Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
DNA vaccination wikipedia , lookup
Immune system wikipedia , lookup
Lymphopoiesis wikipedia , lookup
Polyclonal B cell response wikipedia , lookup
Hepatitis B wikipedia , lookup
Sjögren syndrome wikipedia , lookup
Adaptive immune system wikipedia , lookup
Psychoneuroimmunology wikipedia , lookup
Molecular mimicry wikipedia , lookup
Cancer immunotherapy wikipedia , lookup
1691 Journal of General Virology (1991), 72, 1691-1694. Printed in Great Britain Comparison of the immune response elicited by infectious and inactivated foot-and-mouth disease virus in mice P. G. Piatti,* t A. Berinstein, O. J. Lopez, M. V. Borca, F. Fernandez, A. A. Schudel and A. M. Sadir Institute o f Virology, CICV, INTA-Castelar, Argentina The immune response to foot-and-mouth disease virus (FMDV) elicited by infection or immunization with inactivated virus in adult mice was examined. A model of adoptive transfer of immunocompetent cells was used for this purpose. The results presented here indicate that both short- and long-term secondary immune responses elicited by high doses of inactivated virus are indistinguishable, at the humoral or cellular level, from that observed after infection. The responses to inactivated or infectious virus were both efficiently mediated by B cells. However, immunization with low doses of inactivated virus induced a response which, although effective in aborting infection, was fully dependent on FMDV-specific T cell cooperation. These findings suggest that the different immune responses observed after infection and immunization are mainly the result of the different viral mass presented to the immune system in each case. Foot-and-mouth disease virus (FMDV), the only member of the aphthovirus genus within the Picornaviridae family (Bachrach, 1977), causes an economically important disease affecting cloven-hoofed animals (Cottral et al., 1975). In contrast to natural infection, current FMDV vaccines prepared with inactivated virus and adjuvants elicit short-lived protection (Morgan et aL, 1980). Some of the possible explanations, as yet unsupported by experimental data, could be a constant antigenic boosting due to virus persistence in infected animals (Gebauer et ai., 1988; our unpublished data), the induction of more efficient immune mechanisms after infection (Lopez et al., 1990) or quantitative differences due to variation in the mass of virus presented to the immune system after infection or vaccination. The objective of this research was to evaluate the last possibility by characterizing the humoral and cellular immune responses of adult mice to infection and different doses of inactivated FMDV. The neutralizing antibody response up to 120 days post-infection (p.i.) after intraperitoneal (i.p.) immunization with different amounts of inactivated FMDV (0-1, 0.5, 1 or 10 ~tg) without adjuvant is shown in Fig. 1. Antibody titres were measured by using the constant serum/variable virus method on suckling mice (Cunha et al., 1957). Infection of mice was carried out by i.p. inoculation of 104 suckling mouse 50% lethal doses (SMLDso) of FMDV subtype O1 Campos (O1C) diluted in 0-5 ml of PBS per mouse. FMDV was inactivated with binary ethylenimine, according to Bahnemann (1975) and virus purification was performed as described by Bachrach et al. (1964). Mice were immunized by i.p. inoculation of inactivated virus in PBS. The duration and magnitude of the immune response correlates directly with the mass of inactivated virus used for vaccination. Thus, animals which received 0.1 ~tg of inactivated virus developed transitory and low titres of neutralizing antibodies. Immunization with less than 0-1 ~tg (0.075, 0.025 and 0-01 ~tg; data not shown) demonstrated that 0.1 ~tg was the minimum dose capable of eliciting a reproducible response in this particular system. A clear difference was obtained when 0.5 or 1 ~tg of inactivated virus was used for immunization. Interestingly, the titres of neutralizing antibody and the maintenance of antibody titres in animals which received 10 ~tg of inactivated virus were not significantly different from those observed after inoculation with infectious virus. The dose-response relationship of these results is in agreement with the results of Rweyemamu et al. (1984) obtained in cattle. To gain insight into the cellular mechanisms mediating these antibody responses, an experimental model of adoptive transfer of immunocompetent cells was utilized (Borca et al., 1986). Recipient mice were irradiated with 580 rad 24 h prior to FMDV infection and 107 spleen cells from mice infected 8 days previously or from virusimmunized donors were transferred by i.p. inoculation t Present address: Plum Island Animal Disease Center, USDA, Greenport, New York 11944, U.S.A. 0000-9908 © 1991 SGM Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 14 May 2017 11:55:25 1692 Short communication I I I Table 1. Transference of the short duration secondary i immune response (8 days p.i.) induced by high and low doses of inactivated FMD V 4 Viraemia days post-reconstitution - V / O ~ 3 Group* 5 I 2 3 4 5 6 7 8 9 lo + + + + + + -+ 6 (NI) 13 14 + + + + + + + - 2 \ 0 ! 