Karim Essani

Multiple organ-reactive monoclonal autoantibodies

Autoantibodies directed against a wide range of normal tissue antigens have been found in the sera of patients with autoimmune diseases1–8. It is generally thought that different and specific autoantibodies react with different tissues but the possibility exists that some autoantibodies may react with common antigens found in different tissues and organs. Recently, we showed that mice infected with reovirus developed a polyendocrine disease with autoantibodies to the pancreas, anterior pituitary, thymus and gastric mucosa9,10. Using hybridoma technology, we obtained a number of monoclonal autoantibodies11 which reacted with antigens in single organs. We now report the production and pattern of reactivity of seven multiple organ-reactive monoclonal autoantibodies. By using antibody-affinity columns, autoantigens also have been isloated and their molecular weights determined. The results suggest that monoclonal multiple organ-reactive autoantibodies react either with the same molecule present in several organs or with common antigenic determinants on different molecules in multiple organs. In either case, the existence of multiple organ-reactive antibodies may be a partial explanation for multiple organ autoimmunity.

A secreted high-affinity inhibitor of human TNF from Tanapox virus

A class of secreted poxvirus tumor necrosis factor (TNF)-binding proteins has been isolated from Tanapox-infected cell supernatants. The inhibitor bound to a TNF-affinity column and was identified as the product of the 2L gene. Sequence analysis of 2L family members from other yatapoxviruses and swinepox virus yielded no sequence homology to any known cellular gene. The expressed Tanapox virus 2L protein bound to human TNF with high affinity (Kd = 43 pM) and exhibits an unusually slow off-rate. However, 2L is unable to bind to a wide range of human TNF family members. The 2L protein can inhibit human TNF from binding to TNF receptors I and II as well as block TNF-induced cytolysis. Thus, Tanapox virus 2L represents an inhibitor of human TNF and offers a unique strategy with which to modulate TNF activity.

Amphibian and Piscine Iridoviruses Proposal for Nomenclature and Taxonomy Based on Molecular and Biological Properties

We have compared a number of properties of the well-characterized iridovirus, frog virus 3, with two other iridoviruses from amphibia, bullfrog edema virus and Lucké triturus virus, and with a piscine iridovirus, goldfish virus (GFV), to provide information for developing taxonomic classification of these viruses and establishing their ecological niche. Purified virions had similar size and shape (icosahedral) for each virus, and the genomic DNAs of each virus were methylated by a virus-induced DNA methyltransferase. The three amphibian viruses replicated equally well in fish (FHM), hamster (BHK), and human (WI-38) cell monolayer with identical cytopathology, while GFV failed to replicate in these cell lines. However, GFV replicated albeit at a slow rate, in a goldfish cell line; there was no detectable replication by the amphibian viruses in these cells. The amphibian iridoviruses had virtually similar DNA sequences, while those of GFV were markedly different. Analyses of virus-induced polypeptides in infected cells corroborated the DNA analyses; the polypeptides of the amphibian viruses were similar and distinct from those of the fish virus. Nongenetic reactivation could only be accomplished between the three amphibian viruses but not with the piscine virus. Based on these data, we suggest taxonomic and nomenclature designations of amphibian and piscine iridoviruses.

Human multiple organ-reactive monoclonal autoantibody recognizes growth hormone and a 35,000-molecular weight protein.

