, coimmunoprecipitation and surface plasmon resonance (SPR) analysis. The fibre knobs of the three representative fowl adenoviruses specifically recognized CAR domain 1 (D1), but the recognition of CAR domain 2 (D2) by chicken embryo lethal orphan (CELO) strains was weak. These results provide insights into the differences in adenovirus‒host cell interactions and have important implications for the exploration of viral invasion mechanisms.

Cattle-derived knob paratopes grafted onto peripheral loops of the IgG1 Fc region enable the generation of a novel symmetric bispecific antibody format

In this work we present a novel symmetric bispecific antibody format based on engraftments of cattle-derived knob paratopes onto peripheral loops of the IgG1 Fc region. For this, knob architectures obtained from bovine ultralong CDR-H3 antibodies were inserted into the AB loop or EF loop of the CH3 domain, enabling the introduction of an artificial binding specificity into an IgG molecule. We demonstrate that inserted knob domains largely retain their binding affinities, resulting into bispecific antibody derivatives versatile for effector cell redirection. Essentially, generated bispecifics demonstrated adequate biophysical properties and were not compromised in their Fc mediated functionalities such as FcRn or FcγRIIIa binding.

Distantly related Alteromonas bacteriophages share tail fibers exhibiting properties of transient chaperone caps

The host recognition modules encoding the injection machinery and receptor binding proteins (RBPs) of bacteriophages are predisposed to mutation and recombination to maintain infectivity towards co-evolving bacterial hosts. In this study, we reveal how Alteromonas mediterranea schitovirus A5 shares its host recognition module, including tail fiber and cognate chaperone, with phages from distantly related families including Alteromonas myovirus V22. While the V22 chaperone is essential for producing active tail fibers, here we demonstrate production of functional A5 tail fibers regardless of chaperone co-expression. AlphaFold-generated models of tail fiber and chaperone pairs from phages A5, V22, and other Alteromonas phages reveal how amino acid insertions within both A5-like proteins results in a knob domain duplication in the tail fiber and a chaperone β-hairpin “tentacle” extension. These structural modifications are linked to differences in chaperone dependency between the A5 and V22 tail fibers. Structural similarity between the chaperones and intramolecular chaperone domains of other phage RBPs suggests an additional function of these chaperones as transient fiber “caps”. Finally, our identification of homologous host recognition modules from morphologically distinct phages implies that horizontal gene transfer and recombination events between unrelated phages may be a more common process than previously thought among Caudoviricetes phages.

Serum albumin binding knob domains engineered within a VH framework III bispecific antibody format and as chimeric peptides.

Serum albumin binding is an established mechanism to extend the serum half-life of antibody fragments and peptides. The cysteine rich knob domains, isolated from bovine antibody ultralong CDRH3, are the smallest single chain antibody fragments described to date and versatile tools for protein engineering. Here, we used phage display of bovine immune material to derive knob domains against human and rodent serum albumins. These were used to engineer bispecific Fab fragments, by using the framework III loop as a site for knob domain insertion. By this route, neutralisation of the canonical antigen (TNFα) was retained but extended pharmacokinetics in-vivo were achieved through albumin binding. Structural characterisation revealed correct folding of the knob domain and identified broadly common but non-cross-reactive epitopes. Additionally, we show that these albumin binding knob domains can be chemically synthesised to achieve dual IL-17A neutralisation and albumin binding in a single chemical entity. This study enables antibody and chemical engineering from bovine immune material, via an accessible discovery platform.

Knob domain of Fiber 2 protein provides full protection against fowl adenovirus serotype 4

Highly pathogenic fowl adenovirus serotype 4 (FAdV-4) is an acute infectious disease with severe economic impact, causing chicken hepatitis hydropericardium syndrome (HHS) and high mortality. In the present study, we evaluated the immunogenicity of the recombinant Fiber2-knob protein (F2-Knob) as an FAdV-4 candidate subunit vaccine in 14-day-old SPF chickens. The knob domain is the functional region of the viral surface protein Fiber2. The protein was expressed in Escherichia coli and was administered a single immunization with different vaccine doses. The protective efficacy was evaluated by mortality, clinical symptoms, virus shedding and histopathological examinations after challenged with the FAdV-4. The results showed that the level of ELISA antibodies of the chickens immunized with Fiber2-knob protein was significantly higher than that of the chickens immunized with an inactivated vaccine against FAdV-4. The antibody value of the immunized Fiber2-knob protein was positively correlated with the increase in immunization dose. The challenge experiment showed that the F2-Knob protein provided full protection against virulent FAdV-4 challenge and significantly reduced viral shedding. These results suggest that F2-Knob protein could be a novel vaccine candidate provide insights to control FAdV-4.

