Field study of the safety, immunogenicity, and efficacy of an inactivated equine rotavirus vaccine.

OBJECTIVE: To determine safety, immunogenicity, and efficacy of an inactivated equine rotavirus vaccine. DESIGN: Prospective randomized controlled trial. ANIMALS: 316 pregnant Thoroughbred mares during the first year of the study and 311 during the second year. PROCEDURE: During the first year, mares received 3 doses of vaccine or placebo, IM, at 8, 9, and 10 months of gestation. Serum neutralizing antibody titers were measured before vaccination and 1 and 35 days after foaling. Antibody titers were measured in foals 1, 7, 35, 60, 90, and 120 days after birth. During the second year, mares that had been vaccinated the previous year received a single booster dose of vaccine approximately 1 month prior to parturition. Mares that had received the placebo the previous year and mares new to the study received 3 doses of vaccine or placebo. Serum neutralizing antibody titers were measured in samples taken from mares approximately 1 day after foaling and from foals approximately 1 and 60 days after birth. RESULTS: Adverse reactions were not observed. Antibody titers were significantly increased at the time of foaling and 35 days after foaling in vaccinated, compared with control, mares and for 90 days after birth in foals born to vaccinated, compared with foals born to control, mares. Incidence of rotaviral diarrhea was lower in foals born to vaccinated, compared with foals born to control, mares, but the difference was not significant. CLINICAL IMPLICATIONS: Results suggest that the equine rotavirus vaccine is safe and immunogenic and that reasonable efficacy under field conditions can be expected.

Identification of a putative shared epitope between Coxsackie virus B4 and alpha cardiac myosin heavy chain.

Molecular mimicry is an important postulated mechanism for autoimmunity in viral myocarditis. The 356-1 monoclonal antibody neutralizes Coxsackie virus B4 by binding to the VP1 protein and cross-reacts with mouse alpha cardiac myosin heavy chain. We used this monoclonal antibody to screen a lambda gt11 expression library made from CD-1 mouse hearts. Of the 48 positive plaques/10(6) recombinant phages examined, 14 of the strongest-reacting clones were purified for additional studies. The inserts were amplified by polymerase chain reaction and the amplified products ranged from about 150 to 1400 bp in size. Northern hybridization using these inserts demonstrated that 11 out of 14 reacted with a message equivalent to that of cardiac myosin in size. Additional Southern hybridization studies suggested that these 11 inserts contained overlapping sequences in the light meromyosin fragment of cardiac myosin. Sequence analysis confirmed that these 11 independent, recombinant clones contained a common sequence representing amino acid residues 1299-1647. Within this fragment only one isoform-specific site matched the observed reactivity pattern of 356-1 among hearts from various species. Thus, we were able to identify a putative shared epitope represented by residues 1632-1647.

Molecular cloning of a heart antigen that cross-reacts with a neutralizing antibody to Coxsackievirus B4.

A panel of coxsackievirus B4 (CVB4) neutralizing monoclonal antibodies was tested against a panel of normal mouse tissues. One mAb, 356-1, reacted specifically with murine heart tissue. Examination of the reactivity of 356-1 with CVB4 polypeptides using Western immunoblotting revealed that 356-1 binds to the VP-1 capsid protein. Immunohistochemical studies revealed an A band pattern of staining of the heart by this antibody. Western immunoblotting of sequential differential extracts of heart 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. The monoclonal antibody 356-1 was then used to screen a lambda gt11 CD1 mouse heart cDNA expression library. Forty-eight positive plaques were obtained, 14 of which reacted strongly with the antibody and were selected for additional studies. In 13/14 clones, inserts were amplified using the polymerase chain reaction (PCR). The PCR products ranged in size from approximately 150 to 1400 bp. Northern hybridization using these inserts demonstrated that 10/13 recognized a approximately 6.5 kb message in mouse heart total RNA and not in liver total RNA. These amplified inserts were sequenced and were found to contain sequences which encode approximately 1/3 of the amino-terminal end of light meromyosin. By comparison with known sequences of rat alpha and beta cardiac myosin heavy chains, we were able to identify sequences specific for alpha chain as potential cross-reactive autoantigenic epitope.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell surface expression of the 70-kD component of Ku, a DNA-binding nuclear autoantigen.

The Ku complex, a heterodimer of 86- and 70-kD proteins, is a nuclear DNA-binding autoantigen. However, hydrophobicity analysis of the deduced amino acid sequence of the 70-kD protein had strongly suggested that this might also be a membrane protein. In the present study, using antibodies to synthetic peptides and a polyclonal antiserum to the purified protein, we show that the 70-kD protein of the Ku complex is present in isolated plasma membranes of human cells. By indirect immunofluorescence microscopy and fluorescein-activated cell sorting, we demonstrate that this autoantigen is exposed on the cell surface. In addition, we have identified several domains of the protein that are exposed. Our study provides one of the first demonstrations of a eukaryotic, nuclear DNA-binding protein that is also on the cell membrane. Moreover, our results might help explain how autoantibodies to the Ku autoantigen could target cells for an autoimmune attack.

