Kees Weijer

Early stages in the development of human T, natural killer and thymic dendritic cells

Summary: T‐cell development is initiated when CD34+ pluripotent stem cells or their immediate progeny leave the bone marrow Co migrate to the thymus. Upon arrival in the thymus the stem cell progeny is not yet committed to the T‐cell lineage as it has the capability to develop into T, natural killer (NK) and dendritic cells (DC). Primitive hematopoietic progenitor cells in the human thymus express CD34 and lack CD la. When these progenitor cells develop into T cells they traverse a number of checkpoints. One early checkpoint is the induction of T‐cell commitment, which correlates with appearance of CD la and involves the loss of capacity to develop into NK cells and DC and the initiation of T‐cell receptor (TCR) gene rearrangements, Basic helix‐loop‐helix transcription factors play a role in induction of T‐cell commitment. CDla+CD34+ cells develop into CD4+CD8α+β+ cells by upregulating first CD4, followed by CD8α and then CD8β. Selection for productive TCRβ gene rearrangements (β selection) likely occurs in the CD4+CD8α+β‐ and CD4+CD8α+β+ populations. Although the T and NK‐cell lineages arc closely related to each other, NK cells can develop independently of the thymus. The fetal thymus is most likely one site of NK‐cell development.

From Jenner to Jerne: Towards Idiotype Vaccines

In 1798 Edward Jentier introduced vaccinatioti against an infectious agent, i.e. .smallpox, a discovery whicb was only fully appreciated 80 years later through the demonstration by Louis Pasteur of a vaccine prophylaxis for chicken cholera. Vaccination with both hve and inactivated vaccines has proved to be a major contribution in controlling infectious diseases of man and animals. Vaccination with this first generation of vaccines is, for certain vaccines, still accompanied by serious complications, such as side effects, incomplete protection and problems with large-scale production. An important move towards the creation of safer vaccines was made by Goebel (Goebel 1938) when he designed a synthetic vaccine conferring protective immunity to mice against pneumococci type III. Recently, on the basis of sequence data, peptides structtirally resembling viral antigens have been synthesized, which are capable of inducing antibodies reactive with native viral antigens (Gerin et al. 1983, and see Lerner 1982 for review). More often, however, synthetic peptides proved weak immunogens and their future applicability for vaccination will depend on the development of new methodologies, aiming at the improvement of their immunogenicity (Stewart-Tull 1985).

Serological responses in cats vaccinated with FeLV ISCOM and an inactivated FeLV vaccine.

Various approaches have been considered for generation of effective and safe vaccines against retroviruses, including HIV, with limited success. In the present vaccination study, encompassing 137 household cats, we have composed an experimental ISCOM subunit vaccine containing gp70 of feline leukaemia virus (FeLV)--the external glycosylated envelope protein, and the transmembrane protein p15E, with a commercial available inactivated FeLV vaccine (Leukocell). The two vaccines were estimated to contain approximately the same amount of gp70 antigen and the cats were immunized three times according to the recommendations of the commercial vaccine. A control preparation not containing gp70 or p15E was also included. During the observation period of 200 days all cats remained healthy and no virus was isolated during the isolation attempts. The serological responses were measured in ELISA, membrane immunofluorescence (MIF) and virus neutralization (VN) tests. In contrast to the cats in the other groups almost all ISCOM-vaccinated cats responded by seroconversion or increased titres in the three tests. The development of specific antibodies to gp70 and p15E were confirmed in Western blot. These results clearly illustrate the potential of the ISCOM structure for the development of safe and effective vaccines against retroviruses.

Induction of feline leukaemia virus-neutralizing antibodies by immunization with synthetic peptides derived from the FeLV env gene

The surface glycoprotein, gp70, of feline leukaemia virus (FeLV) contains sites which are important in inducing neutralizing antibodies. Using synthetic peptides corresponding to nucleotide sequence 729-957 (amino acid sequence 243-319) of FeLV-A/Glasgow-1, the antigenicity and immunogenicity of this part of the viral surface glycoprotein were investigated. The region contains two highly conserved domains separated by a variable region (VR4), when compared with FeLV of subgroups B and C, and an epitope known to be involved in virus neutralization is located in the N-terminal conserved domain. Five murine monoclonal antibodies generated by immunization with virus were found to be directed to this domain and four were virus-neutralizing. Polyclonal mouse, rabbit and cat anti-FeLV antisera, which were virus-neutralizing, were directed to B-cell epitopes in the peptides. To determine if those synthetic peptides could induce neutralizing antibodies, rabbits were immunized with the peptides, singly or in combination. Antibody responses were measured by ELISA for anti-peptide, anti-FeLV and anti-gp70 activity, by immunoblotting, by membrane immunofluorescence and by virus-neutralization tests. Virus-neutralizing antibodies were induced by FeLV gp70 peptides and there was a synergistic effect on antibody production when a combination of peptides covering amino acid sequence 243-319 of FeLV-A was used. In a second experiment, six rabbits and six cats were immunized with a combination of two peptides, which covered the above-defined FeLV gp70 sequence. Comparisons were made of the responses to these peptides incorporated into immunostimulating complexes (ISCOMs) via myristic acid tails, inoculated with empty ISCOMs, or with Al(OH)3. Complete Freunds adjuvant (CFA) had a very strong potentiating effect on the induction of antibodies and immunization with peptides incorporated into ISCOMs was superior to immunization using adjuvants other than CFA. It is very promising that not only in rabbits, but also in the natural host of FeLV, the cat, anti-FeLV gp70 (peptides) antibodies could be induced by FeLV gp70 peptides.