Yeast PIC-mediator structure with RNA polymerase II C-terminal domain

For transcription initiation, RNA polymerase II (Pol II) forms a preinitiation complex (PIC) that associates with the general coactivator Mediator. Whereas atomic models of the human PIC-Mediator structure have been reported, structures for its yeast counterpart remain incomplete. Here, we present an atomic model for the yeast PIC with core Mediator, including the Mediator middle module that was previously poorly resolved and including subunit Med1 that was previously lacking. We observe three peptide regions containing eleven of the 26 heptapeptide repeats of the flexible C-terminal repeat domain (CTD) of Pol II. Two of these CTD regions bind between the Mediator head and middle modules and form defined CTD-Mediator interactions. CTD peptide 1 binds between the Med6 shoulder and Med31 knob domains, whereas CTD peptide 2 forms additional contacts with Med4. The third CTD region (peptide 3) binds in the Mediator cradle and associates with the Mediator hook. Comparisons with the human PIC-Mediator structure show that the central region in peptide 1 is similar and forms conserved contacts with Mediator, whereas peptides 2 and 3 exhibit distinct structures and Mediator interactions.

Design and Identification of a Novel Antiviral Affinity Peptide against Fowl Adenovirus Serotype 4 (FAdV-4) by Targeting Fiber2 Protein.

Outbreaks of hydropericardium hepatitis syndrome caused by fowl adenovirus serotype 4 (FAdV-4) with a novel genotype have been reported in China since 2015, with significant economic losses to the poultry industry. Fiber2 is one of the important structural proteins on FAdV-4 virions. In this study, the C-terminal knob domain of the FAdV-4 Fiber2 protein was expressed and purified, and its trimer structure (PDB ID: 7W83) was determined for the first time. A series of affinity peptides targeting the knob domain of the Fiber2 protein were designed and synthesized on the basis of the crystal structure using computer virtual screening technology. A total of eight peptides were screened using an immunoperoxidase monolayer assay and RT-qPCR, and they exhibited strong binding affinities to the knob domain of the FAdV-4 Fiber2 protein in a surface plasmon resonance assay. Treatment with peptide number 15 (P15; WWHEKE) at different concentrations (10, 25, and 50 μM) significantly reduced the expression level of the Fiber2 protein and the viral titer during FAdV-4 infection. P15 was found to be an optimal peptide with antiviral activity against FAdV-4 in vitro with no cytotoxic effect on LMH cells up to 200 μM. This study led to the identification of a class of affinity peptides designed using computer virtual screening technology that targeted the knob domain of the FAdV-4 Fiber2 protein and may be developed as a novel potential and effective antiviral strategy in the prevention and control of FAdV-4.

HDAd6/35++ - A new helper-dependent adenovirus vector platform for in vivo transduction of hematopoietic stem cells

In previous studies, we achieved safe and efficient invivo hematopoietic stem cell (HSC) transduction in mobilized mice and macaques with intravenously injected helper-dependent adenovirus HDAd5/35++ vectors. These vectors are derivatives of serotype Ad5-containing CD46-affinity enhanced Ad35 fiber knob domains. Considering the impact of anti-Ad5/HDAd5/35++ neutralizing serum antibodies present in the human population, we generated HSC-retargeted HDAd6/35++ vectors derived from serotype 6. We found a lower prevalence and titers of serum anti-HDAd6/35++ in human samples compared with HDAd5/35++. HDAd6/35++ vectors efficiently transduced human and rhesus CD34+ cells invitro. Intravenous injection of HDAd5/35++-GFP or HDAd6/35++-GFP vectors after G-CSF/AMD3100 mobilization of mice with established human hematopoiesis or human CD46 transgenic mice resulted in comparable GFP marking rates in HSCs in the bone marrow and spleen. In long-term invivo HSC transduction and selection studies with integrating vectors, stable GFP expression in >75% of PBMCs was show for both vectors. In contrast with HDAd5/35++, undesired transduction of hepatocytes was minimal with HDAd6/35++. Furthermore, HDAd6/35++ allowed for efficient invivo HSC transduction in Ad5-pre-immune mice. These features, together with the straightforward production of HDAd6/35++ vectors at high yield, make this new HDAd vector platform attractive for clinical translation of the invivo approach.

