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
J. gen. Virol. (1985), 66, 2347-2354. Printedin Great Britain 2347 Key words: FMDV/vaccine/oligopeptide/immunologicalpriming Immunological Priming with Synthetic Peptides of Foot-and-Mouth Disease Virus By M. J. F R A N C I S , * C. M. F R Y , D.J.'ROWLANDS, F. B R O W N , J. L. B I T T L E , 1 R. A. H O U G H T E N 1 AND R. A. L E R N E R 1 Wellcome Biotechnology Ltd, F M D Division, Ash Road, Pirbright, Woking, Surrey GU24 ONQ, U.K. and 1Research Institute of Scripps Clinic, La Jolla, California 92037, U.S.A. (Accepted 12 August 1985) SUMMARY A sub-immunizing dose of a synthetic peptide corresponding to the amino acids 141 to 160 region of protein V P 1 from foot-and-mouth disease virus (FMDV), serotype O1, coupled to keyhole limpet haemocyanin (141-160KLH) has been shown to prime the immune system of guinea-pigs for an FMDV serotype-specific neutralizing antibody response to a second sub-immunizing dose of the same peptide. Optimal priming required an interval of 42 days between the priming dose and the booster dose. No priming was observed in the absence of adjuvant. The secondary response was not restricted by the carrier since animals primed with 141-160KLH could be boosted with uncoupled 141-160 or 141-160 coupled to tetanus toxoid. It has also been shown that uncoupled peptide 141-160 will prime for a neutralizing antibody response when it is incorporated into a relatively non-immunogenic carrier such as small unilamellar liposomes. These results indicate that the 141-160 peptide of FMDV, as well as containing an important neutralizing antibody site, can initiate its own T-helper cell response. INTRODUCTION The foot-and-mouth disease virus (FMDV) particle is composed of one molecule of singlestranded RNA (mol. wt. 2.6 × 106) and 60 copies of each of four structural proteins, VP1, VP2, VP3 (mol. wt. 24 x 103) and VP4 (mol. wt. 10 x 103). Of these structural proteins VP1 appears to play a key role in the antigenic and immunogenic activity of the virus particle (Wild et al., 1969; Laporte et al., 1973; Bachrach et al., 1975). Detailed study of both enzymically and chemically cleaved fragments of VP1 from virus of serotype O has identified two regions, between amino acids 138 to 154 and 200 to 213, which are found on the surface of the virus and fragments containing these regions are able to induce neutralizing antibodies against the homologous virus (Strohmaier et al., 1982). Furthermore, studies using chemically synthesized peptides corresponding to several regions of VP1 have led to the identification of similar sites on the molecule (141 to 160 and 200 to 213) which elicit neutralizing antibodies that can protect guinea-pigs against experimental infection (Bittle et al., 1982; Pfaffet al., 1982). Although such peptides have an immunizing activity of only 1 ~o or less of that of the inactivated virus particle on an equal weight basis, the level of neutralizing antibody produced is several orders of magnitude greater than that obtained with the whole VP1 molecule (Bittle et al., 1982). Moreover, anti-peptide antibodies mimic the subtype specificity of the intact virus particle (Clarke et al., 1983; Rowlands et al., 1983). Peptides corresponding to the immunogenic regions of poliovirus VP1 have been used to prime the immune system of rabbits to produce high neutralizing antibody titres after a second injection of a sub-immunizing dose of the intact virus particle (Emini et al., 1983). Peptides of the haemagglutinin molecule of influenza virus will also prime for a secondary response to virus particles (R. Arnon & F. Melchers, personal communications). A similar priming effect has also been observed with anti-idiotype antibodies to hepatitis B which can be used to enhance the 0000-6773 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 12 Jun 2017 00:36:24 2348 M . J . FRANCIS AND OTHERS i m m u n e response to a s u b s e q u e n t injection o f the s a m e anti-idiotype a n t i b o d y or hepatitis B surface antigen ( K e n n e d y & D r e e s m a n , 1984). A l t h o u g h it has not yet b e e n s h o w n w h e t h e r p e p t i d e - p r i m e d animals will be p r o t e c t e d against s u b s e q u e n t infection, it has b e e n p o i n t e d out that the strategy o f p r i m i n g could be used to identify neutralizing e p i t o p e s on the virion ( E m i n i et al., 1983). In a p r e l i m i n a r y study we h a v e d e s c r i b e d a p r i m i n g effect o f p e p t i d e s o f the VP1 protein o f F M D V ( F r a n c i s et al., 1985). T h e s e o b s e r v a t i o n s h a v e led us to investigate p r i m i n g w i t h F M D V p e p t i d e s in m o r e detail w i t h a v i e w to studying the role of carriers and a d j u v a n t s in the antip e p t i d e response. METHODS Syntheticpeptides. 