0 50 100 150 ' • 200 250 Days p.i. Fig. 1. Neutralizing antibody response to different doses of purified inactivated FMDV. Adult BALB/c mice were infected (11) or immunized i.p. with 10 Ixg (A), I lig (V), 0.5 lig (0) or 0.1 ltg (0) of purified inactivated FMDV. Results are expressed as neutralizing indices (NI) and titres represent the mean of two determinations at each time. 24 h after infection. Spleen cells were o b t a i n e d by gently h o m o g e n i z i n g the o r g a n i n 199 m e d i u m ( G i b c o ) c o n t a i n i n g 5 % foetal calf serum. E r y t h r o c y t e s were lysed with T r i s - N H , C1 p H 7.2 a n d a d h e r e n t cells were r e m o v e d b y i n c u b a t i o n for 1 h at 37 °C (5% CO2 atmosphere). T cells a n d B cells were purified from the n o n - a d h e r e n t cells by i n c u b a t i o n for 1 h at 37 °C with r a b b i t a n t i - m o u s e Ig s e r u m or a n t i - T h y 1.2 m o n o c l o n a l a n t i b o d y ( M A b ) , respectively, i n the presence of g u i n e a - p i g c o m p l e m e n t . T h e purity of T- a n d B-enriched p o p u l a t i o n s was assessed by direct i m m u n o f l u o r e s c e n c e using fluorescein isothiocyanate-labelled a n t i - T h y 1.2 M A b a n d antimouse Ig. O n l y cell p r e p a r a t i o n s s h o w i n g m o r e t h a n 97 % p u r i t y were used. V i r a e m i a was d e t e r m i n e d by intram u s c u l a r i n o c u l a t i o n o f a litter o f six 6-day-old suckling mice with 0.05 ml o f blood o b t a i n e d from two reconstituted a n i m a l s each day, diluted 1/10 i n PBS. Results are expressed as positive (at least one out of six mice died) or n e g a t i v e (no mice died). As c a n be seen in T a b l e 1, n o significant difference i n either the p r o d u c t i o n of neutralizing a n t i b o d y or the e l i m i n a t i o n of i n f e c t i o n was observed for a n i m a l s w h i c h received cells 8 days after sensitization f r o m d o n o r s w h i c h were either infected or were i m m u n i z e d with 10 ~tg o f i n a c t i v a t e d virus. I n b o t h cases, r e p o p u l a t i o n o f recipients w i t h u n f r a c t i o n a t e d spleen cells or purified B cells was equally effective i n eliciting the p r o d u c t i o n of n e u t r a l i z i n g a n t i b o d y a n d e l i m i n a t i o n o f viraemia. S i m i l a r results were observed w h e n the d o n o r cells were o b t a i n e d from a n i m a l s infected or i m m u n i z e d with 10 Ixg of i n a c t i v a t e d virus 120 days earlier (Table 2). Both e x p e r i m e n t s show t h a t there is a direct correlation b e t w e e n the ability of t r a n s f e r r e d B cells a n d + + + + + (O.40) (2.50) (2-00) (0.30) (2.I4) (0.24) (0-24) (2.20) (2.20) (o.io) * Group 1 refers to the transference of unfractionated cells from uninfected non-immunized donors; group 2, unfractionated ceils from infected donors; group 3, B cells from infected donors; group 4, T cells from infected donors; group 5, unfractionated cells from donors immunized with 0-1 lig of inactivated FMDV; group 6, B cell from donors immunized as group 5; group 7, T cells from donors immunized as group 5; group 8, unfractionated cells from donors immunized with 10 lig of inactivated FMDV; group 9, B cells from donors immunized as group 8; group 10, not reconstituted. Table 2. Transference of long lasting secondary immune response (120 days p.i.) induced by high and low doses of inactivated FMD V Viraemia days post-reconstitution Group* 5 1 2 3 4 5 6 7 + + + + + 8 + 6 (NI) + + (2.40) (2.50) (2.14) (2.20) (2.20) (0.30) (0-20) + (0.10) 7 . . . . . + + + 8 . . . . . 9 . . . . . . + + + 10 + 14 . . . . NVt ND ND ND ND ND . . . . . + + 11 . . . . + + + * Group 1 refers to transference of unfractionated ceils from infected donors; group 2, B cells from infected donors; group 3, unfractionated cells from immunized donors (10 lig); group 4, B cells from immunized donors (10 lig); group 5, unfractionated cells from immunized donors (0.1 ~tg); group 6, B cells from immunized donors (0.1 lag); group 7, unfractionated cells from untreated mice; group 8, non-repopulated mice. ND, Not determined. u n f r a c t i o n a t e d cells to a b o r t i n f e c t i o n a n d the presence o f n e u t r a l i z i n g a n t i b o d y . However, w h e n 0.1 p.g of i n a c t i v a t e d virus was used to p r i m e donors 8 days before the cell transfer, r e c o n s t i t u t i o n with T cells or B cells alone did n o t result i n a n efficient i m m u n e response Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 14 May 2017 11:55:25 1693 Short communication 14( I.