Vaccine vectors derived from a large collection of simian adenoviruses induce potent cellular immunity across multiple species.

Replication-defective adenovirus vectors based on human serotype 5 (Ad5) induce protective immune responses against diverse pathogens and cancer in animal models, as well as elicit robust and sustained cellular immunity in humans. However, most humans have neutralizing antibodies to Ad5, which can impair the immunological potency of such vaccines. Here, we show that rare serotypes of human adenoviruses, which should not be neutralized in most humans, are far less potent as vaccine vectors than Ad5 in mice and nonhuman primates, casting doubt on their potential efficacy in humans. To identify novel vaccine carriers suitable for vaccine delivery in humans, we isolated and sequenced more than 1000 adenovirus strains from chimpanzees (ChAd). Replication-defective vectors were generated from a subset of these ChAd serotypes and screened to determine whether they were neutralized by human sera and able to grow in human cell lines. We then ranked these ChAd vectors by immunological potency and found up to a thousandfold variation in potency for CD8+ T cell induction in mice. These ChAd vectors were safe and immunologically potent in phase 1 clinical trials, thereby validating our screening approach. These data suggest that the ChAd vectors developed here represent a large collection of non-cross-reactive, potent vectors that may be exploited for the development of new vaccines.

Mechanistic implications for LDL receptor degradation from the PCSK9/LDLR structure at neutral pH.

The protein PCSK9 (proprotein convertase subtilisin/kexin type 9) is a key regulator of low-density lipoprotein receptor (LDLR) levels and cardiovascular health. We have determined the crystal structure of LDLR bound to PCSK9 at neutral pH. The structure shows LDLR in a new extended conformation. The PCSK9 C-terminal domain is solvent exposed, enabling cofactor binding, whereas the catalytic domain and prodomain interact with LDLR epidermal growth factor(A) and ß-propeller domains, respectively. Thus, PCSK9 seems to hold LDLR in an extended conformation and to interfere with conformational rearrangements required for LDLR recycling.

A novel chimpanzee serotype-based adenoviral vector as delivery tool for cancer vaccines.

The use of adenovirus (Ad) as vaccine vectors is hindered by pre-existing immunity to human Ads in most of the human population. In order to overcome this limitation, uncommon alternative Ad serotypes need to be utilized. In this study, an E1-E3 deleted recombinant Ad based on the chimpanzee serotype 3 (ChAd3) was engineered to express human carcinoembryonic antigen (CEA) protein or rat neu extracellular/transmembrane domains (ECD.TM). ChAd3 vectors were tested in CEA transgenic (CEA.Tg) and BALB/NeuT mice, which show immunologic tolerance to these antigens. ChAd3 is capable of inducing an immune response comparable to that of hAd5 serotype-based vectors, thus breaking tolerance to tumor associated antigens (TAAs) and achieving anti-tumor effects. Of importance is that ChAd3 can overcome hAd5 pre-existing immunity and work in conjunction with DNA electroporation (DNA-EP) and other Ad vaccines based on common human serotypes.

Structural and biochemical characterization of the wild type PCSK9-EGF(AB) complex and natural familial hypercholesterolemia mutants.

PCSK9 regulates low density lipoprotein receptor (LDLR) levels and consequently is a target for the prevention of atherosclerosis and coronary heart disease. Here we studied the interaction, of LDLR EGF(A/AB) repeats with PCSK9. We show that PCSK9 binds the EGF(AB) repeats in a pH-dependent manner. Although the PCSK9 C-terminal domain is not involved in LDLR binding, PCSK9 autocleavage is required. Moreover, we report the x-ray structure of the PCSK9DeltaC-EGF(AB) complex at neutral pH. Compared with the low pH PCSK9-EGF(A) structure, the new structure revealed rearrangement of the EGF(A) His-306 side chain and disruption of the salt bridge with PCSK9 Asp-374, thus suggesting the basis for enhanced interaction at low pH. In addition, the structure of PCSK9DeltaC bound to EGF(AB)(H306Y), a mutant associated with familial hypercholesterolemia (FH), reveals that the Tyr-306 side chain forms a hydrogen bond with PCSK9 Asp-374, thus mimicking His-306 in the low pH conformation. Consistently, Tyr-306 confers increased affinity for PCSK9. Importantly, we found that although the EGF(AB)(H306Y)-PCSK9 interaction is pH-independent, LDLR(H306Y) binds PCSK9 50-fold better at low pH, suggesting that factors other than His-306 contribute to the pH dependence of PCSK9-LDLR binding. Further, we determined the structures of EGF(AB) bound to PCSK9DeltaC containing the FH-associated D374Y and D374H mutations, revealing additional interactions with EGF(A) mediated by Tyr-374/His-374 and providing a rationale for their disease phenotypes. Finally, we report the inhibitory properties of EGF repeats in a cellular assay measuring LDL uptake.