Fiber-1 of serotype 4 fowl adenovirus mediates superinfection resistance against serotype 8b fowl adenovirus.

In recent years, hepatitis-hydropericardium syndrome (HHS) and inclusion body hepatitis (IBH) caused by serotype 4 fowl adenovirus (FAdV-4) and serotype 8b fowl adenovirus (FAdV-8b), respectively, are widely prevalent in China, causing huge economic losses to the poultry industry. Numerous studies have revealed the mechanism of the infection and pathogenesis of FAdV-4. However, little is known about the mechanism of infection with FAdV-8b. Among the major structural proteins of fowl adenoviruses, fiber is characterized by the ability to recognize and bind to cellular receptors to mediate the infection of host cells. In this study, through superinfection resistance analysis and an interfering assay, we found that Fiber-1 of FAdV-4, rather than hexon, penton, and fiber of FAdV-8b, conferred efficient superinfection resistance against the infection FAdV-8b in LMH cells. Moreover, truncation analysis depicted that the shaft and knob domains of FAdV-4 Fiber-1 were responsible for the inhibition. However, knockout of the coxsackie and adenovirus receptor (CAR) in LMH cells inhibited the replication of FAdV-8b only at early time points, indicating that CAR might not be the key cell receptor for FAdV-8b. Overall, our findings give novel insights into the infection mechanism of FAdV-8b and provide a new target for the prevention and control of both FAdV-4 and FAdV-8b.

Role of Fiber Shaft Length in Tumor Targeting with Ad5/3 Vectors.

Desmoglein 2 (DSG2) is overexpressed in many epithelial cancers and therefore represents a target receptor for oncolytic viruses, including Ad5/3-based viruses. For most Ad serotypes, the receptor-binding fiber is composed of tail, shaft, and knob domains. Here, we investigated the role of the fiber shaft in Ad5/3 tumor transduction in vitro and in human DSG2-transgenic mice carrying human DSG2high tumors. DSG2tg mice express DSG2 in a pattern similar to humans. We constructed Ad5/3L (with the "long" Ad5 shaft) and Ad5/3S (with the "short" Ad3 shaft) expressing GFP or luciferase. In in vitro studies we found that coagulation factor X, which is known to mediate undesired hepatocyte transduction of Ad5, enhances the transduction of Ad5/3(L), but not the transduction of Ad5/3(S). We therefore hypothesized that Ad5/3(S) would target DSG2high tumors while sparing the liver after intravenous injection. In vivo imaging studies for luciferase and analysis of luciferase activity in isolated organs, showed that Ad5/3(L) vectors efficiently transduced DSG2high tumors and liver but not normal epithelial tissues after intravenous injection. Ad5/3(S) showed minimal liver transduction, however it failed to transduce DSG2high tumors. Further modifications of the Ad5/3(S) capsid are required to compensate for the lower infectivity of Ad5/3(S) vectors.

A Single Dose of a Hybrid hAdV5-Based Anti-COVID-19 Vaccine Induces a Long-Lasting Immune Response and Broad Coverage against VOC.

Most approved vaccines against COVID-19 have to be administered in a prime/boost regimen. We engineered a novel vaccine based on a chimeric human adenovirus 5 (hAdV5) vector. The vaccine (named CoroVaxG.3) is based on three pillars: (i) high expression of Spike to enhance its immunodominance by using a potent promoter and an mRNA stabilizer; (ii) enhanced infection of muscle and dendritic cells by replacing the fiber knob domain of hAdV5 by hAdV3; (iii) use of Spike stabilized in a prefusion conformation. The transduction with CoroVaxG.3-expressing Spike (D614G) dramatically enhanced the Spike expression in human muscle cells, monocytes and dendritic cells compared to CoroVaxG.5 that expressed the native fiber knob domain. A single dose of CoroVaxG.3 induced a potent humoral immunity with a balanced Th1/Th2 ratio and potent T-cell immunity, both lasting for at least 5 months. Sera from CoroVaxG.3-vaccinated mice was able to neutralize pseudoviruses expressing B.1 (wild type D614G), B.1.117 (alpha), P.1 (gamma) and B.1.617.2 (delta) Spikes, as well as an authentic P.1 SARS-CoV-2 isolate. Neutralizing antibodies did not wane even after 5 months, making this kind of vaccine a likely candidate to enter clinical trials.

Subtelomeric Chromatin in the Fission Yeast S. pombe.

