Bioconjugate Chem. 2006, 17, 1508−1513
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Influence of Carrier Proteins on the Immunologic Response to Haptenic Antitetrodotoxin Vaccine Qin-Hui Xu,* Xiu-Nan Zhao, Jun-Ping Cheng, Chang-Hua Wei, Qin-Hua Zhang, and Kang-Tai Rong Beijing Institute of Pharmacology and Toxicology, 27 Taiping Road, Beijing 100850, P.R. China. Received April 4, 2006; Revised Manuscript Received August 29, 2006
Tetrodotoxin (TTX) is a haptenic, highly toxic neurotoxin with no specific antidote available yet. Anti-TTX vaccine is being studied for antitoxin development. The effectiveness of the carrier protein in eliciting TTXspecific antibody response was comparatively studied. TTX was conjugated to Tachypleus tridentatus hemocyanin (TTH), Limulus polyphemus hemocyanin (LPH), tetanus toxoid (TT), diphtheria toxoid (DT), and bovine serum albumin (BSA) chemically to form artificial antigens TTH-TTX, LPH-TTX, TT-TTX, DT-TTX, and BSATTX, respectively, with which BALB/c mice were immunized, and the antibody response and antitoxic efficacy were detected. The serum anti-TTX antibody response and antitoxic efficacy varied markedly with adopted carrier protein. TTH-TTX elicited the best and BSA-TTX the worst TTX-specific antibody response. The proportion of the immunized mice surviving a 3× lethal dose (LD) dose of TTX challenge was 92%, 75%, 42%, 8%, and 0% for TTH-, TT-, LPH-, DT-, and BSA-TTX conjugates, respectively. The rank order of total efficacy of carrier protein for both anti-TTX antibody response and antitoxic effect was TTH > TT > LPH > DT > BSA. As a result of formaldehyde treatment in coupling of TTX carriers, the relative immunogenicity of TTX Vs carrier, that is, the ratio of TTX- to carrier-specific antibody response, evidently varied with respective carrier adopted, in a rank order of TT > BSA > TTH > DT > LPH. The results suggest that the carrier protein used in haptenic TTX vaccine is greatly important in eliciting potent anti-TTX antibody, and both TTH and TT are the preferred carriers for development of excellent experimental TTX vaccine.
INTRODUCTION A small molecular chemical is immunologically inert. So it has to be chemically modified by covalent linking to a large molecular weight carrier protein (CP)1 to form an artificial antigen before it can act as an immunogen (1-6). The selection of the carrier molecule of the haptenic vaccine is paramount for the successful production of hapten-specific antibody, although special research on selection of CP for a haptenic vaccine is very rare (7, 8). Tetrodotoxin (TTX), mainly found in the organs of puffer fish, is one of the highly toxic haptenic neurotoxins. TTX blocks diffusion of sodium through the sodium channel, preventing depolarization and propagation of action potentials in nerve cells. Poisoning with TTX usually occurs after ingestion of improperly prepared TTX-containing puffer fish in China and Japan where the puffer fish is considered a delicacy. Death in seriously poisoned humans often occurs from respiratory failure (9-11). Management of clinical TTX poisoning disease is often difficult, and the main treatment is meticulous supportive care, but it is usually inefficient in serious cases. So far no specific antidote for TTX poisoning has been available (12, 13). Immunological protection, especially the passive immunotherapy of antitoxin, has been considered as a unique measure capable of conquering TTX diseases; studies in this field have been published with varying degrees of success (14-19). As a first stage to producing a potential anti-TTX antitoxin for treating victims, a * Corresponding author. Tel: +86-10-6687-4609. Fax: +86-106821-1656. E-mail address:
[email protected]. 1 Abbreviations: BSA, bovine serum albumin; CP, carrier protein; DT, diphtheria toxoid; ELISA, enzyme-linked immunosorbent assay; Kap, apparent affinity; LPH, Limulus polyphemus hemocyanin; PBS, phosphate-buffered saline; TT, tetanus toxoid; TTH, Tachypleus tridentatus hemocyanin; TTX, tetrodotoxin.
chemo-experimental vaccine of TTX that could highly effectively protect animals from TTX intoxication has been developed (20-22), and a preliminary attempt of passive immunization research for TTX has been performed (23) in our lab. The purpose of this report is to present an anterior research on the selection of CP in anti-TTX experimental vaccine in order to obtain a potent antibody response and then excellent antitoxic effect from TTX vaccine and further to develop a potential antiTTX antitoxin. Five experimental vaccines constructed by coupling TTX to different proteins have been compared in their immunogenic potency and antitoxic efficacy in mice, the possible cause for the effect differences among vaccines has been analyzed experimentally, and the optimal carriers adopted in TTX vaccine have been validated.
