Experimental vaccination Against FMDV with Immunocomplexes

Transcription

1 263 Experimental vaccination Against FMDV with Immunocomplexes Appendix 33 H.Yadin 1, Dalia Chai 1, A. Bril 1, B. Gelman 1 and M. Amadori 2. 1.Kimron Veterinary Institute, Beit Dagan50250, Israel. 2. Instituto Zooprofilattico Sperimentale della Lombardia e dell Emilia, Brescia, Italy. Introduction Vaccination against Foot and Mouth Disease is compulsory in Israel for all domestic ruminants on annual, biannual or triannual basis. The aim of these vaccinations is to enhance immunity against FMD for preventing huge virus amplification from primary outbreaks and reduction the extent of secondary outbreaks. The most susceptible animals on affected premises are the young calves and lambs, which possess maternal antibodies and are reacting slowly or not at all to vaccination. In addition, the duration of immunity is often limited in primo-vaccinated animals, which actually further accrues to the effectiveness of the vaccination campaigns. This work was based on the hypothesis that administration of antigen in complex with specific antibody alters the process of B cell memory formation in germinal centers, thus enhancing the immune response. Preliminary experiments in guinea-pigs (Amadori et al, 1998) would support such a tenet. Practical challenging this hypothesis in view of a future improvement of the immune response of young animals was the aim of this work. Materials and Methods Animals - On a commercial fattening farm a group of 16 calves in the age of 3-4 months was divided into 4 subgroups. Each subgroup was vaccinated s.c. With 2 ml of the corresponding vaccine of the subgroup. Blood samples for Ab testing were collected just before vaccination day 0 (D 0), at day 21 (D 21) and day 45(D 45) post vaccination. Serum - A post vaccination serum was collected from a cow one year after the 4 th shot of a trivalenrt vaccine containing O 1 Geshur Isr. 2/85, O 1 Manisa, A 22 Iran 87 and Asia 1 Shamir. The serum was tested for SN Ab as described in the OIE manual and stored at 20 C in small aliquots; its SN titer was equal to 1:256. Vaccines - A commercial vaccine, containing the antigens O 1 Geshur Isr. 2/85 and O 1 Manisa in Al (OH) 3 adjuvant, was used. For each subgroup, vaccine was formulated as follows: Group 1 : 9.8 ml aqueous Al(OH) 3 vaccine µl PBS (control). Group 2 : 9.8 ml aqueous Al(OH) 3 vaccine µl positive serum (1:50 ratio).

2 264 Group 3 : 9.8 ml aqueous Al(OH) 3 vaccine µl positive serum +100 µl PBS (1:100 ratio). Group 4 : 9.8 ml aqueous Al(OH) 3 vaccine + 50 µl Positive serum +150 µl PBS (1:200 ratio). Serology - The antibody titers were evaluated by SN test using microplates with monolayers of secondary pig kidney cells. Each serum sample was titrated in duplicate against 100 TCID 50 / well of O 1 Geshur. Results were checked after 48 hours according to OIE manual. Results The results of the SN tests are shown in table 1 and figure 1. There was evidence of a bell-shaped dose/response effect of the immunocomplexes: a slight reduction of the Ab response was observed at the 1: 50 ratio; a stronger inhibition was evidenced at the 1:200 ratio with a remarkable heterogenicity: two calves (5522 and 9336) did not respond to the vaccine, one had a high and one a usual response, with a clear tendency to a decrease on day 45 post vaccination; a very effective immunization was obtained instead at the 1:100 ratio with a clear tendency to a further increase of the Ab titers at day 45 post vaccination. The mean Ab titer of group 3 at day 45-post vaccination was significantly different from those of the other groups (P<0.05 in variance analysis).

3 265 TABLE 1 Ab response (SN titers*) of calves to different FMD vaccine formulations Group treatment Calf Nr. D 0 vaccination D 21 P. vacc. D 45 P. vacc. Gr Conventional Vaccine > Mean ± Sd 2.0 ± ± ± 1.2 Gr Vaccine /50 positive serum < Mean ± Sd 1.75 ± ± ± 0.29 Gr >7.0 >9.0 Vaccine +1/ positive serum >7.0 Mean ± Sd 2.75 ± ± ± 2.14 Gr Vaccine +1/ < positive serum Mean ± Sd 2.75 ± ± ± 0.96 * SN titres were expressed as Log 2.