01 Kaufbeuren peptides were synthesized by the solid-phase method using a Beckman model 990B peptide synthesizer at the Scripps Clinic, U.S.A. (Bittle et al., 1982) based on the 213 amino acid sequence of VP1 published by Kurz et al. (1981). Peptides were coupled to keyhole limpet haemocyanin (KLH) through an additional cysteine on the carboxy-terminus using N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) as a coupling agent and to tetanus toxoid (TT) using glutaraldehyde. Animals. Female Dunkin-Hartley guinea-pigs, approximately 12 weeks old and weighing between 450 and 500 g, maintained as a closed randomly bred colony at the Animal Virus Research Institute, Pirbright, U.K., were used. Challenge test. 01 Kaufbeuren virus suspension in a 0.02 ml dose, containing 500 guinea-pig IDs0, was injected intradermally into the left hind footpad and the animals were examined daily for 7 days. Guinea-pigs with no lesions or lesions_only at the injection site were regarded as protected and those with more extensive lesions as unprotected. Neutralization assay. The neutralizing activity of serum samples against 100 TCID5o of FMDV was determined using a microneutralization test (Francis & Black, 1983). Each test was performed in triplicate and the results were recorded as the mean log~0 reciprocal of the serum dilution that gave confluent cell sheets in 50 ~ of the microplate wells (SNso). Enzyme-linked immunosorbent assay (EL1SA). A modification of the indirect ELISA technique described by Voller & Bidwell (1976) was used to assay anti-virus particle and anti-peptide IgG responses. Briefly, microplates were coated overnight at room temperature with purified FMDV (Brown & Cartwright, 1963) or uncoupled synthetic peptide at a concentration of 2 ~tg/ml. The plates were washed and test serum samples, either at a standard 1:50 dilution or a range of doubling dilutions from 1:10, were added. After incubation for 1 h at 37 °C, plates were washed and anti-guinea-pig IgG-peroxidase conjugate was added. After a further hour at 37 °C the plates were washed and an enzyme substrate (0.04% o-phenylenediamine + 0.004% hydrogen peroxide in phosphate/citrate buffer) was added. After 5 to 7 min, colour development was stopped with 12.5~ sulphuric acid and the absorbance at 492 nm was measured in a Titertek Multiskan (Flow Laboratories). For the fixed 1 : 50 dilutions, A492 readings of sera taken at intervals after inoculation of the guinea-pigs were corrected by subtracting from them the corresponding 0 day values (mean 0 day A492value for peptide was 0-07 + 0.02 and for virus was 0-19 _+ 0.03) obtained in the same test. Alternatively, the A,,92 values obtained from doubling dilutions of post-inoculation samples were plotted against the log~0 reciprocal antiserum dilution and the antibody titres were calculated by reference to a negative standard (a 1 : 10 dilution of pre-inoculation serum). The results reported are the means of two tests, using duplicate wells for each serum dilution in each test. Preparation ofliposomes. Liposomes were prepared according to the method described by Souhami et al. (1981). Negatively charged liposomes were made using the molar ratio of phosphatidylcholine (7), cholesterol (2) and dicetylphosphate (1); neutral liposomes contained phosphatidylcholine and cholesterol, in the ratio 4:1. Briefly, dried lipid films obtained by rotary evaporation were shaken off the inner surface of a round-bottom flask with a 30 vtg/ml solution of peptide 141-160 prepared in 0.04 M-phosphate buffer pH 7-6, to give a final suspension of 3 (w/v). This preparation was referred to as large multilamellar liposomes. In order to produce small unilamellar liposomes, the large multilamellar liposomes were sonicated with 20 bursts of 30 s in an ice-bath, using a 'Rapids' sonicator (Ultrasonics, U.K.). RESULTS Timing o f the secondary inoculation Sixteen guinea-pigs were inoculated i n t r a m u s c u l a r l y w i t h 30 ttg o f p e p t i d e 141-160 coupled to K L H and formulated in 0.5 ~ A1 (OH)3, as an adjuvant. T h e y were subsequently d i v i d e d into four groups o f four animals and r e i n o c u l a t e d intramuscularly w i t h the s a m e p r e p a r a t i o n either 14, 28, 42 or 84 days later. S e r u m samples were collected at the start of the e x p e r i m e n t , at the t i m e of reinoculation and then weekly for 5 weeks. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 12 Jun 2017 00:36:24 2349 Immunological priming with FMD V peptides 2.5 0 2-0 ~z 1.5 1.0 0~5 1.o i i (a) I I I I~ I I J¢ I I I* 1 1 (b) Z ._ (a) (e) 0.5 dial i l i l m m n m 0 1-0 • (3 • I I I¢ I ~ __ (f) < 0.5 0 I I* I 1.0 0-5 mm • • ~ 40 80 120J 0 40 80 120 Time after primary inoculation (days) Fig. 1. (a, b) Neutralizing, (c, d) anti-virus particle, (e,J) anti-peptide 141 160 and (g, h) anti-peptide 200-213 antibody responses of guinea-pigs primed with 141 160KLH(O, O), 200-213KLH (A, A) or KLH alone (ai, D) and boosted (T) 42 days later with 141 160KLH (a, c, e, g) or 200-213KLH (b, d,f, h). No anti-peptide or neutralizing antibody was detected in any of the groups at the time of reinoculation. Low levels of anti-peptide 141-160 antibody were produced after reinoculation at either 14 or 28 days after the primary injection. These were associated with neutralizing and anti-virus particle antibodies which were slow to develop and did not reach peak activity for 21 to 28 days. Reinoculation at 42 days, however, produced significant anti-peptide 141-160, antivirus particle and neutralizing antibody responses (see Fig. 1) which reached peak activity within 14 days. A similar booster response was observed when the second inoculation was given at 84 days. Therefore, a 42 day time interval between primary and secondary inoculations was used in all subsequent experiments. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 12 Jun 2017 00:36:24 2350 M.J. FRANCIS AND OTHERS Specificity of priming with peptide Three groups of eight guinea-pigs were inoculated intramuscularly with 30 ktg of peptide 141160 coupled to KLH (141-160KLH), 30 ~tg of peptide 200-213 coupled to KLH (200-213KLH) or 20 ktg of untreated KLH alone, each formulated with 0.5 ~o AI(OH)3. After 42 days they were subdivided into groups of four and reinoculated intramuscularly with either 30 p.g of peptide 141-160KLH or 30 ~tg of peptide 200-213KLH, each formulated with 0.5~ AI(OH)3. Serum samples were collected at regular intervals after both primary and secondary inoculations. No neutralizing activity, anti-virus particle or anti-peptide 141-160 was detected in any of the groups after primary inoculation (Fig. 1). However, after a secondary inoculation the group that had been primed and boosted with peptide 141-160KLH (Fig. 1a) produced significant levels of neutralizing antibodies within 7 days and a peak titre of 2-3 log~0 SNs0 was reached within 14 days. This neutralizing antibody production was associated with the appearance of anti-virus particle (Fig. I c) and anti-peptide 141-160 (Fig. l e) activity. No such neutralizing activity could be detected against the heterotypic viruses A24 Cruzeiro or C Noville. The priming for peptide 141-160KLH did not occur when guinea-pigs were given a primary inoculation of untreated KLH alone. However, a low level of neutralizing activity was observed in the group primed with peptide 200-213KLH and boosted with peptide 141-160KLH (Fig. la). Groups of guinea-pigs primed with peptide 141 160KLH, peptide 200-213KLH or KLH alone which received peptide 200-213KLH as the second injection Produced no detectable neutralizing, anti-virus particle or anti-peptide 141-160 antibody (Fig. 1). However, there was a primary anti-peptide 200-213 antibody response in the group primed with peptide 200-213KLH (Fig. 1g, h). There was also an anti-peptide 200-213 response following the second inoculation with 200-213KLH, although this was less marked in the groups which had received 141160KLH or KLH alone as the primary inoculation (Fig. 1 h). Effect of adjuvant Three groups of four guinea-pigs were primed and boosted after 42 days with 30 ~tg of peptide 141-160KLH, either alone or formulated with 0-5~o AI(OH)3 or 1:1 with incomplete