MATERIALS AND METHODS Materials. TTX (Mr 319.3) was purchased from Fisheries Research Institute of Hebei Province (Qinhuangdao, China); the product was prepared by freeze-drying the original solution containing citrate buffer with pH 4.8-4.9 and with purity of 98%. Bovine serum albumin (BSA, Mr 67 000) and Limulus polyphemus hemocyanin (LPH, Mr 3 300 000) were purchased from Sigma; diphtheria toxoid (DT, Mr 63 000) and tetanus toxoid (TT, Mr 67 000) were obtained from Changchun Institute of Biological Products (Changchun, China); Tachypleus tridentatus hemocyanin (TTH) with molecular weight of 3 000 000 Da by ultracentrifugation analysis was prepared by our lab. All these proteins were lyophilized. Mice. Female BALB/c mice, aged 6-8 weeks, Grade II, Certificate No. SCXK 2003-001, were obtained from the Institute of Jing-Feng Medical Laboratory Animals (IJM) (Beijing, China) and were housed under standard laboratory conditions and fed ad libitum diet and water. All animal study
10.1021/bc060083u CCC: $33.50 © 2006 American Chemical Society Published on Web 11/02/2006
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procedures were approved by the Center on Experimental Animal Care and Research from our Institute. Preparation of Artificial Antigen. TTX was conjugated to CP via formaldehyde as the coupling agent to form artificial antigen as described previously with modification (22-24). Briefly, 1 mg of lyophilized TTX was dissolved in 1 mL of distilled water to yield an aqueous solution consisting of citrate buffer, pH 4.9. CP (8 mg), that is, TTH, TT, LPH, DT, or BSA, was dissolved in 2.0 mL of 20 mM phosphate-buffered saline (PBS) (pH 6.8), and TTX solution was added to yield a weight ratio of TTX to CP of 1:8. Then formaldehyde was added up to final concentration of 1.5%. The reagents were thoroughly mixed and slowly shaken in a 30 °C water bath for 72 h, and then dialyzed for 48 h at 4 °C against six 1 L changes of PBS (pH 7.2) to remove residual free TTX. TTX-CP conjugates, that is, TTH-TTX, TT-TTX, LPH-TTX, DT-TTX, and BSA-TTX, were obtained, and conjugate aliquots were stored at -20 °C before use. In addition, the formal-treated control CPs, that is, f-TTH and f-BSA, were prepared by the above procedure with omission of TTX. Immunization of Mice. Mice were randomly divided into groups of 12 and immunized intraperitoneally (ip) with 50 µg of TTX-CP, emulsified by mixing it with an equal volume of complete Freund’s adjuvant. Booster immunizations were administered at the 15th day after the primary immunization (d15), d30, d55, d85, d115, and d150 with 50 µg of TTX-CP per injection, emulsified in incomplete Freund’s adjuvant or in saline solution alternatively. Each mouse received a total of 350 µg of artificial antigen in terms of the amounts of CP. In addition, mice were immunized with representative CPs instead, that is, DT, TT, and TTH, according to the same scheme acting as control. Determination of TTX-Specific Antibody Response. The antisera were collected on schedule during the immunization and the TTX challenge course by tail-bleeding and stored at -20 °C until tested. Antibody quality, expressed by parameters antibody titer and apparent affinity, was tested as described before (22, 23). Briefly, test antigen BSA-TTX, at a concentration of 2 µg/mL, 100 µL/well, was coated onto 96-well microtiter plates for detection of antibody against all TTX conjugates except BSA-TTX itself, and instead, TTX-DT was used for detection of TTX-BSA, in order to avoid the false positive reaction from the antibody against CP of immunogen reacting on the same CP in test antigen. DT-TTX acting as test antigen was certified essentially identical with BSA-TTX in preliminary experiments. The antisera were tenfold serially diluted in PBS (pH 7.2) containing 1% BSA, and the antibody reactivity with test antigen (BSA-TTX or DT-TTX) was detected by enzyme-linked immunosorbent assay (ELISA). TTX-specific antibody level was expressed as antibody titer (n). The n value was defined as the logarithm of specimen dilution at which the positive reaction was certificated as a result of A490 nm read on a Bio-Rad model 550 microplate reader (Bio-Rad), exceeding those in normal mice sera control 3-fold. Apparent affinity (Kap) was