Inactivation of respiratory syncytial virus by zinc finger reactive compounds

<p>Abstract</p> <p>Background</p> <p>Infectivity of retroviruses such as HIV-1 and MuLV can be abrogated by compounds targeting zinc finger motif in viral nucleocapsid protein (NC), involved in controlling the processivity of reverse transcription and virus infectivity. Although a member of a different viral family (<it>Pneumoviridae</it>), respiratory syncytial virus (RSV) contains a zinc finger protein M2-1 also involved in control of viral polymerase processivity. Given the functional similarity between the two proteins, it was possible that zinc finger-reactive compounds inactivating retroviruses would have a similar effect against RSV by targeting RSV M2-1 protein. Moreover, inactivation of RSV through modification of an internal protein could yield a safer whole virus vaccine than that produced by RSV inactivation with formalin which modifies surface proteins.</p> <p>Results</p> <p>Three compounds were evaluated for their ability to reduce RSV infectivity: 2,2'-dithiodipyridine (AT-2), tetraethylthiuram disulfide and tetramethylthiuram disulfide. All three were capable of inactivating RSV, with AT-2 being the most potent. The mechanism of action of AT-2 was analyzed and it was found that AT-2 treatment indeed results in the modification of RSV M2-1. Altered intramolecular disulfide bond formation in M2-1 protein of AT-2-treated RSV virions might have been responsible for abrogation of RSV infectivity. AT-2-inactivated RSV was found to be moderately immunogenic in the cotton rats <it>S.hispidus </it>and did not cause a vaccine-enhancement seen in animals vaccinated with formalin-inactivated RSV. Increasing immunogenicity of AT-2-inactivated RSV by adjuvant (Ribi), however, led to vaccine-enhanced disease.</p> <p>Conclusions</p> <p>This work presents evidence that compounds that inactivate retroviruses by targeting the zinc finger motif in their nucleocapsid proteins are also effective against RSV. AT-2-inactivated RSV vaccine is not strongly immunogenic in the absence of adjuvants. In the adjuvanted form, however, vaccine induces immunopathologic response. The mere preservation of surface antigens of RSV, therefore may not be sufficient to produce a highly-efficacious inactivated virus vaccine that does not lead to an atypical disease.</p

Molecular imaging of rheumatoid arthritis by radiolabelled monoclonal antibodies: new imaging strategies to guide molecular therapies

The closing of the last century opened a wide variety of approaches for inflammation imaging and treatment of patients with rheumatoid arthritis (RA). The introduction of biological therapies for the management of RA started a revolution in the therapeutic armamentarium with the development of several novel monoclonal antibodies (mAbs), which can be murine, chimeric, humanised and fully human antibodies. Monoclonal antibodies specifically bind to their target, which could be adhesion molecules, activation markers, antigens or receptors, to interfere with specific inflammation pathways at the molecular level, leading to immune-modulation of the underlying pathogenic process. These new generation of mAbs can also be radiolabelled by using direct or indirect method, with a variety of nuclides, depending upon the specific diagnostic application. For studying rheumatoid arthritis patients, several monoclonal antibodies and their fragments, including anti-TNF-α, anti-CD20, anti-CD3, anti-CD4 and anti-E-selectin antibody, have been radiolabelled mainly with 99mTc or 111In. Scintigraphy with these radiolabelled antibodies may offer an exciting possibility for the study of RA patients and holds two types of information: (1) it allows better staging of the disease and diagnosis of the state of activity by early detection of inflamed joints that might be difficult to assess; (2) it might provide a possibility to perform ‘evidence-based biological therapy’ of arthritis with a view to assessing whether an antibody will localise in an inflamed joint before using the same unlabelled antibody therapeutically. This might prove particularly important for the selection of patients to be treated since biological therapies can be associated with severe side-effects and are considerably expensive. This article reviews the use of radiolabelled mAbs in the study of RA with particular emphasis on the use of different radiolabelled monoclonal antibodies for therapy decision-making and follow-up

The effect of aglycosylation on the immunogenicity of a humanized therapeutic CD3 monoclonal antibody.