Telomeres play important roles in safeguarding the genome. The specialized repressive chromatin that assembles at telomeres and subtelomeric domains is key to this protective role. However, in many organisms, the repetitive nature of telomeric and subtelomeric sequences has hindered research efforts. The fission yeast S. pombe has provided an important model system for dissection of chromatin biology due to the relative ease of genetic manipulation and strong conservation of important regulatory proteins with higher eukaryotes. Telomeres and the telomere-binding shelterin complex are highly conserved with mammals, as is the assembly of constitutive heterochromatin at subtelomeres. In this review, we seek to summarize recent work detailing the assembly of distinct chromatin structures within subtelomeric domains in fission yeast. These include the heterochromatic SH subtelomeric domains, the telomere-associated sequences (TAS), and ST chromatin domains that assemble highly condensed chromatin clusters called knobs. Specifically, we review new insights into the sequence of subtelomeric domains, the distinct types of chromatin that assemble on these sequences and how histone H3 K36 modifications influence these chromatin structures. We address the interplay between the subdomains of chromatin structure and how subtelomeric chromatin is influenced by both the telomere-bound shelterin complexes and by euchromatic chromatin regulators internal to the subtelomeric domain. Finally, we demonstrate that telomere clustering, which is mediated via the condensed ST chromatin knob domains, does not depend on knob assembly within these domains but on Set2, which mediates H3K36 methylation.

The Chemical Synthesis of Knob Domain Antibody Fragments.

Cysteine-rich knob domains found in the ultralong complementarity determining regions of a subset of bovine antibodies are capable of functioning autonomously as 3-6 kDa peptides. While they can be expressed recombinantly in cellular systems, in this paper we show that knob domains are also readily amenable to a chemical synthesis, with a co-crystal structure of a chemically synthesized knob domain in complex with an antigen showing structural equivalence to the biological product. For drug discovery, following the immunization of cattle, knob domain peptides can be synthesized directly from antibody sequence data, combining the power and diversity of the bovine immune repertoire with the ability to rapidly incorporate nonbiological modifications. We demonstrate that, through rational design with non-natural amino acids, a paratope diversity can be massively expanded, in this case improving the efficacy of an allosteric peptide. As a potential route to further improve stability, we also performed head-to-tail cyclizations, exploiting the proximity of the N and C termini to synthesize functional, fully cyclic antibody fragments. Lastly, we highlight the stability of knob domains in plasma and, through pharmacokinetic studies, use palmitoylation as a route to extend the plasma half-life of knob domains in vivo. This study presents an antibody-derived medicinal chemistry platform, with protocols for solid-phase synthesis of knob domains, together with the characterization of their molecular structures, in vitro pharmacology, and pharmacokinetics.

A new insight into aggregation of oncolytic adenovirus Ad5-delta-24-RGD during CsCl gradient ultracentrifugation

Two-cycle cesium chloride (2 × CsCl) gradient ultracentrifugation is a conventional approach for purifying recombinant adenoviruses (rAds) for research purposes (gene therapy, vaccines, and oncolytic vectors). However, rAds containing the RGD-4C peptide in the HI loop of the fiber knob domain tend to aggregate during 2 × CsCl gradient ultracentrifugation resulting in a low infectious titer yield or even purification failure. An iodixanol-based purification method preventing aggregation of the RGD4C-modified rAds has been proposed. However, the reason explaining aggregation of the RGD4C-modified rAds during 2 × CsCl but not iodixanol gradient ultracentrifugation has not been revealed. In the present study, we showed that rAds with the RGD-4C peptide in the HI loop but not at the C-terminus of the fiber knob domain were prone to aggregate during 2 × CsCl but not iodixanol gradient ultracentrifugation. The cysteine residues with free thiol groups after the RGD motif within the inserted RGD-4C peptide were responsible for formation of the interparticle disulfide bonds under atmospheric oxygen and aggregation of Ad5-delta-24-RGD4C-based rAds during 2 × CsCl gradient ultracentrifugation, which could be prevented using iodixanol gradient ultracentrifugation, most likely due to antioxidant properties of iodixanol. A cysteine-to-glycine substitution of the cysteine residues with free thiol groups (RGD-2C2G) prevented aggregation during 2 × CsCl gradient purification but in coxsackie and adenovirus receptor (CAR)-low/negative cancer cell lines of human and rodent origin, this reduced cytolytic efficacy to the levels observed for a fiber non-modified control vector. However, both Ad5-delta-24-RGD4C and Ad5-delta-24-RGD2C2G were equally effective in the murine immunocompetent CT-2A glioma model due to a primary role of antitumor immune responses in the therapeutic efficacy of oncolytic virotherapy.