assayed by competition-inhibited enzyme immunoassay. Briefly, the mixtures of a series of molar concentrations of TTX solution and antibody specimens in fixed dilutions ranging from 102-105 were incubated with test antigen at 37 °C for 0.5 h. Then anti-TTX antibody reactivity was determined as above, and IC50, the TTX concentration (M) inhibiting 50% of antibody binding activity to test antigen, was calculated. The apparent affinity, Kap, was defined as the negative logarithm of IC50 (Kap ) pIC50). Determination of CP-Specific Antibody Response. CPspecific antibody in antisera was determined by ELISA procedure same as the above by a modification of using respective
Figure 1. Comparison of the dynamic course of TTX-specific antibody level among TTX vaccines. BALB/c mice in groups were immunized with different TTX-carrier conjugates, and the antiserum specimens were prepared on schedule, initially at day 35 after initial immunization. Antibody titer (n), defined as the negative logarithm of the final dilution of antisera at which positive antibody reactivity appeared, was detected by ELISA procedure and expressed as mean ( SEM, n ) 12 per group initially, and thereafter the survival number is indicated otherwise in parentheses.
CP alone, that is, TT, DT, or TTH, instead of BSA-TTX or DT-TTX conjugate as a test antigen. Calculation of Relative Immunogenicity of TTX Ws CP. The relative immunogenicity of TTX Vs CP was reflected by relative antibody response intensity and expressed as the ratio of TTX- to carrier-specific antibody response in A490 nm read assayed as above. TTX Challenge Experiment. To compare the antitoxic effect of different CP-TTX vaccines, 12 immunized mice from each group were challenged repeatedly with 1× lethal dose (LD) of TTX for the first challenge at d157, 2×LD for the second at d167, 3×LD for the third at d183, and 4×LD for the fourth at d198. Referring to a previous report (25), a dose of 13.5 µg/kg of TTX in 10 mL/kg of normal saline solution was defined as 1×LD of TTX intraperitoneal (ip) challenge for female BALB/c mice in this study. The survivals were determined 24 h after TTX exposure, and the lethal time in the case of decedents was recorded. Statistical Analysis. All replicate data were expressed as mean ( S.E.M. The differences among groups were determined by using Student’s t-test, and P < 0.05 was considered significant as compared with the corresponding control.
RESULTS Comparison of the Dynamic Course of TTX-Specific Antibody Level among Different Vaccines. The circulating TTX-specific antibody level (n) was increased gradually within 3-6 months (Figure 1). Among the five TTX vaccines, the quickest antibody-raising rate occurred in the TTH-TTX group; a moderate rate occurred in both the TT- and LPH-TTX groups; a slower rate occurred in the DT-TTX group; and the slowest rate occurred in the BSA-TTX group. A 3-month period for TTH group but a 5-6-month period for the others was necessary for the antibody titer to ascend to its top level. As a result, the antibody titer difference between the highest in the TTH-TTX and the lowest in the BSA-TTX group approximated two units of n value during the antibody-raising course. The n value from the fourth assay (d125) exemplified in Table 1 statistically differed from each other for all paired comparisons between groups, except TT-TTX Vs LPH-TTX. The sequence of final antibody level magnitude was accordant with the antibody-raising rate, keeping rank as TTH-, TT-/ LPH-, DT-, and BSA-TTX groups (Figure 1). After repeated
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Table 1. Comparison of the Antibody Level among TTX Vaccines with Different Carrier Proteins statistical significance of difference between groupsb immunogen
titer (n)a
TTH-TTX TT-TTX LPH-TTX DT-TTX BSA-TTX
5.4 ( 0.1 4.8 ( 0.1 4.8 ( 0.2 4.2 ( 0.1 3.8 ( 0.1
BSA-TTX
DT-TTX
LPH-TTX
TT-TTX
/// // /
/ †
//
/// /// /// /
TTH-TTX
a Antisera were collected at day 125 after initial immunization. The titer (n) was detected as note in Figure 1 and expressed as mean ( SEM, n ) 12 per group. b Asterisks and daggers indicated statistical significance of difference in n values: † P > 0.05, / P < 0.05, // P < 0.01, and /// P < 0.001, compared with each other group by t-test.