Freud's adjuvant (IFA). Serum samples were collected at weekly intervals. No neutralizing antibody was detected in any of the groups after primary inoculation. Furthermore, no detectable neutralizing antibody was produced following a second injection of peptide 141-160KLH in the absence of adjuvant (Fig. 2). There was also no detectable anti-virus particle (peak A49z of <0.1) or anti-peptide 141-160 (peak A492 of <0"1) antibody in these animals. However, the groups inoculated with either AI(OH)3- or IFA-adjuvanted preparations produced significant levels of neutralizing, anti-virus particle (peak A492 of 0"6 to 1'3) and antipeptide 141-160 (peak A492 of 0"8 to l'0) antibodies following the second injection (Fig. 2). Effect of carrier on secondary response Three groups of four guinea-pigs were inoculated with 30 ~tg of peptide 141-160KLH plus 0.5~ AI(OH)3, and reinoculated with either 30 ~g of peptide 141-160KLH, 30 ~g of peptide 141-160 coupled to TT (141-160TT) or 30 ~g of uncoupled peptide 141-160, each formulated with 0"5~o AI(OH)3. One additional group was given 30 p.g of 141-160TT with 0-5~ AI(OH)3 as both the primary and secondary inoculation. Serum samples were collected at weekly intervals. No primary neutralizing antibody response was observed in any of the groups. Each of the peptide preparations used for secondary inoculation produced significant levels of neutralizing antibody (Fig. 3) and this was associated with the appearance of comparable levels of anti-virus particle (peak A492 of 0.2 to 1.0) and anti-peptide 141-160 (peak A492 of 0.5 to 1-0) antibody. Furthermore, the neutralization titres in the group reinoculated with uncoupled peptide 141-160 were similar to those in the group which was reinoculated with peptide 141-160KLH. The group primed with peptide 141-160KLH which was reinoculated with peptide 141160TT produced lower levels of neutralizing antibody. However, these levels were not significantly lower than those present in the group primed and boosted with peptide 141-160TT. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 12 Jun 2017 00:36:24 2351 Immunological priming with FMD V peptides 2.0 Z 1.8! 2 ._~ 1.6: 2-0 Z r~ 1.8 ~ 1.6 , i f I i i i , , , , , , , 1.4 ~ 1.4 0 .8 .~ 1.2 "~ 1.2 .~ 1.0 .~ 1.0 M N ~'0.8 "~ 0.8 = -1 ~ 0.6 0 I 5 I 1o I 15 l 20 2'5 3'o ;~ 0-61 0 [ Time after second inoculation (days) 5 I0 ! 20 2 30 35 Time after second inoculation (days) Fig. 2 Fig. 3 Fig. 2. Secondary neutralizing antibody response of guinea-pigs primed and boosted after 42 days with 141 160KLH, using either IFA (A) or AI(OH) 3 ( 0 ) as adjuvant, or no adjuvant ( I ) . Fig. 3. Secondary neutralizing antibody response of guinea-pigs primed with 141-160KLH (O, A, I ) or 141 160TT (A) and boosted after 42 days with 141-160KLH (O), 141 160TT (A, A) or 141-160 alone ( I ) . The lower levels obtained with this peptide indicated that it had lower intrinsic activity than the KLH-coupled peptide. Effect of incorporating peptide into liposomes Four groups of four guinea-pigs were primed and boosted with 30 ktg of uncoupled peptide 141-160 incorporated into liposomes. The samples consisted of either negatively charged, or neutral, large multilamellar liposomes or small unilamellar tiposomes, derived by sonication of the multilamellar preparations. Two further groups of animals, injected with either 30 ~tg of peptide 141-160 which had been mixed with preformed negatively charged small unilamellar liposomes, or 30 p.g of peptide 141-160 alone, were included as controls. Serum samples were collected at regular intervals after the primary and secondary inoculations. No neutralizing activity was detectable in any group after the primary inoculation (Fig. 4). However, following a second inoculation neutralizing activity was produced in the groups which received small unilamellar liposomes, either neutral or negatively charged, which incorporated the peptide (Fig. 4a, b). This was associated with the appearance of anti-virus particle (2.8 to 3-0 log10) and anti-peptide 141-160 (3.0 to 3.4 logto) activity. Furthermore, 75 to 100~o of the animals from these groups were protected against challenge 56 days after secondary inoculation. No detectable neutralizing activity was observed in the pooled sera from groups which received large multilamellar liposomes incorporating peptide (Fig. 4a, b) or