The cryo-electron microscopy structure of huntingtin.

Huntingtin (HTT) is a large (348 kDa) protein that is essential for embryonic development and is involved in diverse cellular activities such as vesicular transport, endocytosis, autophagy and the regulation of transcription. Although an integrative understanding of the biological functions of HTT is lacking, the large number of identified HTT interactors suggests that it serves as a protein-protein interaction hub. Furthermore, Huntington's disease is caused by a mutation in the HTT gene, resulting in a pathogenic expansion of a polyglutamine repeat at the amino terminus of HTT. However, only limited structural information regarding HTT is currently available. Here we use cryo-electron microscopy to determine the structure of full-length human HTT in a complex with HTT-associated protein 40 (HAP40; encoded by three F8A genes in humans) to an overall resolution of 4 Å. HTT is largely α-helical and consists of three major domains. The amino- and carboxy-terminal domains contain multiple HEAT (huntingtin, elongation factor 3, protein phosphatase 2A and lipid kinase TOR) repeats arranged in a solenoid fashion. These domains are connected by a smaller bridge domain containing different types of tandem repeats. HAP40 is also largely α-helical and has a tetratricopeptide repeat-like organization. HAP40 binds in a cleft and contacts the three HTT domains by hydrophobic and electrostatic interactions, thereby stabilizing the conformation of HTT. These data rationalize previous biochemical results and pave the way for improved understanding of the diverse cellular functions of HTT.

HAP40 protein levels are huntingtin-dependent and decrease in Huntington disease.

The huntingtin-associated protein 40 (HAP40) is an abundant interactor of huntingtin (HTT). In complexes of these proteins, HAP40 tightly binds to HTT in a cleft formed by two larger domains rich in HEAT repeats, and a smaller bridge domain connecting the two. We show that HAP40 steady-state protein levels are directly dependent on HTT (both normal and mutant HTT) and that HAP40 is strongly stabilized by the interaction with HTT resulting in an at least 5-fold increase in HAP40's half-life when bound to HTT. Cellular HAP40 protein levels were reduced in primary fibroblasts and lymphoblasts of Huntington Disease (HD) patients and in brain tissue of a full-length HTT mouse model of HD, concomitant with decreased soluble HTT levels in these cell types. This data and our previous demonstration of coevolution between HTT and HAP40 and evolutionary conservation of their interaction suggest that HAP40 is an obligate interaction partner of HTT. Our observation of reduced HAP40 levels in HD invites further studies, whether HAP40 loss-of-function contributes to the pathophysiology of HD.

The evolution of the huntingtin-associated protein 40 (HAP40) in conjunction with huntingtin.

BACKGROUND: The huntingtin-associated protein 40 (HAP40) abundantly interacts with huntingtin (HTT), the protein that is altered in Huntington's disease (HD). Therefore, we analysed the evolution of HAP40 and its interaction with HTT. RESULTS: We found that in amniotes HAP40 is encoded by a single-exon gene, whereas in all other organisms it is expressed from multi-exon genes. HAP40 co-occurs with HTT in unikonts, including filastereans such as Capsaspora owczarzaki and the amoebozoan Dictyostelium discoideum, but both proteins are absent from fungi. Outside unikonts, a few species, such as the free-living amoeboflagellate Naegleria gruberi, contain putative HTT and HAP40 orthologs. Biochemically we show that the interaction between HTT and HAP40 extends to fish, and bioinformatic analyses provide evidence for evolutionary conservation of this interaction. The closest homologue of HAP40 in current protein databases is the family of soluble N-ethylmaleimide-sensitive factor attachment proteins (SNAPs). CONCLUSION: Our results indicate that the transition from a multi-exon to a single-exon gene appears to have taken place by retroposition during the divergence of amphibians and amniotes, followed by the loss of the parental multi-exon gene. Furthermore, it appears that the two proteins probably originated at the root of eukaryotes. Conservation of the interaction between HAP40 and HTT and their likely coevolution strongly indicate functional importance of this interaction.

miRNA-mediated silencing in hepatocytes can increase adaptive immune responses to adenovirus vector-delivered transgenic antigens.