Figure 2. Comparison of TTX-specific antibody affinity among TTX vaccines. Antisera were prepared before the first TTX challenge in groups. Apparent affinity, Kap, defined as the negative logarithm of IC50, the TTX concentration (M) inhibiting 50% of antisera binding activity to test antigen (Kap ) pIC50), was tested by competition-inhibited enzyme immunoassay procedure and expressed as mean ( SEM, n ) 12 per group.
TTX challenges, the antibody level declined slightly in both the TTH and TT vaccines and sharply in the LPH vaccine, while all mice in the DT- and BSA-TTX groups had died off, implying that the circulating antibody had been neutralized by TTX in ViVo (Figure 1). No TTX-specific antibody certainly was detected in the sera from mice immunized with CP alone. Comparison of Antibody Affinity among TTX Vaccines. The apparent affinity (Kap) for TTX-specific antibody, assayed before the first TTX challenge, also varied with CP adopted in these vaccines (Figure 2). The difference between the highest in the TTH-TTX and the lowest in BSA-TTX group was as high as approximately two units of Kap value; the average magnitude of Kap sequenced basically same as that of antibody level with the exception of the DT-TTX group, which showed slightly higher Kap value. In the TTH-TTX group, a lower standard deviation of Kap value implied that the antibody affinity matured more rapidly and then more readily tended to uniformity than in other four vaccines. Comparison of Antitoxic Efficacy among TTX Vaccines. TTX challenge experiments began at d157 when the antibody level in all tested groups approximately approached the top level and was repeated at intervals of 10-16 days with gradually increasing doses (Figure 3). The majority of the immunized mice were able to tolerate more than one TTX exposure. The group TTH-TTX showed the most excellent effect, all mice surviving a 2×LD single dose and the majority surviving increasing TTX doses, and the group TT-TTX showed a slightly lower effect, all surviving a 1×LD single TTX dose. In turn, LPH-TTX presented a moderate antitoxic potency, the majority surviving 1×LD and the survival rate descending sharply with the increasing TTX doses. The DT-TTX showed a poorer effect; and especially, the BSA-TTX showed the poorest effect, none
Figure 3. Comparison of antitoxic efficiency among TTX vaccines. Immunized mice in groups of 12 were intraperitoneally challenged repeatedly with 1×LD of TTX for the first challenge at the 157th day after initial immunization (d157), 2×LD for the second at d167, 3×LD for the third at d183, and 4×LD for the fourth at d198. All control mice, uninoculated or inoculated with TTH or TT alone, died of 1×LD (n ) 22) and 2×LD (n ) 6) TTX challenge; 1×LD ) 13.5 µg/kg, in 10 mL/kg of normal saline. The survivors were counted 24 h after TTX exposure. Table 2. Comparison of Lethal Time for Victims among TTX Vaccines immunogen uninoculated control TTH or TT control TTH-TTX TT-TTX LPH-TTX DT-TTX BSA-TTX
number of mice 12 6 10 6 7 10 12 12
TTX dosea (×LD) 1 2 1 3-4 2-4 1-4 1-4 1-3
lethal timeb (min) 13 ( 2 5(0 10 ( 1 22 ( 3*,‡ 56 ( 26*,† 20 ( 6 21 ( 4† 10 ( 1
a The animals were challenged as described in Figure 3. b Lethal times are means ( SEM. *P < 0.05 vs control of 1×LD and †P < 0.05, ‡P < 0.01 vs BSA-TTX by t-test.