4 266 FIGURE 1 Kinetics of Ab response in FMD-vaccinated calves 7 6 Lg Gr. 1 Gr. 2 Gr. 3 Gr. 4 0 D 0 D. 21 PV D. 45 PV Discussion The immune response to a vaccine injected parenterally usually starts with the step wise capture of antigen by dendritic cells, which transport it to the local draining lymph node. This process goes along with a gradual change of the main features of dendritic cells under the influence of Ag capture and of the local inflammatory environment: they stop the intense pinocytotic activity and are now able to achieve a high and stable expression of Ag/MHC II and co-stimulatory molecules on the cell surface. Under such conditions they can now adequately attract T cells by chemokines once arrived in the lymph node paracortex and stimulate them. Instead, free or released Ag is usually deposited on specialised Follicular Dendritic Cells (FDCs) in the form of Ag/Ab/C'3 complexes. In this case, the "natural" antibodies probably play a role in naive animals, as opposed to specific Ig in primed animals. B cells recognize

5 267 their target on such FdCs and become activated. Thus, they can secrete chemokines, which attract the nearby activated T cells from the paracortex and give rise to a cognate mutual recognition between B and T cells and to a Th function leading to the maturation and differentiation of B cells; a part of these cells become Ab-secreting with/without change of the expressed Ab isotype and mostly settle in the bone marrow; another part is submitted to the germinal centre reaction, leading to isotype switch and somatic hypermutation; this leads in turn to the appearance of memory B cells with increased Ab affinity. The administration of Ag in the form of Ag/Ab immunocomplexes is likely to interfere with the speficic phase of Ag recognition by B cells; the size, type and stability of the immunocomplexes deposited on FDCs would be probably different; as a consequence, there could be a longer persistence of the germinal centre reaction; this could lead to the selection of high-affinity B cell clones able to persist under condition of low Ag concentration, or to perform a long-term Ab secretion in the bone marrow, as previously reported in other experimental models. A direct role of immunocomplexes in the regulation of the GC reaction has been demonstrated in a murine model (Nie X. et al. 1997): as compared to the control Ag, there was a GC reaction characterised by a wider array of Ig populations and a higher frequency of point mutations/vh gene. Notice however that the interactions of Ag and Ab may lead to quite different results in the immunization; on the one hand, it is widely known that pre-existing maternal antibody are quite inhibitory; the same holds true of Ab injected before the administration of a rabies vaccine in mice (Schumacher C.L. et al, Vaccine), whereby the magnitude and the duration of such suppression are related to concentration and half-life of Ig, respectively. The inhibiting role of pre-formed antibodies has been also indicated in models of mucosal immunization against Rotavirus in calves (Van Zaane D., 1986); on the contrary an immunocomplex Rotavirus vaccine administered parenterally (Hess R.G. et al., 1982) and an immunocomplex Hepatitis B vaccine (McCluskie MJ et al, 1998) administered intranasally can be quite effective. It seems therefore that the presence of large amounts of pre-formed antibodies are often inhibiting, whereas a small amount of antibody complexed with antigen is generally instrumental in generating a vigorous Ab response. A preferential interaction with the low affinity Fc gammarii (CD32) in the case of a large excess of pre-formed antibodies could partly explain this apparent paradox, since Fc gammarii-knock-out mice mount a much more vigorous response to Ag/Ab complexes (Wernersson, 1999); notice however that F(ab )2 fragments of antibodies can also block the Ab response to sheep erythrocytes (Karlsson MC, 1999). The Ag/Ab ratios in the vaccine are another crucial factor for the final outcome of the immunising procedure; thus, for instance, a slight Ab excess was reported in the Rotavirus model (Hess et al.). Such a tenet would be confirmed by our study, in which different ratios were tested at the same time. The 1:100 ratio, which gave excellent results, might correspond the slight Ab excess described by Hess et al. (1982) in the Rotavirus model. Please notice that the antiserum was added to antigens adsorbed onto aluminium hydroxide; this could affect the type, size and form of the resulting immunocomplexes. The duration of the Ab response in the 1:100 group is unusual for aqueous vaccines in primo-vaccinated animals: this is reminiscent of the Ab kinetic after injection of W/O vaccines and points to a fundamental adjuvanticity of the Ag/Ab complexes: this has been confirmed in a murine model whereby toxins fused to S. aureus protein A and linked to antibodies to surface components of APC can induce an Ab response without the need for adjuvants (Leonetti M, 1998).

