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Comparative immunogenicities of tobacco mosaic virus, protein subunits, and reaggregated protein subunits
VIROLOGY
33, 215-220
Comparative
(1967)
lmmunogenicities Subunits,
and
of Tobacco
Reaggregated
Mosaic
Protein
Virus,
Protein
Subunits’
F. LOOR* Laboratory
of Animal
Physiology, Accepted
When TMV protein, jected into rabbits, the virus is %8 times smaller are injected. Duration month instead of more deposition and rate of organs.
University June
The immunogenicities of intact tobacco mosaic virus (TMV), of dissociated protein subunits (TMVP), and of reconstituted protein rods (TMVRP) were studied by Marbrook and Matthews (1966) in mice and independently by me in rabbits. My results were published as a prereport
(Loor,
1966)
and
are
detailed here. In contrast, to Marbrook and Matthews, I have followed antibody production during a period ranging from a few days to many months and have attempted to correlate the differential immunogenicity observed with the fate of injected antigens. MATERIALS
AND
Belgium
either dissociated subunits or reconstituted protein rods, is inamount of precipitating antibodies directed against the intact at the peak of synthesis than when similar amounts of TMV of the synthesis of these TMV antibodies is also shorter (one than one year). These differences seem to be related to unequal degradation of the three antigens in the antibody-producing
INTRODUCTION
liminary
of Brussels,
6, 1967
METHODS
Antigens. TMV was isolated from an extract, of infected leaves of Nicotianiu tabacum, by the method of Jeener (1965) using several cycles of isoelectric precipitation and fractionation by UItracentrifugation. The virus was further purified by CM-cellulose chromatography (Cochran et al., 1957). The purity of such TMV 1 Project carried out under a Euratom contract the University of Brussels (007-61-10 ABIB). 2 Aspirant de recherches du Fonds National de la Recherche Scientifique. with
215
preparations was checked by electron microscopy. Uniformly and highly 14C-labeled TMV was obtained by a new method (to be published in detail elsewhere). TMV was grown in tobacco plants that had incorporated W02 by photosynthesis. Young tobacco plants, deprived of C02, were grown for 5 weeks at 26-28” in the presence of ‘*CO2 in an air-tight Lucite box. They were infected with TMV after the first, week. A concentration of 0.2% v/v of 14C02 was obtained and maintained in the air-tight box by the reaction of lactic acid on Ba14C03. After purification of the virus, 3-4 mg of 14C TMV per gram of infected leaves was obtained. About 5% of the 14C used was incorporated in the virus. The specific radioactivity of 14C TMV used in the following experiments was 5.25 pC/mg, corresponding to about 3500 cpm per microgram of virus. TMV protein subunits (TMVP) were prepared by the acetic acid method (Fraenkel-Conrat, 1957). Reaggregated TMV protein (TMVRP) was obtained in the following way. A purified TMVP solution was first, treated with ribonuclease (RNase) to ensure the complete removal of viral RNA. TMVP was then reassociated into protein rods devoid
216
LOOR
of RNA by dialysis for 2 hours against ammonium acetat’e buffer (5 X low3 ill, 19.59). TMVRP was pH 5.6) (Anderer, finally washed several times by differential ultracentrifugation. In some experiments, TMV and TMVRP were first treated with carboxypeptidase A (Worthington CoA DFP) (Harris and Knight, 1955) to remove the C-terminal threoninc before injection. Extent of Cterminal threonine removal was checked by t’he method of Jeener (1965). These carboxypept,idase-treated antigens are designated thr-, and the others are designated thr+. Immunization 0s rabbits. To study the immunogenicity of the different antigens, rabbits were subjected to five injections of 20 mg of purified ant,igen (10 mg intravenously and 5 mg in each hind food pad) (see Results). To study the persistence of antigens in lymphoid organs, rabbit,s received one or two injections of about 75 pg of 14Clabeled antigen in the hind foot pad (see Resu1t.s). Antibody content. The antibodies were precipitated by successive additions to the antiserum of small amounts of 14C-labeled antigen until no more precipitation occurred. The successive precipitates were pooled, washed 3 times with 0.15 A4 NaCI, and dissolved in 0.1 N NaOH. The amount of antibody in the final precipitate was calculated from the difference between the total amount of protein and the amount of viral protein. The total amount’ of protein was determined by Folin-Ciocalteau measurements performed with an Auto-Analyzer Technicon apparatus. The amount of viral protein was determined from the specific radioactivity of 1% TMV and the radioactivity of the final precipitate. Radioactivities were measured with a lowbackground Nuclear detector. ilssays for antigens in tissues. 14C-labeled antigens were injected into rabbits, and the radioactivity fixed by the cells of the lymphoid organs was determined as follows. Different organs were cut in small several Gmes with 1% pieces, washed ammonium acetate buffer (pH 7.5), and
