Bellur S. Prabhakar

Virus-Induced Autoimmunity

Many important human diseases of undetermined etiology have an autoimmune component. In some diseases, the autoimmune component is very broad, involving a number of different organs and tissue antigens. For example, in the case of systemic lupus erythematosus, autoantibodies are found that react with DNA, RNA, cytoplasmic proteins, lymphocytes, and erythrocytes. Similarly, in patients with polyendocrinopathy, autoantibodies that react with the pancreas, thyroid, pituitary, and gastric mucosa have been detected. In contrast, in diseases such as myasthenia gravis, the autoimmune component is far more restricted and predominantly directed against the acetylcholine receptor.

Organ reactive autoantibodies from non-immunized adult BALB/c mice are polyreactive and express non-biased VH gene usage

To examine the naturally activated autoreactive B cell repertoire, we analyzed a panel of hybridomas from unmanipulated adult BALB/c spleen cells for reactivity patterns and VH gene usage. We found a pattern of VH usage that was diverse and appeared to reflect the germline repertoire. Although all but one natural antibody hybridoma (NAb) were initially selected for organ rather than antigen binding, the majority of organ reactive IgM NAbs were polyreactive, expressing a broad range of reactivity patterns for both self and foreign antigens, that were unique for each NAb and were not indiscriminate. Our results are consistent with the hypothesis that many naturally activated adult B cells are highly polyreactive and that autoreactivity is a consequence of polyreactivity. We suggest that the population of NAbs exhibiting organ reactivity overlaps the populations of other IgM autoantibodies that have been described previously, and that these all derive from a pool of polyreactive IgM antibodies which are polyclonally activated in the early immune response. These polyreactive natural antibodies may represent a first line of defense and offer protection for the host against a variety of foreign agents.

Characterization of Multireactive Autoantibodies and Identification of LEU-1+ B Lymphocytes as Cells Making Antibodies Binding Multiple Self and Exogenous Molecules

(1988). Characterization of Multireactive Autoantibodies and Identification of LEU-1+ B Lymphocytes as Cells Making Antibodies Binding Multiple Self and Exogenous Molecules. International Reviews of Immunology: Vol. 3, No. 1-2, pp. 17-45.

Binding and functional effects of thyroid stimulating hormone on human immune cells

The expression and functional relevance of thyroid stimulating hormone (TSH) receptors on human immune cells were studied. Flow cytometric analysis was used to study the binding of biotinylated TSH to human peripheral blood mononuclear cells (PBMC) and various purified lymphoid populations. Our results indicate that the hormone binds well to monocytes and natural killer (NK) cells and marginally to purified tonsillar T and B lymphocytes. There was a significant increase in the binding of TSH to purified B cells that were activatedin vitro withStaphylococcus aureaus Cowan. In contrast, the binding of TSH to T cells was unaltered when they were stimulated with phytohemagglutinin (PHA). While TSH increases DNA synthesis and intracellular cAMP levels of FRTL-5 rat thyroid cells, it did not have such stimulatory effects on lymphocytes. However, there was a moderate increase in Ig production by activated B lymphocytes when they were cultured in the presence of the hormone. A possible function for TSH as a link between the immune system and the thyroid is discussed.

Detection of conserved and nonconserved epitopes on coxsackievirus B4: Frequency of antigenic change

A sensitive microneutralization assay was used to detect antigenic variants of Coxsackievirus B4. Analysis of 47 clinical isolates with 16 monoclonal neutralizing antibodies revealed highly conserved, moderately conserved, and poorly conserved epitopes. Upon passage of Coxsackievirus B4 in tissue culture, certain epitopes disappeared and others appeared in the apparent absence of selective pressure. Changes in epitopes often exceeded a frequency of 10(-2). Despite considerable variation in individual epitopes, the overall antigenic composition of Coxsackievirus B4, as measured by the capacity of hyperimmune serum to neutralize clinical isolates, remains fairly constant.