surviving the 3×LD TTX dose. The survival proportion of the immunized mice, for example, challenged with a single TTX dose of 3×LD, was 92%, 75%, 42%, 8%, and 0% for TTH-, TT-, LPH-, DT-, and BSA-TTX, respectively. The group sequence in anti-TTX poisoning effect was approximately consistent with that for both antibody-raising rate and antibody level (Figure 1), that is, in the order TTH, TT, LPH, DT, and BSA carrier. All control mice, uninoculated or immunized with TTH or TT alone, died of 1× or 2×LD TTX exposure. In addition, the decedents in all groups except BSA-TTX, especially in TTH- and TT-TTX groups, survived longer than those in control groups, even those who were challenged with higher TTX doses, whereas the victims in the BSA-TTX group behaved almost the same as the control (Table 2). Influence of Formaldehyde Treatment of CP on Its Antigenicity. In an attempt to explain the cause of the difference in TTX-antibody response among TTX vaccines, the influence of formaldehyde treatment in coupling of TTX-CP on CP
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individual anti-CP antisera and unrelated CP, implying that only TTH and LPH may share common antigenicity. Cross-Reactivity of Anti-TTX Vaccine Antisera to CP and TTX. To compare the relative immunogenicity of TTX Vs CP in individual TTX vaccines, both CP-specific and TTX-specific antibodies contained in antisera were measured by the ELISA procedure (Table 4). The antisera elicited by the TT-TTX vaccine strongly reacted with TTX ligand but weakly with TT carrier. Similarly, the anti-TTH-TTX antisera reacted very strongly with TTX but considerably less strongly with TTH, and anti-BSA-TTX antisera moderately to TTX but negligibly to BSA. In contrast, the antisera against both LPH-TTX and DT-TTX reacted with the respective CPs more strongly than with TTX. Furthermore, the antisera from both TTH-TTX and LPH-TTX obviously cross-reacted each other, whether the CP was in a free or coupled state, which was somewhat consistent with the case of anti-TTH antisera being cross-reactive to LPH or LPH-TTX (Table 3). Based on these data, the relative intensity of anti-TTX to antiCP antibody response was analyzed (Table 5). The ratio of TTXto CP-specific antibody response ranks in sequence of TT-, BSA-, TTH-, DT-, and LPH-TTX vaccine.
Table 3. Cross-reactivity (A490 nm) of Anti-CP Antisera against CP and CP-TTX Conjugatea anti-CP antisera eliciting immunogenc test
antigenb
BSA f-BSA DT LPH TT TTH f-TTH DT-TTX LPH-TTX TT-TTX TTH-TTX
TT (n ) 10)
DT (n ) 8)
TTH (n ) 9)
0.68 ( 0.05 0.36 ( 0.05** -
0.38 ( 0.02 0.42 ( 0.02 -
0.73 ( 0.04 0.94 ( 0.05 0.15 ( 0.01‡ 1.11 ( 0.12 ** 0.98 ( 0.06
aA 490 nm values are means ( SEM; n indicates the number of specimens tested. **p < 0.01 vs respective CP and ‡p < 0.01 vs TTH, by t-test; denotes a value of A490 nm < 0.1, same as that in normal mice sera control. b f-BSA and f-TTH refer to formaldehyde-treated BSA and TTH, the procedure being the same as the preparation of CP-TTX conjugates by omission of TTX. c Antisera, collected at day 120 from mice immunized with CP, were tested by ELISA procedure at a dilution of 1:1000.