gag- and env-specific serum antibodies in cats after natural and experimental infection with feline immunodeficiency virus

In order to monitor the antibody response to feline immunodeficiency virus (FIV) in cats, following experimental and natural infection, enzyme-linked immunosorbent assays (ELISAs) were developed using recombinant env and gag proteins and p24-specific monoclonal antibodies. It was shown that in experimentally infected cats an env protein-specific antibody response was directly followed by a gag protein-specific response. Furthermore, an ELISA for the detection of env protein-specific serum antibodies proved more sensitive in identifying experimentally and naturally infected cats than ELISAs demonstrating gag protein-specific antibodies. It was concluded that, like in HIV infection of humans, the detection of env protein-specific serum antibodies in addition to gag protein-specific antibodies is not only an important tool in the diagnosis of the infection but also in studies concerning the pathogenesis of the disease.

Tyrosinase gene expression in clear cell sarcoma indicates a melanocyte origin: insight from the first reported canine case

The aim of this study was to characterize a metastasizing soft tissue tumor in a dog, which clinically, grossly and histologically showed a close resemblance to human clear cell sarcoma, a soft tissue variant of malignant melanoma. Ultrastructurally, melanosomes were found, indicating a melanocytic origin of the tumor. Using reverse‐transcription polymerase chain reaction, expression of the gene encoding tyrosinase was determined in tumor cells. With this first case of canine clear cell sarcoma, as well as the earlier report from our laboratory on amelanotic melanomas in the cat, we demonstrate that expression of the tyrosinase gene may occur in a broader range of less differentiated melanocytic tumors in different species, including man.

Control of feline leukaemia virus.

Feline leukaemia virus (FeLV) usually occurs in its natural species, the domestic cat. FeLV is also important to human individuals as a comparative model, as it may cause a variety of diseases, some malignant and some benign, such as immunosuppression, which bears a resemblance to AIDS (acquired immune deficiency syndrome) in man. FeLV is transmitted among cats by contagion. The main sources of infection are persistently infected carrier cats which continuously excrete virus. Dissemination of FeLV among cats may be prevented by identifying infected carrier cats and removing them from contact with non-infected cats. Removal programmes using indirect immunofluorescence antibody tests were applied successfully in The Netherlands. The proportion of FeLV-positive cats decreased from 9% in 1974 to approximately 3% in 1985 during such a programme. The results of a removal programme carried out in a catbreeders society were even better: the incidence of cats positive for FeLV decreased from 11% in 1974 to less than 2% within 4 years. None of the cats tested in this society has been found to be positive for FeLV since 1984. Besides removal programmes, other methods of control, such as pre-exposure treatment, were developed to prevent the spread of FeLV. We attempted to protect kittens against oronasal infection with FeLV by treatment with virus-neutralizing (VN) monoclonal antibodies (MoAbs) directed against an epitope on the viral glycoprotein gp70. However, no protection was achieved. It is unlikely that the amount of VN antibodies, the mode and route of their application or the infectious dose of FeLV used can account for this failure. Other possible explanations for the lack of protective effect are that (i) the restricted epitope specificity of the MoAb preparation used may have led to selection of neutralization-resistant virus mutants, or (ii) other mechanisms than virus neutralization (complement-mediated lysis, antibody-dependent cell cytotoxicity), that may be involved in protection, function less efficiently with MoAb. However, in the light of our finding that an early anti-idiotypic response is observed in all cats following administration of the MoAb preparation, the rapid clearance of anti-FeLV MoAb from the circulation is a more likely explanation. Efforts were further made to develop a vaccine for controlling FeLV infection. The immunostimulating complex vaccine (FeLV-ISCOM vaccine), a subunit vaccine in which FeLV gp70 is presented in a particular manner, looks promising. The protective effect of FeLV-ISCOM vaccine was studied by vaccinating six 8-week-old SPF cats with ISCOM, followed by oronasal challenge with FeLV.(ABSTRACT TRUNCATED AT 400 WORDS)