The effect of certolizumab drug concentration and anti-drug antibodies on TNF neutralisation.

OBJECTIVES: Tumour necrosis factor (TNF) inhibitors like certolizumab, elicit an immunogenic response leading to the formation of anti-drug antibodies (ADAs). We sought to mechanistically investigate the relationship between certolizumab concentrations, ADAs, and the effective TNF neutralising capacity in sera of rheumatoid arthritis (RA) patients. TNF neutralising capacity of certolizumab was compared to the neutralising capacity of adalimumab. METHODS: Serum samples were collected from 40 consecutive certolizumab-treated RA patients at baseline and 4, 16, 28 and 52 weeks after treatment initiation [Dutch Trial Register NTR (Nederlands Trial Register) Trial NL2824 no. 2965]. Certolizumab concentration and ADA titre were measured with a certolizumab bridging enzyme-linked immunosorbent assay (ELISA) and a drug-tolerant radioimmunoassay (RIA), respectively. TNF neutralisation by certolizumab and adalimumab, in presence or absence of ADAs, was analysed with the TNF-sensitive WEHI bioassay. RESULTS: Despite a high incidence of ADAs during one year of follow-up (65%; 26/40 patients), certolizumab levels of >10 µg/ml were measured in most patients. The capacity for TNF neutralisation highly correlated with certolizumab serum concentration, whereas no association with ADAs was observed. Similar results were obtained for adalimumab. The relative in vitro neutralising potency was higher for certolizumab compared to adalimumab. CONCLUSIONS: Anti-certolizumab antibodies were detected in a large proportion of patients, but in most cases where ADAs were detected, certolizumab was also present in high concentrations, directly correlating with in vitro neutralising capacity. These results indicate that measurement of certolizumab drug levels, rather than ADAs, have direct clinical significance.

Targeted chromosomal insertion of large DNA into the human genome by a fiber-modified high-capacity adenovirus-based vector system.

A prominent goal in gene therapy research concerns the development of gene transfer vehicles that can integrate exogenous DNA at specific chromosomal loci to prevent insertional oncogenesis and provide for long-term transgene expression. Adenovirus (Ad) vectors arguably represent the most efficient delivery systems of episomal DNA into eukaryotic cell nuclei. The most advanced recombinant Ads lack all adenoviral genes. This renders these so-called high-capacity (hc) Ad vectors less cytotoxic/immunogenic than those only deleted in early regions and creates space for the insertion of large/multiple transgenes. The versatility of hcAd vectors is been increased by capsid modifications to alter their tropism and by the incorporation into their genomes of sequences promoting chromosomal insertion of exogenous DNA. Adeno-associated virus (AAV) can insert its genome into a specific human locus designated AAVS1. Trans- and cis-acting elements needed for this reaction are the AAV Rep78/68 proteins and Rep78/68-binding sequences, respectively. Here, we describe the generation, characterization and testing of fiber-modified dual hcAd/AAV hybrid vectors (dHVs) containing both these elements. Due to the inhibitory effects of Rep78/68 on Ad-dependent DNA replication, we deployed a recombinase-inducible gene switch to repress Rep68 synthesis during vector rescue and propagation. Flow cytometric analyses revealed that rep68-positive dHVs can be produced similarly well as rep68-negative control vectors. Western blot experiments and immunofluorescence microscopy analyses demonstrated transfer of recombinase-dependent rep68 genes into target cells. Studies in HeLa cells and in the dystrophin-deficient myoblasts from a Duchenne muscular dystrophy (DMD) patient showed that induction of Rep68 synthesis in cells transduced with fiber-modified and rep68-positive dHVs leads to increased stable transduction levels and AAVS1-targeted integration of vector DNA. These results warrant further investigation especially considering the paucity of vector systems allowing permanent phenotypic correction of patient-own cell types with large DNA (e.g. recombinant full-length DMD genes).

Structural protein requirements in equine arteritis virus assembly.

Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae of the order Nidovirales. EAV particles contain seven structural proteins: the nucleocapsid protein N, the unglycosylated envelope proteins M and E, and the N-glycosylated membrane proteins GP(2b) (previously named G(S)), GP(3), GP(4), and GP(5) (previously named G(L)). Proteins N, M, and GP(5) are major virion components, E occurs in virus particles in intermediate amounts, and GP(4), GP(3), and GP(2b) are minor structural proteins. The M and GP(5) proteins occur in virus particles as disulfide-linked heterodimers while the GP(4), GP(3), and GP(2b) proteins are incorporated into virions as a heterotrimeric complex. Here, we studied the effect on virus assembly of inactivating the structural protein genes one by one in the context of a (full-length) EAV cDNA clone. It appeared that the three major structural proteins are essential for particle formation, while the other four virion proteins are dispensable. When one of the GP(2b), GP(3), or GP(4) proteins was missing, the incorporation of the remaining two minor envelope glycoproteins was completely blocked while that of the E protein was greatly reduced. The absence of E entirely prevented the incorporation of the GP(2b), GP(3), and GP(4) proteins into viral particles. EAV particles lacking GP(2b), GP(3), GP(4), and E did not markedly differ from wild-type virions in buoyant density, major structural protein composition, electron microscopic appearance, and genomic RNA content. On the basis of these results, we propose a model for the EAV particle in which the GP(2b)/GP(3)/GP(4) heterotrimers are positioned, in association with a defined number of E molecules, above the vertices of the putatively icosahedral nucleocapsid.

Intra- and intermolecular disulfide bonds of the GP2b glycoprotein of equine arteritis virus: relevance for virus assembly and infectivity.

Equine arteritis virus (EAV) is an enveloped, positive-strand RNA virus belonging to the family Arteriviridae of the order NIDOVIRALES: EAV virions contain six different envelope proteins. The glycoprotein GP(5) (previously named G(L)) and the unglycosylated membrane protein M are the major envelope proteins, while the glycoproteins GP(2b) (previously named G(S)), GP(3), and GP(4) are minor structural proteins. The unglycosylated small hydrophobic envelope protein E is present in virus particles in intermediate molar amounts compared to the other transmembrane proteins. The GP(5) and M proteins are both essential for particle assembly. They occur as covalently linked heterodimers that constitute the basic protein matrix of the envelope. The GP(2b), GP(3), and GP(4) proteins occur as a heterotrimeric complex in which disulfide bonds play an important role. The function of this complex has not been established yet, but the available data suggest it to be involved in the viral entry process. Here we investigated the role of the four cysteine residues of the mature GP(2b) protein in the assembly of the GP(2b)/GP(3)/GP(4) complex. Open reading frames encoding cysteine-to-serine mutants of the GP(2b) protein were expressed independently or from a full-length infectious EAV cDNA clone. The results of these experiments support a model in which the cysteine residue at position 102 of GP(2b) forms an intermolecular cystine bridge with one of the cysteines of the GP(4) protein, while the cysteine residues at positions 48 and 137 of GP(2b) are linked by an intrachain disulfide bond. In this model, another cysteine residue in the GP(4) protein is responsible for the covalent association of GP(3) with the disulfide-linked GP(2b)/GP(4) heterodimer. In addition, our data highlight the importance of the correct association of the minor EAV envelope glycoproteins for their efficient incorporation into viral particles and for virus infectivity.

Generation of a candidate live marker vaccine for equine arteritis virus by deletion of the major virus neutralization domain.

Equine arteritis virus (EAV) is an enveloped plus-strand RNA virus of the family Arteriviridae (order Nidovirales) that causes respiratory and reproductive disease in equids. Protective, virus-neutralizing antibodies (VNAb) elicited by infection are directed predominantly against an immunodominant region in the membrane-proximal domain of the viral envelope glycoprotein G(L), allowing recently the establishment of a sensitive peptide enzyme-linked immunosorbent assay (ELISA) based on this particular domain (J. Nugent et al., J. Virol. Methods 90:167-183, 2000). By using an infectious cDNA we have now generated, in the controlled background of a nonvirulent virus, a mutant EAV from which this immunodominant domain was deleted. This virus, EAV-G(L)Delta, replicated to normal titers in culture cells, although at a slower rate than wild-type EAV, and caused an asymptomatic infection in ponies. The antibodies induced neutralized the mutant virus efficiently in vitro but reacted poorly to wild-type EAV strains. Nevertheless, when inoculated subsequently with virulent EAV, the immunized animals, in contrast to nonvaccinated controls, were fully protected against disease; replication of the challenge virus occurred briefly at low though detectable levels. The levels of protection achieved suggest that an immune effector mechanism other than VNAb plays an important role in protection against infection. As expected, infection with EAV-G(L)Delta did not induce a measurable response in our G(L)-peptide ELISA while the challenge infection of the animals clearly did. EAV-G(L)Delta or similar mutants are therefore attractive marker vaccine candidates, enabling serological discrimination between vaccinated and wild-type virus-infected animals.