antigenicity and the cross-antigenicity between CPs have been assayed using mice antisera elicited by three representative CPs, that is, TT, DT, and TTH (Table 3). The anti-TT antisera reacted with TT-TTX far more weakly than with TT alone, whereas both anti-TTH and anti-DT antisera reacted similarly with their respective TTX conjugates and CP alone. Moreover, the antiTTH antisera reacted very weakly with f-TTH, a product from aggregation of TTH molecules by formaldehyde treatment. These results indicated that the change of antigenicity for CP involved in the TTX conjugate due to formaldehyde treatment varied markedly, that is, significant loss in the case of TT and negligible change in DT and TTH, and that formaldehyde might bring on the loss of most antigenicity in free TTH but had negligible effect on TTH involved in its TTX conjugate. In addition, except anti-TTH antisera, which reacted with LPH and LPH-TTX, no positive cross-reaction occurred between
DISCUSSION It is considered that proteins of large molecular size and originating from outside the mammalian order are strongly immunogenic and could be preferred as CPs of haptenic vaccine. Among the proteins studied, both TTH and LPH are excellent immunogenic agents themselves (26-28) and have been effectively used as carriers of other haptenic poisons (29, 30); TT and DT have been clinically used as active immunizing agents for a long time; BSA has the advantage of being easily available in pure form and proven to be an effective carrier (4, 31, 32), whereas some reports showed it being a weak carrier in other poison conjugates (33, 34). The present research showed that the anti-TTX antibody response elicited by TTX vaccines varied greatly with vaccine-
Table 4. Cross-Reactivity (A490 nm) of Antisera Elicited by TTX Vaccines to Both Haptenic TTX and CPa antisera-eliciting immunogen test antigen
BSA-TTX (n ) 8)
DT-TTX (n ) 9)
LPH-TTX (n ) 10)
TT-TTX (n ) 12)
TTH-TTX (n ) 12)
BSA-TTX DT-TTX LPH-TTX TT-TTX TTH-TTX BSA DT LPH TT TTH f-TTH
0.75 ( 0.15 0.57 ( 0.10 † † † 0.20 ( 0.02 -
0.66 ( 0.14 1.94 ( 0.19 † † † 1.12 ( 0.15 -
1.02 ( 0.15 0.93 ( 0.18 1.72 ( 0.14 † 1.50 ( 0.05 1.85 ( 0.11 1.17 ( 0.07 -
1.20 ( 0.08 1.36 ( 0.06 † 1.27 ( 0.08 † 0.25 ( 0.02 -
1.69 ( 0.07 1.90 ( 0.08 2.52 ( 0.12 † 1.56 ( 0.09 1.66 ( 0.12 1.20 ( 0.10 0.18 ( 0.02
a Cross-reactivity was assayed by ELISA procedure in which antisera were diluted 1000-fold with PBS containing 1% BSA (pH 7.2). The results (A 490 nm) are means ( SEM; the control value of nonspecific binding of normal mice sera (1:1000) to test antigens was 0.09 ( 0.00, n ) 22. †, not detected; denotes a value of A490 nm < 0.1, same as that in normal mice sera control. No TTX-antibody reaction was detected from antisera from control mice inoculated with TTH or TT alone (n ) 12). f-TTH refers to formaldehyde-treated TTH.
Table 5. Analysis for Relative Intensity of Antibody Reaction to TTX and CP antisera-eliciting immunogen reactivity to CPa reactivity to TTXa reactivity ratio of TTX/CPb sequence of ratioc
BSA-TTX
DT-TTX
LPH-TTX
TT-TTX
TTH-TTX
(/+ ++ 4.3 ( 1.0 2
+++ ++ 0.8 ( 0.2 4
++++ +++ 0.5 ( 0.1 5
+ +++ 5.0 ( 0.4 1
+++ ++++ 1.6 ( 0.1 3
a The grade of intensity was defined according to data in Table 4: (, A 490 nm < 0.2; +, 0.2 < A490 nm < 0.5; ++, 0.5 < A490 nm < 1.0; +++, 1.0 < A490 nm < 1.5; ++++, A490 nm > 1.5. b The reactivity ratio means the ratio of TTX-specific antibody reaction with test antigen in which the CP was different from that in immunogen vs CP-specific antibody reaction with respective CP alone. Values are means ( SEM; n ) 8-12 per group. c The numbers denote the sequence of ratios listed above, indicating relative potency of TTX-specific antibody response as 1 > 2 > 3 > 4 > 5.