a small unilamellar liposome/peptide mixture up to 42 days after secondary inoculation (Fig. 4c). Individual sera collected at 56 days after the second inoculation had neutralizing titres up to 1.2 loglo SNso and this resulted in a low degree of protection (0 to 25~o). Peptide alone produced no detectable neutralizing activity (Fig. 4c) and no protection. DISCUSSION It is well established that, following primary immunization, animals will generally produce a rapid and enhanced immune response to a second stimulus (Glenny & Sudmersen, 1921). Furthermore, this so-called memory state can be adoptively transferred by transplanting cells from previously immunized donors into isologous hosts (Makela & Mitchison, 1965). An analysis of immunological memory demonstrated that animals may be primed without producing detectable levels of primary antibody formation (Nossal et al., 1965). The present study has shown that a sub-immunizing dose of FMDV peptide 141-160KLH can prime the immune system of guinea-pigs for a serotype-specific neutralizing antibody response to a second Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 12 Jun 2017 00:36:24 2352 M. J. FRANCIS AND OTHERS 1.9 I I I I t "~ 7 I I I (a) 1.6 1.3 1.0 Z rJ 3 0.7 e~ v 1.6 (b) O 1.3 1.0 0.7" 1.6 I , , (c) 1.3 1.0 0+7" 0 + , ~ . Pl, ,,', ,,', P',. ~ , ,', 10 20 30 40 50 60 70 80 90 Time after primary inoculation (days) Fig. 4. Neutralizing antibody response of guinea-pigs primed and boosted (T) after 42 days with (a) negatively charged multilamellar (O) or small unilamellar (0) liposomes incorporating peptide 141160, (b) neutral mulfilamellar (A) or small unilamellar (A) liposomes incorporating peptide 141 160,or (c) small unilamellar liposomes mixed with peptide 141 160 (D) or peptide 141 160 alone (m). dose of the same peptide. An interval of at least 42 days between primary and secondary inoculations is required to produce the optimal boosting effect. This may be explained by the observation that B-cell memory appears to require several weeks for full development (Cunningham & Sercarz, 1971) The presence of a low level of neutralizing activity in the sera of guinea-pigs primed with 200213KLH and boosted with 141 160KLH would indicate some sequence homology between the two peptides. Examination of the published sequence of VP1 for this virus (Strohmaier et al., 1982) reveals that the sequences of amino acids 151 to 155 and 209 to 213 are similar but in reverse order. However, it is difficult to understand this observation because this sequence relationship is unlikely to be reflected in any structural similarity. Moreover, 141-160KLH did not appear to prime for 200-213KLH, and whereas 200-213KLH produced a good primary antipeptide 200-213 response no neutralizing activity was detected. This provides further support to the published observations on the greater neutralizing efficiency of antibodies to the 141-160 region over antibodies to the 200 213 region (Bittle et al., 1982). The fact that no detectable neutralizing antibody response was produced in the absence of AI(OH)3 or I F A indicates that an adjuvant is required either for priming or boosting a neutralizing antibody response to the F M D V peptides. However, adequate priming was obtained using either AI(OH)3 or I F A as the adjuvant. In previous studies with a variety of viral peptides a number of different protein carriers and coupling techniques have been used to enhance their immunizing activity (for review, see Palfreyman et al., 1984). However, relatively little attention appears to have been directed towards the role of the carrier in producing an enhanced anti-peptide response. Studies with hapten-protein conjugates have shown that T-cells recognize, and respond to, carrier determinants on the protein in order to enable hapten-reactive B-cells to proliferate and Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 12 Jun 2017 00:36:24 Immunological priming with F M D V peptides 2353 differentiate into antibody-forming cells (Raft, 1970; Mitchison, 1971). Furthermore, an optimal boosting effect requires the hapten to be coupled to the same carrier as that used for priming, or the animals to have been previously primed with the same carrier, non-haptenized (Ovary & Benacerraf, 1963; Rajewsky et al., 1969). While these studies have provided valuable information on