Adenovirus vectors based on human serotype 5 can induce potent CD8 T cell responses to vector-encoded transgenic antigens. However, the individual contribution of different cell types expressing antigen upon adenovirus vector injection to the generation of antigen-directed adaptive immune responses is poorly understood so far. We investigated the role of hepatocytes, skeletal muscle, and hematopoietic cells for the induction of cellular and humoral immune responses by miRNA-mediated tissue-specific silencing of antigen expression. Using hepatitis B small surface antigen (HBsAg) as the vector-encoded transgene we show that adenovirus vector dissemination from an intramuscular (i.m.) injection site into the liver followed by HBsAg expression in hepatocytes can limit early priming of CD8 T cells and the generation of anti-HBsAg antibody responses. However, hepatocyte-specific miRNA122a-mediated silencing of HBsAg expression overcame these limitations. Early clonal expansion of K(b)/S(190-197)-specific CD8 T cells was significantly enhanced and improved polyfunctionality of CD8 T cells was found. Furthermore, miRNA122a-mediated antigen silencing induced significantly higher anti-HBsAg antibody titers allowing an up to 100-fold vector dose reduction. These results indicate that miRNA-mediated regulation of antigen expression in the context of adenovirus vectors can significantly improve transgene product-directed immune responses. This finding could be of interest for future adenovirus vaccine vector development.

Modifications of adenovirus hexon allow for either hepatocyte detargeting or targeting with potential evasion from Kupffer cells.

In vivo gene transfer with adenovirus vectors would significantly benefit from a tight control of the adenovirus-inherent liver tropism. For efficient hepatocyte transduction, adenovirus vectors need to evade from Kupffer cell scavenging while delivery to peripheral tissues or tumors could be improved if both scavenging by Kupffer cells and uptake by hepatocytes were blocked. Here, we provide evidence that a single point mutation in the hexon capsomere designed to enable defined chemical capsid modifications may permit both detargeting from and targeting to hepatocytes with evasion from Kupffer cell scavenging. Vector particles modified with small polyethylene glycol (PEG) moieties specifically on hexon exhibited decreased transduction of hepatocytes by shielding from blood coagulation factor binding. Vector particles modified with transferrin or, surprisingly, 5,000 Da PEG or dextran increased hepatocyte transduction up to 18-fold independent of the presence of Kupffer cells. We further show that our strategy can be used to target high-capacity adenovirus vectors to hepatocytes emphasizing the potential for therapeutic liver-directed gene transfer. Our approach may lead to a detailed understanding of the interactions between adenovirus vectors and Kupffer cells, one of the most important barriers for adenovirus-mediated gene delivery.

Process- and product-related impurities in the ChAdOx1 nCov-19 vaccine.

ChAdOx1 nCov-19 and Ad26.COV2.S are approved vaccines inducing protective immunity against SARS-CoV-2 infection in humans by expressing the Spike protein of SARS-CoV-2. We analyzed protein content and protein composition of ChAdOx1 nCov-19 and Ad26.COV2.S by biochemical methods and by mass spectrometry. Four out of four tested lots of ChAdOx1 nCoV-19 contained significantly higher than expected levels of host cell proteins (HCPs) and of free viral proteins. The most abundant contaminating HCPs belonged to the heat-shock protein and cytoskeletal protein families. The HCP content exceeded the 400 ng specification limit per vaccine dose, as set by the European Medicines Agency (EMA) for this vaccine, by at least 25-fold and the manufacturer's batch-release data in some of the lots by several hundred-fold. In contrast, three tested lots of the Ad26.COV2.S vaccine contained only very low amounts of HCPs. As shown for Ad26.COV2.S production of clinical grade adenovirus vaccines of high purity is feasible at an industrial scale. Correspondingly, purification procedures of the ChAdOx1 nCov-19 vaccine should be modified to remove protein impurities as good as possible. Our data also indicate that standard quality assays, as they are used in the manufacturing of proteins, have to be adapted for vectored vaccines.

Vaccine-induced COVID-19 mimicry syndrome.

To fight the COVID-19 pandemic caused by the RNA virus SARS-CoV-2, a global vaccination campaign is in progress to achieve the immunization of billions of people mainly with adenoviral vector- or mRNA-based vaccines, all of which encode the SARS-CoV-2 Spike protein. In some rare cases, cerebral venous sinus thromboses (CVST) have been reported as a severe side effect occurring 4-14 days after the first vaccination and were often accompanied by thrombocytopenia. Besides CVST, splanchnic vein thromboses (SVT) and other thromboembolic events have been observed. These events only occurred following vaccination with adenoviral vector-based vaccines but not following vaccination with mRNA-based vaccines. Meanwhile, scientists have proposed an immune-based pathomechanism and the condition has been coined vaccine-induced immune thrombotic thrombocytopenia (VITT). Here, we describe an unexpected mechanism that could explain thromboembolic events occurring with DNA-based but not with RNA-based vaccines. We show that DNA-encoded mRNA coding for Spike protein can be spliced in a way that the transmembrane anchor of Spike is lost, so that nearly full-length Spike is secreted from cells. Secreted Spike variants could potentially initiate severe side effects when binding to cells via the ACE2 receptor. Avoiding such splicing events should become part of a rational vaccine design to increase safety of prospective vaccines.