By fusing peripheral leukocytes from a patient with insulin-dependent diabetes with mouse myeloma cells, a heterohybridoma was isolated that, for over one year, has secreted a human monoclonal autoantibody, designated MOR-h1 (multiple organ-reactive human 1). This antibody reacts with antigens in several endocrine organs including the pituitary, thyroid, stomach, and pancreas. By double immunofluorescence, MOR-h1 was found to react specifically with growth hormone (GH)-containing cells in the anterior pituitary and, by enzyme-linked immunosorbent assay, MOR-h1 was shown to react with both natural and biosynthetic GH. Absorption experiments revealed that GH could remove the capacity of MOR-h1 to react not only with cells in the anterior pituitary, but also with cells in the thyroid, stomach, and pancreas. The demonstration with hyperimmune serum that these organs do not contain GH indicated that MOR-h1 was reacting with a different molecule(s) in these organs. By passing extracts of pituitary, thyroid, and stomach through an MOR-h1 affinity column and analyzing the eluted antigens by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a 35,000-mol wt polypeptide was isolated from each of these organs. In addition, a 21,500-mol wt polypeptide with an electrophoretic mobility identical to purified human GH was isolated from the pituitary, but not the other organs. It is concluded that MOR-h1 reacts with a 35,000-mol wt polypeptide present in the pituitary, thyroid, and stomach and that this antibody also recognizes a determinant on GH.

Oncolytic tanapoxvirus expressing FliC causes regression of human colorectal cancer xenografts in nude mice

Colorectal cancers are significant causes of morbidity and mortality and existing therapies often perform poorly for individuals afflicted with advanced disease. Oncolytic virotherapy is an emerging therapeutic modality with great promise for addressing this medical need. Herein we describe the in vivo testing of recombinant variants of the tanapoxvirus (TPV). Recombinant viruses were made ablated for either the 66R gene (encoding a thymidine kinase), the 2L gene (encoding a TNF-binding protein), or both. Some of the recombinants were armed to express mouse chemotactic protein 1 (mCCL2/mMCP-1), mouse granulocyte-monocyte colony stimulating factor (mGM-CSF), or bacterial flagellin (FliC). Tumors were induced in athymic nude mice by implantation of HCT 116 cells and subsequently treated by a single intratumoral injection of one of the recombinant TPVs. Histological examination showed a common neoplastic cell type and a range of immune cell infiltration, necrosis, and tumor cell organization. Significant regression was seen in tumors treated with virus TPV/Δ2L/Δ66R/fliC, and to a lesser extent the recombinants TPV/Δ2L and TPV/Δ66R. Our results suggest that oncolytic recombinants of the TPV armed with activators of the innate immune response may be effective virotherapeutic agents for colorectal cancers in humans and should be explored further to fully realize their potential.

Interaction of human TNF and β2-microglobulin with Tanapox virus-encoded TNF inhibitor, TPV-2L

Tanapox virus (TPV) encodes and expresses a secreted TNF-binding protein, TPV-2L or gp38, that displays inhibitory properties against TNF from diverse mammalian species, including human, monkey, canine and rabbit. TPV-2L also has sequence similarity with the MHC-class I heavy chain and interacts differently with human TNF as compared to the known cellular TNF receptors or any of the known virus-encoded TNF receptor homologs derived from many poxviruses. In order to determine the TNF binding region in TPV-2L, various TPV-2L C-terminal truncations and internal deletions were created and the muteins were expressed using recombinant baculovirus vectors. C-terminal deletions from TPV-2L resulted in reduced binding affinity for human TNF and specific mutants of TNF that discriminate between TNF-R1 and TNF-R2. However, deletion of C-terminal 42 amino acid residues totally abolished the binding of human TNF and its mutants. Removal of any of the predicted internal domains resulted in a mutant TPV-2L protein incapable of binding to human TNF. Deletion of C-terminal residues also affected the ability of TPV-2L to block TNF-induced cellular cytotoxicity. In addition to TNF, TPV-2L can also form complexes with human beta2-microglobulin to form a novel macromolecular complex. In summary, the TPV-2L protein is a bona fide MHC-1 heavy chain family member that binds and inhibits human TNF in a fashion very distinct from other known poxvirus-encoded TNF inhibitors, and also can form a novel complex with the human MHC-1 light chain, beta2-microglobulin.