Characterization of the 70kDa component of the human Ku autoantigen expressed in insect cell nuclei using a recombinant baculovirus vector

The Ku autoantigen is a human nuclear, DNA-binding heterodimer of 70kDa and 86kDa proteins. It is the target of autoantibodies in several autoimmune diseases. We now report the expression of a cDNA encoding the 70kDa Ku protein. Large amounts of protein were obtained using a recombinant baculovirus vector, in contrast with earlier unsuccessful attempts using other expression systems. We demonstrate that the 70kDa Ku protein is targeted to the nucleus and is associated with the nuclear matrix when expressed in the absence of the 86kDa Ku component. No post-translational modifications were observed. The 70kDa protein binds double and single-stranded DNA with very high affinity. Our results suggest that the baculovirus expression system may be of widespread use in the production and characterization of human autoantigens.

Monoclonal Autoantibodies That React with Multiple Organs Basis for Reactivity

We have been able to make monoclonal MOR antibodies in several different ways. First, we have been able to prepare MOR antibodies from mice with autoimmune disease and from humans with autoimmune disease. Second, we have prepared MOR antibodies from normal mice without autoimmune disease and from healthy humans without evidence of autoimmune disease. Third, we have prepared MOR antibodies by hybridoma technology and by transformation of lymphocytes with EBV. We have shown that MOR antibodies are common and are part of the hosts normal B-cell repertoire. Our studies raise the possibility that most monoclonal autoantibodies, when extensively screened, will to a greater or lesser degree be of the MOR type.

Monoclonal antibodies against osteonectin show conservation of epitopes across species

SummarySeveral monoclonal antibodies were produced to bovine osteonectin, a major noncollagenous protein in the extracellular matrix of bone, and four were characterized. These antibodies showed different reactivities in Western immunoblots, immunoprecipitation, and indirect immunofluorescence, indicating that they recognize different epitopes on the protein. The data indicate that an epitope recognized by one of the antibodies is masked in interactions of osteonectin within cells and in the extracellular matrix. The high degree of cross-species immunoreactivity observed against bone osteonectin with these monoclonal antibodies indicates that these common epitopes have been conserved during evolution.

Anti-idiotypic antibodies to monoclonal antibodies that neutralize coxsackievirus B4 do not recognize viral receptors

We have made anti-idiotypic antibodies in rabbits against three mouse monoclonal neutralizing antibodies with specificities for independent epitopes on Coxsackievirus B4. Each of these anti-idiotypic antibodies was found to react specifically with the immunizing monoclonal antibody in radioimmunoassays and did not react with the other monoclonal antibodies. In addition, the anti-idiotypic antibodies specifically inhibited the function (i.e., virus neutralization) of the immunizing antibody. These anti-idiotypic antibodies were tested for their ability to recognize receptors for Coxsackievirus B4 as measured by their ability to inhibit the attachment of radiolabeled virus to cellular receptors. The anti-idiotypic antibodies did not block the binding of Coxsackievirus B4 to monkey kidney cells. Moreover, when tested for their ability to bind to other receptor positive cells, none of the anti-idiotypic antibodies bound above control levels. Anti-idiotypic antibodies did induce a small anti-anti-idiotypic antibody response in mice when tested by radioimmunoassay; however, little if any virus neutralizing activity was found in the sera of these mice. Our results contrast to those reported for anti-idiotypic antibodies in several other virus systems and suggest that not all anti-idiotypic antibodies made against neutralizing antibodies are capable of eliciting an antiviral immune response or binding to viral viral receptors on cells.

Monoclonal Antibody Techniques Applied to Viruses

Publisher Summary A polyclonal serum against a given virus may have the ability to neutralize the virus, fix complement, inhibit hemagglutination by the virus, or facilitate killing of virus-infected cells by macrophages and lymphocytes. Monoclonal antibodies against a large number of viruses have already been generated, and some of them have been characterized as to their biological properties. Monoclonal antibodies are useful in discerning the structural and functional properties of various viral proteins and have been instrumental in the identification of a large number of antigenic variants that were not previously known. This chapter presents a list of a number of representative viruses against which monoclonal antibodies have been generated, including the nature of antigens used for immunization, the assay for detection of antibodies, and the type of antibodies obtained. It discusses the principles of hybridoma techniques with a broad technical description. The chapter discusses the general strategy for immunizing animals, the fusion protocol, various methods for antibody screening, characterization of antibodies and viral antigens, and the generation of viral variants. It also presents a specific protocol for preparing various reagents.