6 268 In the field of FMD, the technology of immunocomplexes could be possibly exploited to achieve three goals: 1. A longer duration of the immunity conferred by aqueous vaccines. 2. A wider protection against heterologous strains, as previously suggested (Amadori M, FAO, 1998). 3. An effective vaccination of young animals vìs-a-vìs maternal antibodies; this mighy be the case of calf 2873 (see table 1). This latter possibility is of paramount importance in the areas of prophylactic vaccination characterised by high infectious pressure, where the highest vaccination coverage is badly needed and repeated vaccinations of livestock are impractical; in this respect, the use of immunocomplexes could complement that of oil vaccines, which are less affected by maternally-derived antibody ( Spath et al, 1995 ). Further investigations are needed to understand the correlation between type of immunocomplexes and stimulatory vs inhibiting effect on the immune response in the FMD field; in this respect, the guinea-pig model (Amadori M. 1998) could actually provide a certain degree of prediction. Acknowledgements The skilful technical assistance of Mrs. Ida Reda is gratefully acknowledged. References Amadori, M. 1998: Influence of FMD vaccine potency and formulation on the immune response to heterologous FMDV strains. European Commission for the Control of Foot and mouth Disease, session of the Research Group of the standing Technical Committee, Alderhot, UK September 1998, Hess, R.G., Bachmann,P.A., Eichhorn, W., Frahm, K., and Plank, P., 1982: Stimulierung der laktogenen Immunitat des Rindes gegenuber Rotavirusinfekionen. Fortsch. Vet. Med., 35, Karlsson, M.C., Werersson, S., Diaz de Stahl, T., Gustavsson, S., Heyman, B., 1999: Efficient IgG-mediated suppression of primary antibody responses in Fcgamma receptor-deficient mice. Proc. Natl. Acad. Sci. USA. 2; 96(5); Leonetti, M., Thai, R., Cotton, J., Leroy, S., drevet, P., Ducancel, F., Boulain, J.C., Menez, A., 1998: Increasing immunogenicity of antigens fused to Ig-binding proteins by cell surface targeting. J. Immunol. 160(8); McCluskie, M.J., Wen Ym, Di Q, Davis HL., 1998: Immunization against hepatitis B virus by mucosal administration of antigen-antibody complexes. Viral. Immunol. 11(4); Nie, X., Basu, S., and Cerny, J., 1997: Immunization with immune complex alters the reperoire of antigen-reactive <bcells in the germinal centers. Eur. J. Immunol. 27;

7 269 Schumacher, C.L. Ertl, HC., Koprowski, H., Dietzschold, B., 1992: Inhibition of immune responses against rabies virus by monoclonal antibodies directed against rabies virus antigens. Vaccine;10(11); Spath, E.,J., Smitsaart, E., Casaro, AP., Fodevila, N., Fernandez, F., Leunda, Mr., Compaired, D., Buffarini, M., Pessi, H., 1995: Immune response of calves to Footand-Mouth disease virus vaccine emulsified with oil adjuvant. Strategies of vaccination. Vaccine, 13(10); Van Zaaane, D., Ijzerman, J., De Leeuw, P.W., 1986: Intestinal antibody response after vaccination and infection with rotavirus of calves fed colostrum with or without rota virus antibodies. Vet. Immunol. Immunopathol. 11(1); Wernersson, S., Karlsson, M.C., Dahlstrom, J., Mattsson, r., Verbeek, J.S., Heyman,B., 1999: Igg-mediated enhacement of antibody resposes is low in Fc receptor gamma chain-deficient mice and increased in Fc gamma RII-deficient mice. J. Immunol. 15;163(2);