the induction of humoral responses the function of the carrier in a peptide response may be different since peptides containing 10 to 30 amino acids, although they appear to be poor immunogens, cannot be regarded as true haptens. Furthermore, the results of this study have shown that the role of K L H in priming for F M D V peptide is fundamentally different from its role in hapten priming since no carrier was required to boost the response of peptide-KLH-primed animals. The lower boost observed using TT-coupled peptide may have been due to minor differences in the conformation of K L H - and TT-coupled peptides resulting in the lower intrinsic activity of the latter coupled peptide, since if 141-160 coupled to TT is used to prime and boost guinea-pigs a similar reduced response is observed. This conformational difference could be a result of the different methods used to couple peptide to the protein carrier (see Methods). Therefore, it seems likely that the carrier is simply acting as a delivery system for the peptide by increasing its size, the epitope density and/or by influencing the conformation in which it is presented to the immune system. The potent protein carriers frequently used in studying the immunogenic activity of viral peptides may, in any case, not be necessary for stimulating a satisfactory immune response since polymerized F M D V peptides will induce a neutralizing antibody response in the absence of carrier (Bittle et al., 1984). In the present study, liposomes which are themselves very poor antigens have been successfully used as carriers in order to enhance the immunogenicity of the 141-160 peptide. Although no attempt was made to control the epitope density it was clear that the size of the liposomes was of major importance since sonication to a small unilamellar form was required to optimize their adjuvanticity. The charge of the liposomes did not appear to have any marked influence on their immunizing activity. However, it was necessary to incorporate the peptide within the liposomes, since a similar weight of peptide added to preformed small unilamellar liposomes was less immunogenic. The low level of antibody activity which was observed in this control group may have been due to the free peptide associating with the 'empty' liposomes. It is possible that the small unilamellar liposomes are 'targeting' the peptide directly to antigen-presenting cells, for example follicular dendritic cells of the germinal centres. These results emphasize the potential value of small unilamellar liposomes as adjuvants for synthetic vaccines. In conclusion, a low-immunizing dose of peptide 141-160 will prime the immune system of guinea-pigs for a F M D V neutralizing antibody response. Furthermore, the observations that this priming can be achieved with a relatively non-immunogenic carrier and that the secondary response is not carrier-restricted indicates that the 14l 160 peptide can initiate its own T-helper cell activity without the involvement of antigenic carrier molecules. Therefore, the 141-160 peptide contains both B-cell, neutralizing antibody recognition sites, and T-cell determinants. The authors wish to thank Dr H. M. Patel of the Department of Biochemistry, Charing Cross Hospital Medical School, London, U.K. for his practical help and guidance on the techniques of liposome preparation. REFERENCES (1975). Immune and antibody responses to an isolated capsid protein of foot and mouth disease virus. Journal of Immunology 115, 1635-1641. BACHRACH, H. L., MOORE, D. M., McKERCHER, P. D. & POLATNICK, J. BITTLE, J. L., HOUGHTEN, R. A., ALEXANDER, J., SHINNICK, T. M., SUTCLIFFE, J. G., LERNER, R. A., ROWLANDS, D. J. & BROWN,F. (1982). Protection against foot and mouth disease by immunization with a chemically synthesized peptide predicted from the viral nucleotide sequence Nature, London 298, 30 33. BITTLE, J. L., WORRELL, P., HOUGHTEN, R. A., LERNER, R. A,, ROWLANDS, D. J. & BROWN, F. (1984). Immunization against foot and mouth disease with a chemically synthesized peptide. In Modern Approaches to Vaccines, pp. 103-107. Edited by R. M. Chanock & R. A. Lerner. New York: Cold Spring Harbor Laboratory. BROWN,r. &CARTWRIGHT,B. (1963). Purification of radioactive foot and mouth disease virus. Nature, London 199, 1168 1170. CLARKE, B. E., CARROLL, A. R., ROWLANDS, D. J., NICHOLSON, B. H., HOUGHTEN, R. A., LERNER, R. A. & BROWN, F. (1983). Synthetic peptides mimic subtype specificity of foot and mouth disease virus. FEBS Letters 157, 261 264. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 12 Jun 2017 00:36:24 2354 M. J. F R A N C I S A N D O T H E R S CUNNINGHAM, A. J. & SERCARZ, E. E. (1971). The asynchronous development o f immunological memory in helper (T) and precursor (B) cell lines. European Journal of Immunology 1, 413-421. EMINI, E. A., JAMESON, B. A. & WINNER, E. (1983). Priming for and induction of anti-poliovirus neutralizing antibodies by synthetic peptides. Nature, London 304, 699-703. FRANCIS,M. J. & BLACK,L. (1983). Antibody response in pig nasal fluid and serum following foot and m o u t h disease infection or vaccination. Journal of Hygiene 91, 329-334. FRANCIS, M. J., FRY, C. M., ROWLANDS, D. J., BROWN, F., BITTLE, J. L., HOUGHTEN, R. A. & LERNER, R. A. (1985). Priming with peptides of foot and m o u t h disease virus. In Vaccines1985, pp. 203-210. Edited by R. A. Lerner, R. Chanock & F. Brown. New York: Cold Spring Harbor Laboratory. GLENNY, A. T. & SUDMERSEN, H. J. (1921). Notes on the production of immunity to diphtheria toxin. Journal of Hygiene 20, 176-220. KENNEDY, R. C. & DREESMAN, G. R. (1984). E n h a n c e m e n t of the i m m u n e response to hepatitis B surface antigen. Journal of Experimental Medicine 159, 655-665. KURZ, C., FORSS, S., KUPPER, H., STROHMAIER,K. & SCHALLER,H. (1981). Nucleotide sequence and corresponding amino acid sequence of the gene for the major antigen of foot and m o u t h disease virus. NucleicAcids Research 9, 1919-1931. LAPORTE, J., GROSCLAUDE,J., WANTYGHEM,J., BERNARD,S. & ROUZE, P. (1973). Neutralization en culture cellulaire du pouvoie infectieux du virus de la fievre aphteuse par les serums provenant de porcs immunis6s a l'aide d'une proteine virale purifi6e. Comptes rendus hebdomadaires des seances de l'Academie des sciences, sdrie D 276, 3399 3401. MAKELA,O. & MITCHISON, N. A. (1965). The role of cell n u m b e r and source in adoptive immunity. Immunology 8, 539-548. MITCHISON, N. A. (1971). The carrier effect in the secondary response to hapten protein conjugates. II. Cellular cooperation. European Journal of Immunology 1, 18-27. NOSSAL,G. J. V., AUSTIN,C. M. & ADA, G. L. (1965). Antigens in immunity. VII. Analysis of immunological memory. Immunology 9, 333-348. OVARY, Z. & BENACERRAF,B. B. (1963). Immunological specificity of the secondary response with dinitrophenylated proteins. Proceedings of the SocietyJor Experimental Biology and Medicine 114, 72-76. PALFREYMAN, J. W., AITCHESON,T. C. & TAYLOR, P. (1984). Guidelines for the production of polypeptide specific antisera using small synthetic oligopeptides as immunogens. Journalof Immunological Methods 75, 383-393. PFAFF, E., MUSSGAY,i . , BOHM,H. O., SCHULZ,G. E. & SCHALLER,H. (1982). Antibodies against a preselected peptide recognize and neutralize foot and m o u t h disease virus. EMBO Journal 1, 869-874. RAFF, M. C. (1970). Role of thymus-derived lymphocytes in the secondary humoral i m m u n e response in mice. Nature, London 226, 1257-1258. RAJEWSKY, K., SCHIRRMACHER, M., NASE, S. & JERNE, N. K. (1969). The requirement of more than one antigenic determinant for immunogenicity. Journal of Experimental Medicine 129, 1131-1143. ROWLANDS, D. J., CLARKE, B. E., CARROLL,A. R., BROWN, F. NICHOLSON,B. H., BITTLE, J. L. & LERNER, R. A. (1983). Chemical basis of antigenic variation in foot and m o u t h disease virus. Nature, London 306, 694-697. SOUHAMI,R. L., PATEL,H. M. & RYMAN,B. E. (1981 ). The effect of reticuloendothelial blockade on the blood clearance and tissue distribution of liposomes. Biochimica et biophysica acta 674, 354-371. STROHMAIBR,K., FRANZE, R. & ADAM,K. H. (1982). Location and characterization of the antigenic portion of the F M D V immunizing protein. Journal oJ General Virology 59, 295-306. VOLLER, A. & BIDWELL, D. E. (1976). Enzyme immunoassays for antibodies in measles, cytomegalovirus infections and after rubella vaccinations. British Journal of Experimental Pathology 57, 243-247. WILD, T. F., BURROUGHS,J. N. & BROWN, F. (1969). Surface structure of foot-and-mouth disease virus. Journal of General Virology 4, 313-320. (Received 1 July 1985) Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 12 Jun 2017 00:36:24