Biogenesis of vaccinia: Involvement of spicules of the envelope during virion assembly examined by means of conditional lethal mutants and serology☆

Abstract Infection with vaccinia mutant is 6757 under restrictive conditions results in formation of large quantities of viral envelopes. Unlike envelopes which normally surround immature virions, aberrant envelopes assembled under control of this mutant, designated IE, were formed apart from the internal constituents. However, like the envelopes of normal immature virions, IE possessed an external layer of spicules. Following isolation and concentration of IE, antisera were prepared in rabbits which specifically immuno-precipitated a major IE polypeptide of MW 65,000 labeled p65E. Experimental evidence was obtained by several approaches in identifying the spicules as the p65E polypeptide. Data obtained with antisera specific against p65E, combined with biochemical and electron-microscopic observations on the development of several ts mutants, including 6757, are consistent with the view that spicules attached onto envelopes function to provide transitory scaffolding. During virion maturation, the spicules are replaced by the surface tubular elements or STE, characterized by Stern and Dales ((1976), Virology, 75, 232–241).

A neutralizing monoclonal antibody against Coxsackievirus B4 cross-reacts with contractile muscle proteins☆

A panel of Coxsackievirus B4 (CVB4) neutralizing monoclonal antibodies (mAbs) were tested against a panel of normal mouse tissues. One mAb, 356-1, reacted specifically with murine heart tissue. Immunohistochemical studies revealed an A band pattern of staining of the heart. Examination of sequential differential extracts of heart by Western immunoblotting showed that 356-1 predominantly reacted with the murine cardiac myosin heavy chain. A rather weak cross-reaction was found with actin. These observations were confirmed by the binding of 356-1 to purified cardiac myosin and actin. This antibody showed a higher affinity for murine cardiac muscle myosin than for skeletal muscle myosin. Examination of the reactivity of 356-1 with CVB4 polypeptides using Western immunoblotting revealed that 356-1 binds to the VP-1 capsid protein. These studies imply that molecular mimicry is one mechanism by which autoimmunity could develop during CVB4 induced myocarditis.

Transcription of methylated viral DNA by eukaryotic RNA polymerase II

The genome of the large icosahedral DNA virus, frog virus 3 (FV3), is heavily methylated at the cytosine residues of dCdG dinucleotide pairs, with more than 22% of the total cytosine residues in the form of 5-methylcytosine (5mC). This methylation is carried out postreplicatively in the cytoplasm of infected cells by a virus-encoded DNA methyltransferase. DNA methyltransferase activity was shown to copurify with a 26 kD virus-induced, DNA-binding protein that had an altered mobility in extracts from cells infected with a DNA-methyltransferase deficient mutant of FV3. Immediately after infection, the highly methylated parental DNA is transcribed in the nucleus by the host cell RNA polymerase II. As FV3 induces the synthesis of a protein that can override the inhibitory effect of methylation on the transcription of exogenous promoters methylatedin vitro, we suggest that this protein is a factor evolved by this virus to allow transcription from methylated promoters by eukaryotic RNA polymerase II.

Mutation in a DNA-binding protein reveals an association between DNA-methyltransferase activity and a 26,000-Da polypeptide in frog virus 3-infected cells

The DNA of frog virus 3 (FV3), an iridovirus, is highly methylated; more than 20% of the cytosine bases are methylated at the 5-carbon position by an FV3-induced DNA methyltransferase (DNA-mt). To determine the role of this enzyme in virus replication and regulation of gene expression, we have analyzed an FV3 mutant that lacks DNA-mt activity and is resistant to 5-azacytidine (an inhibitor of DNA-mt). Comparative polypeptide analysis, using cytoplasmic extracts from the wild-type FV3 and mutant-infected cells, revealed that a single protein of 26,000 (26K) molecular weight was altered in the mutant-infected cells. The altered polypeptide migrated faster in SDS-polyacrylamide gel as compared to the wild-type FV3 26K protein. Five spontaneous revertants derived from the mutant regained the migrational characteristic of the wild-type 26K protein, DNA-mt activity, and methylation of their DNA. We further show that the 26K polypeptide is a DNA-binding protein and that 80% of the enzyme activity can be eluted from an ssDNA affinity column. Taken together, these data support the conclusion that the 26K polypeptide is associated with DNA-mt activity.