ΔE1 and high-capacity adenoviral vectors expressing full-length codon-optimized merozoite surface protein 1 for vaccination against Plasmodium falciparum.

BACKGROUND: The merozoite surface protein (MSP)-1 of Plasmodium falciparum, the causative agent of malaria tropica, is considered to be a promising vaccine candidate. Although its stable cloning and expression has been difficult in the past, adenoviral vectors expressing the complex protein are described in the present study. METHODS: Codon-optimized msp-1 was used to construct a set of first generation (ΔE1Ad) and high-capacity adenovirus (HC-Ad) vectors, and cellular and humoral immune responses induced by the vectors were characterized in detail in mice. RESULTS: Generation of stable ΔE1Ad and HC-Ad vectors expressing full-length MSP-1 and their production to high vector titers was found to be feasible. Epitope identification and analysis of frequencies of specific CD8 T-cells revealed that MSP-1 expressing HC-Ad vectors induced higher frequencies of interferon-γ + CD8 T-cells than ΔE1 vectors. Irrespective of the vector format, higher titers of MSP-1 specific antibodies were generated by Ad vectors expressing MSP-1 from a chicken ß-actin (CAG) promoter comprising the cytomegalovirus early enhancer element and the chicken ß-actin promoter. CONCLUSIONS: The findings of the present study suggest that Ad vectors expressing full-length codon-optimized MSP-1 are promising candidate vaccines against P. falciparum infections. Use of the HC-Ad vector type for delivery, as well as the CAG promoter to control MSP-1 expression, may further increase the efficacy of this vaccine candidate.

High-capacity adenoviral vectors circumvent the limitations of ΔE1 and ΔE1/ΔE3 adenovirus vectors to induce multispecific transgene product-directed CD8 T-cell responses.

BACKGROUND: The ability to induce cytotoxic T lymphocyte (CTL) responses that are multispecific is considered to comprise an essential feature for an efficacious genetic vaccine against many pathogens including HIV and hepatitis C virus. ΔE1Ad vectors are promising vectored vaccines but have been shown to induce antigen-specific CTLs with only limited multispecificity. In the present study, we investigated the applicability of gene-deleted high-capacity adenovirus (HC-Ad) vectors and focused on the induction of multispecific CTL responses. METHODS: We generated Δ E1 and HC-Ad vectors expressing hepatitis B virus small surface antigen (HBsAg). We comparatively analyzed the CTL profiles against various transgene product- and vector-derived epitopes in several mouse strains and HBsAg- and vector-directed antibody responses. RESULTS: HC-Ad vectors efficiently induced multispecific HBsAg-directed CTLs. By contrast, ΔE1Ad vectors mainly primed CTLs against one immunodominant epitope of HBsAg. This absence of multispecific CTL responses correlated with the induction of CTLs against viral epitopes generated by de novo expression of Ad genes from the ΔE1Ad vector. However, Ad-specific CTLs induced in trans did not impair HC-AdS-induced multispecific CTL responses against HBsAg. Finally, HC-Ad vectors also induced higher HBsAg antibody titers compared to ΔE1Ad vectors. CONCLUSIONS: De novo expression of viral genes from ΔE1Ad vector genomes restricts the multispecificity of transgene product-specific CTLs by immunodominance effects. HC-Ad vectors devoid of Ad genes are favorable for the induction of both multispecific CD8 T-cell responses and high antibody responses. Our results suggest the deletion of Ad genes as an important means for developing potent Ad-based vectored vaccines.

Pathological polyQ expansion does not alter the conformation of the Huntingtin-HAP40 complex.

The abnormal amplification of a CAG repeat in the gene coding for huntingtin (HTT) leads to Huntington's disease (HD). At the protein level, this translates into the expansion of a polyglutamine (polyQ) stretch located at the HTT N terminus, which renders HTT aggregation prone by unknown mechanisms. Here we investigated the effects of polyQ expansion on HTT in a complex with its stabilizing interaction partner huntingtin-associated protein 40 (HAP40). Surprisingly, our comprehensive biophysical, crosslinking mass spectrometry and cryo-EM experiments revealed no major differences in the conformation of HTT-HAP40 complexes of various polyQ length, including 17QHTT-HAP40 (wild type), 46QHTT-HAP40 (typical polyQ length in HD patients), and 128QHTT-HAP40 (extreme polyQ length). Thus, HTT polyQ expansion does not alter the global conformation of HTT when associated with HAP40.