Structural and functional analysis of the human HDAC4 catalytic domain reveals a regulatory structural zinc-binding domain.

Histone deacetylases (HDACs) regulate chromatin status and gene expression, and their inhibition is of significant therapeutic interest. To date, no biological substrate for class IIa HDACs has been identified, and only low activity on acetylated lysines has been demonstrated. Here, we describe inhibitor-bound and inhibitor-free structures of the histone deacetylase-4 catalytic domain (HDAC4cd) and of an HDAC4cd active site mutant with enhanced enzymatic activity toward acetylated lysines. The structures presented, coupled with activity data, provide the molecular basis for the intrinsically low enzymatic activity of class IIa HDACs toward acetylated lysines and reveal active site features that may guide the design of class-specific inhibitors. In addition, these structures reveal a conformationally flexible structural zinc-binding domain conserved in all class IIa enzymes. Importantly, either the mutation of residues coordinating the structural zinc ion or the binding of a class IIa selective inhibitor prevented the association of HDAC4 with the N-CoR.HDAC3 repressor complex. Together, these data suggest a key role of the structural zinc-binding domain in the regulation of class IIa HDAC functions.

Differential screening of phage-ab libraries by oligonucleotide microarray technology.

A novel and efficient tagArray technology was developed that allows rapid identification of antibodies which bind to receptors with a specific expression profile, in the absence of biological information. This method is based on the cloning of a specific, short nucleotide sequence (tag) in the phagemid coding for each phage-displayed antibody fragment (phage-Ab) present in a library. In order to set up and validate the method we identified about 10,000 different phage-Abs binding to receptors expressed in their native form on the cell surface (10 k Membranome collection) and tagged each individual phage-Ab. The frequency of each phage-Ab in a given population can at this point be inferred by measuring the frequency of its associated tag sequence through standard DNA hybridization methods. Using tiny amounts of biological samples we identified phage-Abs binding to receptors preferentially expressed on primary tumor cells rather than on cells obtained from matched normal tissues. These antibodies inhibited cell proliferation in vitro and tumor development in vivo, thus representing therapeutic lead candidates.

An adenovirus type 5 (Ad5) amplicon-based packaging cell line for production of high-capacity helper-independent deltaE1-E2-E3-E4 Ad5 vectors.

Production of multiply deleted adenoviral (Ad) vectors with increased cloning capacity and reduced immunogenicity to adenovirus gene products requires the concomitant generation of efficient packaging cell lines. High expression levels of the complementing genes must be achieved in a coordinated fashion with viral replication. This is a particularly difficult task in light of the significant cytotoxicity displayed by adenoviral proteins. To this end, we developed a novel adenovirus-based amplicon with an Epstein-Barr virus origin of replication, Ad type 5 (Ad5) inverted terminal repeats, all Ad5 early region 2 (E2) genes, and the early region 4 (E4) open reading frame 6 (ORF6) under the control of a tetracycline-dependent promoter. The amplicon (pE2) was stably maintained in multiple copies in the nuclei of 293 cells stably expressing the Epstein-Barr virus nuclear antigen 1 (EBNA1) and allowed replication as a linear DNA upon induction of E2 and ORF6 gene expression. A stable cell line (2E2) was generated by introducing pE2 into 293EBNATet cells expressing the tetracycline-dependent transcriptional silencer and the reverse Tet transactivator (rtTA2). Upon induction with doxicycline, 2E2 cells produced higher levels of polymerase, precursor terminal protein (pTP), and DNA binding protein than noninduced 2E2 cells infected with first-generation Ad5 vector and supported efficient amplification of a multiply deleted Ad5 vector lacking E1, E2, E3, and E4 genes (Ad5DeltaE(1-4)). The high cloning capacity of Ad5DeltaE(1-4) (up to 12.6 kb) was exploited to construct a vector encoding the entire hepatitis C virus (HCV) polyprotein. Infection of HeLa cells by the resulting vector showed high levels of correctly processed HCV proteins.