Safety, tolerability, and pharmacodynamics of an anti-interleukin-1α/ß dual variable domain immunoglobulin in patients with osteoarthritis of the knee: a randomized phase 1 study.

OBJECTIVE: To investigate the safety, tolerability, pharmacokinetics, and pharmacodynamics of ABT-981, a human dual variable domain immunoglobulin simultaneously targeting interleukin (IL)-1α and IL-1ß, in patients with knee osteoarthritis (OA). METHOD: This was a randomized, double-blind, placebo-controlled, single-center study of multiple subcutaneous (SC) injections of ABT-981 in patients with mild-to-moderate OA of the knee (NCT01668511). Three cohorts received ABT-981 (0.3, 1, or 3 mg/kg) or placebo every other week for a total of four SC injections, and one cohort received ABT-981 (3 mg/kg) or placebo every 4 weeks for a total of three SC injections. Assessment of safety and tolerability were the primary objectives. A panel of serum and urine biomarkers of inflammation and joint degradation were evaluated. RESULTS: A total of 36 patients were randomized (ABT-981, n = 28; placebo, n = 8); 31 (86%) completed the study. Adverse event (AE) rates were comparable between ABT-981 and placebo (54% vs 63%). The most common AE reported with ABT-981 vs placebo was injection site erythema (14% vs 0%). ABT-981 significantly reduced absolute neutrophil count and serum concentrations of IL-1α/IL-1ß, high-sensitivity C-reactive protein, and matrix metalloproteinase (MMP)-derived type 1 collagen. Serum concentrations of MMP-derived type 3 collagen and MMP-degraded C-reactive protein demonstrated decreasing trends with ABT-981. Antidrug antibodies were found in 37% of patients but were not associated with the incidence or severity of AEs. CONCLUSION: ABT-981 was generally well tolerated in patients with knee OA and engaged relevant tissue targets, eliciting an anti-inflammatory response. Consequently, ABT-981 may provide clinical benefit to patients with inflammation-driven OA.

Analysis of human immunodeficiency virus type 1 mRNA splicing patterns during disease progression in peripheral blood mononuclear cells from infected individuals.

HIV-1 produces more than 20 mRNAs encoding the viral proteins. We have used a sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) approach to determine HIV-1 transcriptional patterns during the course of viral infection in unstimulated peripheral blood mononuclear cells (PBMCs) from different patients. Several sets of PCR primers, used in parallel reactions, allowed the amplification and specific detection of almost all individual HIV-1 transcripts. We investigated the transcriptional profile in two individuals during primary acute and early chronic infection. In these individuals, HIV-1 mRNA expression was elevated at the first time points examined and declined over time. In addition, we performed a detailed study of HIV-1 expression in several individuals over a minimum of 7 years following seroconversion. We found that long-term asymptomatic individuals had undetectable or low levels of the three classes of HIV-1 transcripts (unspliced, singly spliced, and multiply spliced). Individuals who demonstrated disease progression showed either a general increase in the amount of expression of all transcripts or elevated levels of unspliced transcripts in late-stage disease. The splicing pattern in each patient was conserved over the years and differed among the different individuals. No evidence of major changes in the splicing pattern was found during disease progression within the same individual. Thus, HIV-1 transcriptional patterns are viral strain specific rather than disease stage specific. These results indicate that high-level expression of any class of HIV-1 transcripts is associated with clinical progression. Our analysis also demonstrates the importance of using more than one set of primers to evaluate HIV-1 RNA expression, since virus in patient PBMCs showed sequence heterogeneity in conserved regions.

A Rev protein is expressed in caprine arthritis encephalitis virus (CAEV)-infected cells and is required for efficient viral replication.