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constituting CP species. The TTH-TTX was excellent, showing strong antibody response and high antitoxic efficacy; the TTTTX was just slightly inferior to TTH-TTX in turn; the DTTTX showed a weak immune effect and some toxic side effects; and the BSA-TTX showed poor immune response. The LPHTTX vaccine showed only a moderate anti-TTX effect, although LPH is also macromolecular and shares common antigenicity with TTH, as proved by cross-reactivity to anti-TTH antisera (Table 3). Previous reports demonstrated that only 46% of the amino acid sequence of the N-terminal segment of the LPH molecule is identical with that of the R-chain of TTH (35, 36) and that their characteristics are different in ultracentrifugation, chromatography, electrophoresis, electron microscopy, and immunogenicity (26-28, 37, 38). These differences in structure and characteristics might be responsible for their different effect in application to TTX vaccines. Consequently, TTH chiefly and also TT were selected as optimal CP candidates and approved by further study (22). The antitoxic potency for all TTX vaccines was basically consistent with the respective antibody response. The highest antibody quality induced the best antitoxic efficacy in TTHTTX; the lowest antibody response produced the poorest antitoxic potency in BSA-TTX. This fact demonstrated that the antibody response was consequentially the basis of the antitoxic effect. The results also suggested that an early quick antibody response must result in a higher final circulating antibody level and quicker affinity maturation. Usually the CP involved in an artificial antigen conjugate may be more immunogenic than the hapten, and the conjugate could induce inhomogeneous antisera containing both CP- and hapten-specific antibodies. So the binary antitoxic sera, for example, against both TT or DT and TTX, would be possibly elicited by immunization with TT- or DT-TTX conjugates. Nevertheless, the results in this study showed that the relative intensity of antibody response to CP and to TTX varied with CP species used in the vaccine (Table 5). Animals responded strongly to the TTX ligand but weakly to the TT carrier for the TT vaccine. Similarly, higher TTX/CP antibody response ratio appeared both in the TTH vaccine, very powerful response, and in the BSA vaccine, very weak response. But, in a contrary mode, a stronger response to CP than to TTX was observed in DT and LPH vaccines. The phenomenon that the immunogenicity of some proteins involved in artificial antigens was inferior to that of haptenic TTX is interesting and inexplicable yet. Whether it is true for other CP-hapten conjugates remains to be studied. The experiments on cross-reactivity of anti-CP antisera showed that formaldehyde treatment caused the majority of antigenicity of free TTH to be lost. Probably some immunologically active group becomes inactive because of intraand intermolecule cross-linking (39, 40). But, in the case of the TTX-CP conjugate coupled via formaldehyde, the change in CP antigenicity varied according to adoptive protein species, decreasing for TT, invariable for TTH, and increasing for DT and LPH (Table 3). This was even consistent with and thereby might be able partially to interpret the sequence of TTX/CP antibody response ratios (Table 5). The decrease of CP antigenicity might be beneficial to the prominence of anti-TTX antibody response. This imbalance of immune response against CP and TTX may be one of the direct causes of diverse TTXspecific antibody production. There is much more to be learned whether and how the species and structure of CPs bring on different haptenic stimulus properties. This first work will serve as a basis for further elucidation of influences of CP on haptenic vaccine. The classical coupling technique of TTX to proteins by formaldehyde treatment in which amino groups of proteins were cross-linked with guanidyl groups of the TTX molecule (24,
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41) was to some extent reversible; thereby the free toxin resulting from possible decomposition during the storage, management, or even after injection would poison animals as indicated by the early case of paralytic shellfish poison (24). By virtue of improvement of preparation and storage of vaccine, all TTX vaccines except DT-TTX showed no perceivable side effects. The majority of mice in the DT-TTX group became thin (P < 0.01 Vs control), developed sag and lackluster hair, and quivered, whereas no visible side effects were found in mice immunized with DT itself. The results suggested that the side effects were induced by the DT vaccine; perhaps it might release free toxin more easily than other CP conjugates by reversible decomposition. The variability in rates of decomposition might be very different from protein to protein. In conclusion, The TTX vaccines constructed with various CPs brought on obviously different TTX- to CP-specific antibody response ratios. The CP functionally affected the TTXspecific antibody intensity and then the antitoxic effect. The rank order of total efficacy of CP for these respects is TTH > TT > LPH > DT > BSA. Both TTH and TT were selected as optimal CP candidates for a TTX vaccine because of their excellent TTX-specific antibody-eliciting capacity and few side effects. The use of optimal CP may be able greatly to improve the effect of an anti-TTX vaccine and be beneficial to antiTTX antitoxin research.
ACKNOWLEDGMENT This work was supported by both the ninth and tenth Fiveyear Plan Foundation of Military Medical Science, the Chinese P.L.A. The authors are highly grateful to Dr. Z. G. Xue for her help with preparation of TTH and to Prof. Y. F. Ren for critical review of this manuscript.
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