Targeting of adenovirus vectors to the LRP receptor family with the high-affinity ligand RAP via combined genetic and chemical modification of the pIX capsomere.

Adenovirus (Ad) vector targeting requires presentation of specific ligands on the virion's surface. Geneti-chemical targeting is based on the genetic introduction of cysteine residues bearing reactive thiol groups into solvent-accessible capsomeres of the virion and subsequent chemical coupling of ligands. Here, we exploited this technology to modify the pIX capsomere with high-affinity ligands. Genetic introduction of C-terminal cysteines to pIX allowed for specific coupling of full-length proteins to the virion, while not affecting vector production. Direct comparison of the two high-affinity ligands receptor- associated protein (RAP) and transferrin (Tf) revealed that targeting after coupling of a high-affinity ligand to pIX presumably requires release of the ligand from its receptor after cell entry. In addition, data obtained by live cell imaging of labeled vector particles demonstrated that coupling of very large proteins to pIX can impair intracellular vector particle trafficking. Finally, we demonstrate that the geneti-chemical targeting technology is suitable for in vivo targeting to liver after intravenous injection. Our data provide significant insight into basic requirements for successful targeting of pIX-modified Ad vectors.

Fully detargeted polyethylene glycol-coated adenovirus vectors are potent genetic vaccines and escape from pre-existing anti-adenovirus antibodies.

Genetic vaccination with adenoviral (Ad) gene transfer vectors requires transduction of professional antigen-presenting cells. However, because the natural Ad receptors are expressed on many cell types, the Ad vectors currently in use are characterized by high promiscuity. In fact, the majority of injected Ad vector particles are likely to transduce non-target cells. We have analyzed various sizes of polyethylene glycol (PEG) molecules for vector particle detargeting, and our data provide evidence that the size of the PEG determines detargeting efficiency. With the use of appropriately large PEG molecules, vector particles were detargeted from muscle after local delivery and from liver after systemic delivery in mouse models. Surprisingly, fully detargeted PEGylated Ad vectors still induced strong cellular and humoral immune responses to vector-encoded transgene products. Also, injection of PEGylated and non-PEGylated vector particles resulted in similar kinetics of transgene product-specific cytotoxic immune responses, thereby suggesting that the same cell types were involved in their induction. Furthermore, we showed that PEGylated vectors evade neutralizing anti-Ad antibodies in vivo. This feature might help circumvent the recognized limitation imposed by the widespread occurrence of anti-Ad immunity in the human population. We suggest that PEGylated Ad particles with significantly reduced promiscuity may qualify as a novel and safe vector format for genetic vaccination.

Reductive amination as a strategy to reduce adenovirus vector promiscuity by chemical capsid modification with large polysaccharides.

BACKGROUND: Chemical capsid modification of adenovirus vectors with synthetic polymers has been shown to aid in overcoming typical barriers for adenovirus vector-mediated gene transfer. Carbohydrate-based polymers for covalent modification of adenovirus vectors have been largely neglected so far. We utilized a reductive amination strategy to generate a novel class of adenovirus-based glycovectors with a mannan derivative. METHODS: Reductive amination to covalently couple polysaccharides to the capsid surface of adenovirus serotype 5-based vectors was investigated utilizing an oxidized derivative of mannan. After biochemical and physical characterization of mannanylated vectors, their performance was analysed in vitro in cell lines and primary human cells, and in vivo in mice after local and systemic vector injection. RESULTS: We describe the successful modification of adenovirus vectors with large polysaccharides by reductive amination. The particles were efficiently modified, physically intact and, importantly, detargeted from the natural Coxsackie and adenovirus receptor/integrin pathway in vitro. In addition, they exhibited significantly decreased transduction of muscle after local delivery and of liver after systemic delivery in mice. However, despite the modification of 60% of capsid surface amino groups, mannanylated particles were unable to evade neutralizing anti-Ad5 antibodies. CONCLUSIONS: Mannanylated vectors are a paradigm for a novel class of glycoviruses modified with large polysaccharides. Vector promiscuity as one of the important hurdles for Ad-mediated gene transfer could be significantly decreased in vivo, whereas mannanylated vectors were unable to escape from anti-adenovirus antibodies. Our studies provide a detailed analysis of mannan-modified Ad vectors and suggest further improvements for this novel class of glycovectors.

Traceless bioresponsive shielding of adenovirus hexon with HPMA copolymers maintains transduction capacity in vitro and in vivo.