The factors affecting the immunogenicity of a humanized gamma 1 CD3 monoclonal antibody (mAb) were investigated in transgenic mice that express the human CD3 antigen epsilon polypeptide (the mAb target antigen). Two derivatives of the mAb were employed, one with a normal, glycosylated Fc region (gamma 1 CD3 mAb), and the other with an aglycosylated Fc region (aglycosyl gamma 1 CD3 mAb). Comparisons of the antiglobulin responses elicited by the two derivatives in transgenic and nontransgenic mice demonstrated that Fab-mediated cell binding activity, dependent on target antigen expression, was a major positive determinant of CD3 mAb immunogenicity. A second positive factor was mAb Fc region glycosylation. At low dose levels the gamma 1 CD3 mAb consistently produced a higher antiglobulin response than the aglycosyl gamma 1 CD3 mAb. This was probably a result of the nonspecific, in vivo T cell activating property of the gamma 1 CD3 mAb, a consequence of its ability to cross-link T cells to Fc gamma receptor-bearing cells. (The aglycosyl gamma 1 CD3 mAb has a reduced Fc binding affinity for Fc gamma receptors and so does not activate T cells in vivo.) In support of this hypothesis, the gamma 1 CD3 mAb was able to nonspecifically enhance humoral immunity to an unrelated, coadministered antigen, whereas the aglycosyl gamma 1 CD3 mAb was not. The lower immunogenicity of the aglycosyl gamma 1 CD3 mAb correlated with a longer in vivo half-life and an improved capacity to block the target CD3 antigen. These results suggest that, as well as reducing the cytokine-induced side effects normally associated with CD3 mAb therapy, the nonactivating aglycosyl gamma 1 CD3 mAb will be less likely than the activating gamma 1 CD3 mAb to stimulate a neutralizing antiglobulin response

A humanized monovalent CD3 antibody which can activate homologous complement.

The rat monoclonal antibody (mAb) YTH12.5, specific for the CD3 antigen complex on human T cells has been modified in order to improve its efficacy in human therapy. With the aim of rendering it less immunogenic, it has been humanized using the method of framework grafting. During this process sequence analysis of the YTH12.5 VL gene indicated that it was of the lambda subclass, however, it was markedly dissimilar from previously published rat and mouse V lambda gene sequences and may represent a new V lambda gene family. The humanization of this light chain represents the first successful reshaping of a lambda light chain V region. To improve the effector function of the antibody we have created a monovalent form (1 Fab, 1 Fc) using a novel method involving the introduction of an N-terminally truncated human IgG1 heavy chain gene into cells producing the humanized CD3 mAb. Comparison of the mono- and bivalent humanized mAb in a complement-mediated cell lysis assay revealed that the monovalent antibody mediated lysis of human T cell blasts whereas the bivalent form did not. The availability of a humanized, complement-fixing CD3 mAb may improve opportunities for human therapy, in the management of organ rejection, autoimmunity and the treatment of T cell lymphoma

Engineering multiple-domain forms of the therapeutic antibody CAMPATH-1H: effects on complement lysis.

Antibody-mediated lysis of cells involves a complex interaction between the cell, the target antigen, the antibody and host effector mechanisms. One such mechanism, complement-mediated cell lysis, requires the interaction of C1q with the antibody heavy chain constant regions, and in particular the CH2 domain. Here we investigate the potential benefit of multiple-domain forms of the therapeutic monoclonal antibody CAMPATH-1H. This antibody is directed against the CDw52 antigen expressed by human lymphocytes and has proven lytic abilities both in vitro and in vivo. Using target cells with either high or low antigen density, engineered antibodies that contained additional domains in tandem (CH2, hinge-CH2 or Fc intramolecular repeats) showed no improvement in complement-mediated lysis when compared with controls. However, a homodimeric form of the antibody that was engineered by mutation of a serine residue to cysteine near the carboxy-terminal of the CH3 domain, exhibited markedly improved lysis using target cells expressing antigen at low density. Interestingly, no improvement was seen using cells expressing antigen at high density. These results suggest that dimeric forms of antibodies could be useful for converting cells with low density antigens into useful targets for therapy