Caprine arthritis encephalitis virus (CAEV) is a lentivirus that is closely related to visna virus and more distantly related to the human lentivirus human immunodeficiency virus 1 (HIV-1). Like other lentiviruses, the genome of CAEV contains multiple small ORFs that encode viral regulatory proteins. Sequence analysis of the CAEV genome and cDNAs generated from mRNA in infected cells has suggested that one of these ORFs encodes a protein (Rev-C) that is analogous to Rev of visna virus and HIV. Antibodies generated to a carboxy-terminal peptide of the rev ORF immunoprecipitate an 18-kDa protein from cells transfected with the Rev cDNA clone. Immunoprecipitation and immunofluorescence analysis of CAEV-infected ovine primary cells show that the product of the rev ORF is expressed during infection and localizes to the nucleolus of infected cells. Also, sera from CAEV-infected goats specifically immunoprecipitates an in vitro-translated product from the full-length Rev cDNA clone as well as that from the unique second open reading frame of Rev-C which shows that the Rev-C protein is expressed during natural CAEV infection of animals. Insertion of either a mutation that creates two stop codons in the unique second open reading frame of Rev-C or a mutation in the basic domain of Rev-C into the CAEV infectious molecular clone renders the virus unable to replicate in primary goat synovial membrane cells. Analysis of the RNA and proteins produced from both Rev-deficient clones indicates that they are defective in the accumulation of structural gene mRNAs in the cytoplasm as well as in synthesis of structural proteins compared to the wild-type CAEV clone. These data indicate that CAEV encodes a Rev protein that is required for efficient viral replication in culture.

Identification of the caprine arthritis encephalitis virus Rev protein and its cis-acting Rev-responsive element.

Caprine Arthritis Encephalitis Virus (CAEV) is a lentivirus closely related to visna virus of sheep and more distantly related to the human lentivirus HIV-1. The genomes of lentiviruses contain additional genes that regulate the lentivirus gene expression; one of these is Rev, a protein that regulates the expression of viral proteins via post-transcriptional mechanisms. A cDNA clone was isolated from CAEV infected cells and shown to encode the 18-kDa Rev protein of CAEV. Antibodies against CAEV Rev (Rev-C) demonstrated that the CAEV Rev protein accumulated in the nucleus and in particular in the nucleolus of transiently transfected cells. Mutation of a basic region in the CAEV Rev protein resulted in loss of nucleolar localization. A highly structured RNA element has been identified in the env gene of CAEV (nt 7850-8150); its structure and location suggested that it was analogous to the Rev-responsive element (RRE) of HIV-1 and visna virus. A 300-bp fragment (nt 7850-8150) spanning this region was substituted for the HIV-1 RRE in an HIV-1 Gag expression vector. Expression of the Gag protein was dramatically increased when Rev-C was added in trans, indicating that this fragment contained the cis-acting CAEV Rev Responsive Element. Cross-activation by the Rev/Rex proteins of other lentiviruses and members of the HTLV-I family indicated that this RRE could interact with Rev or Rex proteins of other viruses. This suggests that the highly divergent lentiviruses share similar mechanisms and cofactors regulating post-transcriptional viral gene expression. The Rev/RRE mechanism is thus the most conserved regulatory mechanism in lentiviruses and other complex retroviruses.

The CAEV tat gene trans-activates the viral LTR and is necessary for efficient viral replication.

Caprine arthritis-encephalitis virus (CAEV) is a lentivirus which is closely related by nucleotide sequence and biological properties to visna virus. Sequence analysis of the CAEV genome revealed the presence of a small open reading frame (ORF) which shares amino acid identity with the visna virus tat gene. Using an infectious molecular clone of CAEV the role of the tat ORF in viral replication was examined. Mutations were made in the tat ORF that introduced two in frame stop codons six amino acids downstream of the tat AUG; in addition, a deletion mutant was made that removed most of the tat ORF. Both of these mutants had greatly reduced virus titers (> 1000-fold less than the wild type infectious clone). Co-transfection of a tat expressing plasmid with these viruses containing the tat ORF mutations resulted in higher levels of virus production demonstrating that the effects of both mutants are tat specific. These mutants provide data that the CAEV tat gene is necessary for efficient virus replication. Analysis of the RNA in these transfected cells showed that complementation of the tat gene was in trans and not the result of recombination. Analysis of the gag and rev proteins in the transfected cells demonstrated that these proteins were not detectable in cells transfected with the tat mutants but could be readily detected when the mutations were complemented in trans with a tat expression vector. To test for tat mediated trans-activation a plasmid expressing the CAEV tat ORF was co-transfected with plasmids containing either the CAEV or visna virus LTR driving transcription of the bacterial chloramphenicol acetyltransferase gene (CAT). These experiments indicate that one function of the CAEV tat protein is to trans-activate gene expression from the viral promoter. RNase protection analysis of CAT mRNA from co-transfected cells demonstrated that CAEV Tat trans-activates gene expression by increasing steady-state levels of mRNA.