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.

Scrapie-induced diabetes mellitus in hamsters.

Scrapie-infected hamsters had slightly elevated non-fasting plasma glucose levels, markedly abnormal glucose tolerance tests, and impaired release of insulin in response to a glucose load. Plasma cortisol levels were essentially the same in infected and uninfected animals. Histological examination of the pancreas revealed no morphological changes in infected animals with no alteration in distribution of cells secreting insulin, glucagon and somatostatin. In contrast, brains of scrapie-infected animals had the diffuse vacuolation typical of spongiform encephalopathy. These experiments suggest that scrapie-induced diabetes mellitus in hamsters may result from damage to the central nervous system.

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.

Spontaneously proliferating human B lymphocytes make autoantibodies.

Peripheral blood lymphocytes from 29 patients with autoimmune diseases and eight normal controls were cultured in vitro. Several weeks later, lymphocyte proliferation was observed in many of these cultures. Cell lines were obtained from about half of the donors, at a frequency of approximately 1 in 10(7) lymphocytes. Outgrowth occurred only in cultures from patients who had antibodies to the viral capsid antigen of Epstein-Barr virus (EBV). The proliferating cells also contained EBV nuclear antigen, an occurrence confirming that proliferation was a result of latent EBV infection of the donors. Supernatants from these cell lines were tested for autoantibodies by screening against normal tissues. About 30% of the cell lines made autoantibodies, many of which reacted with smooth muscle or epithelial cells. Because EBV is harbored in a latent form in the majority of the adult population, reactivation of latent EBV infection in vivo may explain the production of autoantibodies seen in a variety of immunoregulatory disorders.

Virus-induced thyroiditis.

Mice infected with reovirus type 1 developed a mild thyroiditis characterized by focal destruction of acinar tissue, infiltration of inflammatory cells, and autoantibodies to thyroglobulin and microsomal antigens. Thyroid involvement appears to be part of a more generalized virus-induced polyendocrine disease.

Selection of coxsackievirus B4 variants with monoclonal antibodies results in attenuation.

Inoculation of suckling mice with coxsackievirus B4 (CB4) results in the death of a majority of the animals. In this study we selected antigenic variants of CB4 in the presence of neutralizing monoclonal antibodies and tested them to see whether they were attenuated. Antigenic variants selected with a single antibody showed little or no attenuation by producing a high mortality (60 to 100%). A double variant selected with two antibodies showed considerable attenuation by causing only 25% mortality. A triple variant selected with three antibodies was almost completely attenuated (killed only 5% of the animals). Polypeptides from these variants were tested for their ability to interact with the monoclonal antibodies used for their selection. These studies showed that resistance of variant virus to neutralization in general was due to the inability of the antibody to bind to the virus. However, one of the antibodies could bind but not neutralize the virus, perhaps due to an alteration in the epitope. It is concluded that selection of CB4 variants using more than one neutralizing monoclonal antibody can lead to attenuation of the virus.

Multiple organ-reactive IgG antibody induced by an antiidiotypic antibody to a human monoclonal IgM autoantibody.

MOR-h1 is a human multiple organ-reactive (MOR) monoclonal autoantibody (Ab1) that reacts with human growth hormone (hGH) and a 35 kD protein found in the anterior pituitary, thyroid, stomach, and pancreas. 4E6 is a mouse monoclonal anti-idiotypic antibody (Ab2) that reacts with the paratope of MOR-h1 and is ligand inhibitable. In the present study, we immunized a rabbit with 4E6 and purified an IgG fraction (anti-4E6) from the sera. Competitive inhibition experiments showed that anti-4E6 (Ab3) binds to the same epitope on 4E6 and to the same antigens (i.e., hGH and 35 kD protein) as does MOR-h1. By immunofluorescence, anti-4E6, an IgG antibody, shows the same multiple organ reactivity with tissues as does MOR-h1, an IgM antibody. From these and other studies, we conclude that the 4E6 paratope (Ab2) has a conformational resemblance to an epitope on hGH and the 35 kD protein. This raises the possibility that antibodies made in response to certain anti-idiotypic antibodies may be one of the mechanisms for triggering an autoimmune response.

Molecular mimicry: frequency of reactivity of monoclonal antiviral antibodies with normal tissues.

More than 600 monoclonal antiviral antibodies made against 11 different viruses were screened against 14 different organs from normal uninfected mice. Of these antiviral antibodies, 21, or approximately 3.5%, reacted with specific cells in these organs. Several of these antibodies were of the multiple-organ-reactive type and recognized antigens in more than one organ. It was concluded that the reactivity of monoclonal antiviral antibodies with normal tissues is a common phenomenon.