Formation of disulfide-linked complexes between the three minor envelope glycoproteins (GP2b, GP3, and GP4) of equine arteritis virus.

Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae of the order NIDOVIRALES: Six transmembrane proteins have been identified in EAV particles: the nonglycosylated membrane protein M and the glycoprotein GP(5) (previously named G(L)), which occur as disulfide-bonded heterodimers and are the major viral envelope proteins; the unglycosylated small envelope protein E; and the minor glycoproteins GP(2b) (formerly designated G(S)), GP(3), and GP(4). Analysis of the appearance of the GP(2b), GP(3), and GP(4) proteins in viral particles by gel electrophoresis under reducing and nonreducing conditions revealed the occurrence of two different covalently linked oligomeric complexes between these proteins, i.e., heterodimers of GP(2b) and GP(4) and heterotrimers of GP(2b), GP(3), and GP(4). Shortly after their release from infected cells, virions contained mainly cystine-linked GP(2b)/GP(4) heterodimers, which were subsequently converted into disulfide-bonded GP(2b)/GP(3)/GP(4) trimers through the covalent recruitment of GP(3). This process occurred faster at a higher pH but was arrested at 4 degrees C. Furthermore, the conversion was almost instantaneous in the presence of the thiol oxidant diamide. In contrast, the sulfhydryl-modifying agent N-ethylmaleimide inhibited the formation of disulfide-bonded GP(2b)/GP(3)/GP(4) trimers. Using sucrose density gradients, we could not demonstrate a noncovalent association of GP(3) with the cystine-linked GP(2b)/GP(4) dimer in freshly released virions, nor did we observe higher-order structures of the GP(2b)/GP(4) or GP(2b)/GP(3)/GP(4) complexes. Nevertheless, the instantaneous diamide-induced formation of disulfide-bonded GP(2b)/GP(3)/GP(4) heterotrimers at 4 degrees C suggests that the three minor glycoproteins of EAV are assembled as trimeric complexes. The existence of a noncovalent interaction between the cystine-linked GP(2b)/GP(4) dimer and GP(3) was also inferred from coexpression experiments showing that the presence of GP(3) increased the electrophoretic mobility of the disulfide-bonded GP(2b)/GP(4) dimers. Our study reveals that the minor envelope proteins of arteriviruses enter into both covalent and noncovalent interactions, the function of which has yet to be established.

Characterization of two new structural glycoproteins, GP(3) and GP(4), of equine arteritis virus.

Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae of the order Nidovirales. Four envelope proteins have hitherto been identified in EAV particles: the predominant membrane proteins M and G(L), the unglycosylated small envelope protein E, and the nonabundant membrane glycoprotein G(S). In this study, we established that the products of EAV open reading frame 3 (ORF3) and ORF4 (designated GP(3) and GP(4), respectively) are also minor structural glycoproteins. The proteins were first characterized by various analyses after in vitro translation of RNA transcripts in a rabbit reticulocyte lysate in the presence and absence of microsomal membranes. We subsequently expressed ORF3 and -4 in baby hamster kidney cells by using the vaccinia virus expression system and, finally, analyzed the GP(3) and GP(4) proteins synthesized in EAV-infected cells. The results showed that GP(4) is a class I integral membrane protein of 28 kDa with three functional N-glycosylation sites and with little, if any, of its carboxy terminus exposed. Both after independent expression and in EAV-infected cells, the protein localizes in the endoplasmic reticulum (ER), as demonstrated biochemically by analysis of its oligosaccharide side chains and as visualized directly by immunofluorescence studies. GP(3), on the other hand, is a heavily glycosylated protein whose hydrophobic amino terminus is not cleaved off. It is an integral membrane protein anchored by either or both of its hydrophobic terminal domains and with no parts detectably exposed cytoplasmically. Also, GP(3) localizes in the ER when expressed independently and in the context of an EAV infection. Only a small fraction of the GP(3) and GP(4) proteins synthesized in infected cells ends up in virions. Most, but not all, of the oligosaccharides of these virion glycoproteins are biochemically mature. Our results bring the number of EAV envelope proteins to six.