Capsid surface shielding of adenovirus vectors with synthetic polymers is an emerging technology to reduce unwanted interactions of the vector particles with cellular and non-cellular host components. While it has been shown that attachment of shielding polymers allows prevention of undesired interactions, it has become evident that a shield which is covalently attached to the vector surface can negatively affect gene transfer efficiency. Reasons are not only a limited receptor-binding ability of the shielded vectors but also a disturbance of intracellular trafficking processes, the latter depending on the interaction of the vector surface with the cellular transport machinery. A solution might be the development of bioresponsive shields that are stably maintained outside the host cell but released upon cell entry to allow for efficient gene delivery to the nucleus. Here we provide a systematic comparison of irreversible versus bioresponsive shields based on synthetic N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers. In addition, the chemical strategy used for generation of the shield allowed for a traceless bioresponsive shielding, i.e., polymers could be released from the vector particles without leaving residual linker residues. Our data demonstrated that only a bioresponsive shield maintained the high gene transfer efficiency of adenovirus vectors both in vitro and in vivo. As an example for bioresponsive HPMA copolymer release, we analyzed the in vivo gene transfer in the liver. We demonstrated that both the copolymer's charge and the mode of shielding (irreversible versus traceless bioresponsive) profoundly affected liver gene transfer and that traceless bioresponsive shielding with positively charged HPMA copolymers mediated FX independent transduction of hepatocytes. In addition, we demonstrated that shielding with HPMA copolymers can mediate a prolonged blood circulation of vector particles in mice. Our results have significant implications for the future design of polymer-shielded Ad and provide a deeper insight into the interaction of shielded adenovirus vector particles with the host after systemic delivery.

Transcellular targeting of fiber- and hexon-modified adenovirus vectors across the brain microvascular endothelial cells in vitro.

In central nervous system (CNS)-directed gene therapy, efficient targeting of brain parenchyma through the vascular route is prevented by the endothelium and the epithelium of the blood-brain and the blood-cerebrospinal fluid barriers, respectively. In this study, we evaluated the feasibility of the combined genetic and chemical adenovirus capsid modification technology to enable transcellular delivery of targeted adenovirus (Ad) vectors across the blood-brain barrier (BBB) in vitro models. As a proof-of-principle ligand, maleimide-activated full-length human transferrin (hTf) was covalently attached to cysteine-modified Ad serotype 5 vectors either to its fiber or hexon protein. In transcytosis experiments, hTf-coupled vectors were shown to be redirected across the BBB models, the transcytosis activity of the vectors being dependent on the location of the capsid modification and the in vitro model used. The transduction efficiency of hTf-targeted vectors decreased significantly in confluent, polarized cells, indicating that the intracellular route of the vectors differed between unpolarized and polarized cells. After transcellular delivery the majority of the hTf-modified vectors remained intact and partly capable of gene transfer. Altogether, our results demonstrate that i) covalent attachment of a ligand to Ad capsid can mediate transcellular targeting across the cerebral endothelium in vitro, ii) the attachment site of the ligand influences its transcytosis efficiency and iii) combined genetic/chemical modification of Ad vector can be used as a versatile platform for the development of Ad vectors for transcellular targeting.

Capsomer-specific fluorescent labeling of adenoviral vector particles allows for detailed analysis of intracellular particle trafficking and the performance of bioresponsive bonds for vector capsid modifications.

Adenoviral (Ad) vectors are widely used for gene therapy approaches. Because of the high abundance of the natural adenoviral receptors (coxsackievirus-adenovirus receptor and integrins) on a wide variety of cells, numerous methods have been developed to redirect the virions to specific receptors on target cell surfaces. Importantly, an increasing number of publications have shown that the success of targeting not only depends on receptor binding and cellular uptake, but also on intracellular trafficking processes. Therefore, improved knowledge about the intracellular fate of targeted Ad vector particles is mandatory for a rational design of targeted Ad vectors. However, the technologies currently available for fluorescent labeling of Ad vectors have significant limitations: (1) at present capsids are labeled all over the particle surface, and this imposes the risk of interference with particle infectivity; (2) capsomer-specific labeling requires extensive genetic modifications and has been demonstrated only at protein IX; and (3) two-color labeling approaches are not available. Here we present a novel, robust, and straightforward labeling procedure that overcomes these limitations. It allows for specific labeling of the capsomer's fiber, protein IX, or hexon and permits two-color labeling. We demonstrate the potential of this labeling technology by analyzing two different bioresponsive bonds that can be used for the attachment of shielding or targeting moieties to the capsids: disulfide and hydrazone bonds. We demonstrate that in contrast to disulfide bonds, hydrazone bonds are quickly hydrolyzed after uptake of the virions and are thus favorable for the generation of bioresponsive vectors.