~ I I I I I Table 3. Collaborative effect of T cells in mice immunized with small doses of inactivated FMD V (0.1 I~g) at 8 days p.i. 12 Viraemia days post-reconstitution •~ lO -~ O 8 6q • O. ~0 4 2 0 0 I I 10 20 I 30 T cells (%) I I 40 50 60 Fig. 2. Relationship between immunizing dose and neutralizing antibody response in T cell-B cell collaboration. Donor mice were infected (O) or i m m u n i z e d with 0.1 ~tg of inactivated purified virus (O) 8 days before reconstitution. Receptors were reconstituted with 107 cells using different proportions of T cells. The presence or absence of viraemia was determined as described in the text. (Table 1). To determine whether this was due to the absence of T cells, mice were reconstituted with purified B cells and different proportions of T cells. The addition of T lymphocytes to the cell suspension used for repopulation reduced the period of viraemia to a degree which correlated directly with the proportion of T ceils present. When equal amounts of T cells and B cells were used (5 x 106 T cells plus 5 x 106 B cells), viraemia became undetectable at the same time post-reconstitution as observed for animals repopulated with untreated cells obtained from infected animals (Fig. 2). This T cell-dependence in a dose-response fashion raised the question of how effective the T cells could be in amplifying the B cell response. Purified B ceils and T cells were obtained from mice 8 days post-immunization with 0-1 p.g of purified inactivated antigen. Different quantities of B cells were-added to 5 x 106 T cells (this number o f T cells was shown to be sufficient to reduce viraemia to 6 days in the experiment described in Fig. 2) and the mixtures were used for repopulation. As shown in Table 3, even the smallest number of B cells tested (5 x 105) was able to eliminate the infection. Thus, doses of B ceils 20 times lower than those shown to be insufficient to abort viraemia by themselves, are effective when T cells are present. These data indicate that for 0.1 Ixg of inactivated virus the mechanism of virus elimination is mediated by B cells, since purified T cells were previously shown to be ineffective, but that T cells play an important role in enhancing the B cell response. Thus the quantity of B cells sensitized by immunization with 0.1 p.g of inactivated virus is insufficient to produce an effective immune response and requires amplification which is Group* 5 6 1 + + 2 3 + + . + 4 . 5 6 + + . . 7 + + 8 + . 9 + + . 7 8 + + . . + . . + . . . + . . . + 10 11 + + + + -- -- + + . + . . . . + . + + . . . . . 9 . . -- . . + * G r o u p 1 refers to transference o f 107 unfractionated cells from noni m m u n i z e d donors; group 2, 107 unfractionated ceils from F M D V immunized donors; group 3, 107 B cells from F M D V - i m m u n i z e d donors; group 4 to group 6, different a m o u n t s of B and T cells taken from F M D V - i m m u n i z e d donors ( 4 : 5 x 106 B and 5 x 106 T; 5: 2.5 x 106 B and 2.5 x 106 T; 6 : 5 x l0 s B and 5 x 106 T); group 7, 5 x 106 B cells from F M D V - i m m u n i z e d donors plus 5 x 106 T cells from K L H - i m m u n i z e d donors; group 8, 5 x 106 B cells from K L H i m m u n i z e d donors plus 5 x 106 T cells from F M D V - i m m u n i z e d donors; group 9, not reconstituted. Groups 7 and 8 were inoculated with 30 ~tg of K L H in PBS i.p. simultaneously with the virus challenge. mediated by T cells. Surprisingly, T cells sensitized with 0.1 txg of FMDV were also able to help cells obtained from donors 21 days post-immunization with 30 ~tg of keyhole limpet haemocyanin (KLH) in Freund's incomplete adjuvant in eliminating the infection. This result emphasizes the importance of T cells when low doses of inactivated FMDV are used for immunization. Thus, FMDV-primed T cells could alter the response from that of a primary reaction (group 7, Table 3) to a secondary one (groups 5, 6 and 8, Table 3). This fact could indicate that even at a very low frequency FMDV B precursor cells are effective in eliminating the infection if FMDVsensitized T cells are present. The analysis of T celldependence of the secondary response to 0.1 Ixg of inactivated virus was extended to determine whether this event requires T cells and B cells to be sensitized by the same antigen (FMDV-specific response) or whether it could be mediated by soluble factors released by T cells sensitized by an unrelated T cell-dependent antigen (KLH). As can be seen in Table 3, T cells sensitized to KLH did not drastically modify the neutralizing antibody response observed in mice reconstituted with purified B cells from animals immunized with 0.1 Ixg of inactivated FMDV, suggesting that the dependence is virus-specific. The need for T cell collaboration observed when low doses of inactivated virus are used can be compared to those results obtained in an in vitro system by Collen et al. (1989). Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 14 May 2017 11:55:25 1694 Short communication In summary, we have shown that there does not seem to be any difference in the immune response elicited by experimental infection or by immunization with high doses of inactivated FMDV, both being relatively T cellindependent. On the other hand, the response to infection mediated by cells sensitized to low doses of inactivated FMDV is efficient only when T cells are specifically sensitized. These T cells can in turn help B cells to eliminate infection, even if they were not previously exposed to FMDV antigen. Although the role of T cells after infection or immunization with 10 ~tg of inactivated virus was not examined, its importance as an amplification factor can not be discounted. Unfortunately no data are available with regard to either the mass of viral antigen produced during infection of cattle or the immune response elicited in this species by the administration of massive amounts of inactivated virus. The amount of 140S fraction present in commercial vaccines is approximately 10 ~tg and the amount of viral particles produced during the FMDV infection is probably far greater than this. Under the experimental conditions presented here it seems likely that the differences observed between the immunization with inactivated virus could be due solely to the virus mass that is in contact with the immune system in either case. References BACHRACH, H. L., TRALrrMAN, R. & BREESE, S. S., JR (1964). Chemical and physical properties of virtually pure foot-and-mouth disease virus. American Journal of Veterinary Research 25, 333-342. BAHNEMANN, H. G. (1975). Binary ethylenimine as an inactivant for foot-and-mouth disease virus and its application for vaccine production. Archives of Virology 47, 47-56. BORCA, M. V., FERNANDEZ, F. M., SADIR, A. M., BRAUN, M. & SCHUDEL, A. A. (1986). Immune response to foot-and-mouth disease virus in an experimental murine model. Effective thymus-independent primary and secondary immune reaction. Immunology 59, 261-267. COLLEN, T., PULLEN, L. & DOEL, T. R. (1989). T cell-dependent induction of antibody against foot-and-mouth disease virus in a mouse model. Journal of General Virology 70, 395-403. COYrRAt,, G. E. (1975). Foot-and-mouth disease virus. In Foreign Animal Disease, 3rd edn., pp. 109-128. Edited by T. G. Murnane. Richmond: Committee in Foreign Animal Disease, United States Animal Health Association. CUNHA, R. G., JUNIOR, J. A. B., SERRARO, W. & TORRUELA, I. (1957). E1 uso de los ratones lactantes en la evaluacion de los anticuerpos contra el virus de la fiebre aftosa y su significancia inmunologica. Gaceta veterinaria 110, 1-27. GEBAUER~ F., DE LA TORRE, J. C., GOMES, I., MATEU, M. G., BARAHONA,H., TIRABOSCHI,B., BERGMANN,I., AUGE DE MELLO, P. & DOMINC,O, E. (1988). Rapid selection of genetic and antigenic variants of foot-and-mouth disease virus during persistence in cattle. Journal of Virology 62, 2041-2049. LOPEZ, O. J., BORCA, M. V., FERNANDEZ, F. M., BRAUN, M. & SCHUDEL, A. A. (1990). Immune response to foot-and-mouth virus in an experimental murine model. II. Basis of persistent antibody reaction. Veterinary Immunology and lmmunopathology 24, 313-321. MORGAN, D. O., MOORE, D. M. & MCKERCHER, P. D. (1980). Vaccination against foot-and-mouth disease. In New Developments with Human and Veterinary Vaccines, pp. 169-178. Edited by A. Mizsahi, I. Hertman, M. A. Klingberg & A. Kohn. New York: Alan R. Liss. RWEYEMAMU, M. M., BLACK, L., BOGE, A., THORNE, A. C. & TERRY, G. M. (1984). The relationship between the 140S antigen dose in aqueous foot-and-mouth disease vaccine and the serum antibody in cattle. Journal of Biological Standardization 12, 111-120. BACHRACH, H. L. (1977). Foot-and-mouth disease virus: properties, molecular biology and immunogenicity. In Beltsville Symposia in Agricultural Research. L Virology in Agriculture, pp. 3-32. Edited by J. A. Romberger. Monclair: Allanheld-Osmun. (Received 10 September 1990; Accepted 13 March 1991) The authors wish to acknowledge the excellent technical assistance of Ana M. Hernandez, Carmen Maciel and Antonio Varone. P.G.P. and A.B. are research fellows of the Consejo Nacional de Investigaciones Cientificas y Tecnicas. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 14 May 2017 11:55:25