homogenized at 0°C in a Virtis enizer. RESULTS Comparatioe Immunogenicity VP, and TMVRP
“45” homog-
of TM V, TM-
Antibodies synthesized after injection of antigen thr+ or thr were precipitated with TMV thr+ or TMV thr-, respectively. Results of qualitative and yuant)itative tests are summarized in Fig. 1 and in Table 1. After injection of TMV thrf or TMV thr-, high titers of TMV antibodies persisted in the serum for many months. For example, in one rabbit injected with TMV thr+, antibody content reached 7.5 mg per milliliter of serum, and was still 1 mg/ml 400 days after the last injection of antigen. In contrast, after injection of TMVP or of TMVRP, the TMV antibody titer was smaller (at most 1 mg/ml), and antibody synthesis seemed to have stopped a few days after the last injection of antigen. One month after the last injection of t’hese could be found. antigens, no antibody At the peak of synthesis, the antibody titer aft’er inject’ion of TMV was 2-8 times greater than after injecbion of TMVP or TMVRP. However, the difference in immunogenicity is still more evident, when the amount of antibodies synthesized during the total period of antibody production is compared; indeed t,he antibody synthesis is much longer after injection of TMV than of TMVP 01’ TMVRP (Fig. 1). The removal of C-terminal threonine did not affect the immunogenicity of TMV. In fact, no significant difference in t,he antibody titer appeared after injection of TMV t,hr+ and TMV thr-. Until now, differences in immunogenicit)y were observed by studying all antibodies that reacted with TMV. I have attempted to determine whether the amounts of some antibodies of very restricted specificity (Loor, 1966) were also different after injection of the different antigens (with or without RNA4). Preliminary experiments agree wit’h the results described.
IMMUNOGENICITIES
OF
TMV
injected
x-x
FIG. 1. TMV antibody synthesis jected with 20 mg antigen on days after the last injection of TMVkP
in rabbits injected with TMV, TMVP, 1, 3, 8, 12, and 15. No TMV antibody or of TMVP.
Comparison of the Deposition and the Persistence of Di$erent Antigens in Popliteal Nodes
217
PROTEINS
antigen
I
TMVP
or TMVRP. Rabbits were incould be detected one month
after immunization 14C subunits were eliminated faster than 14CTMV. l*C TMV and 14CTMVRP. Each rabbit received either 14C TMV or an identical 14CTMV and 14C TMVP. Rabbits were quantity of 14C TMVRP into the hind injected with identical quantities of 14C TMVP in the left hind foot pad and of foot pads. After a few hours, one of the intact 14C TMV in the right one. Some popliteal nodes was aseptically removed, rabbits received only one of these injections. later the second node and the spleen were At different intervals after injection, radio- removed; the radioactivity associated with activity associated with the cells of the their cells was then determined. Results popliteal lymph nodes and of the spleenwas are summarized in Table 4. Radioactivity determined. Results are summarized in fixed by the lymph node cells was at least 20 times greater and decreased two times Table 2. The control experiment (rabbits 207 slower after injection of intact 14C TMV and 208) shows that transfer of the antigen than of 14CTMVRP. For example (Table from one foot to the other, at least during 4), in 1 week the radioactivity decreased the first 24 hours following injection, may by a factor of 5 after injection of 14CTMV be neglected (Table 3). Much more 14C (rabbit 3) and by a factor of 10 after inTMV was deposited in the nodes than jection of 14C TMVRP (rabbit’ 6). In all 14C TMV subunits. During the first week experiments, the radioactivity associated
218
LOOR TABLE COMPARATIVE
IMMUNOGENICITY
1
OF TMV Days
Antigen
TMV
injected
Rabbit
thr-
TMVRP
No.
AND
TMVRP
thr-
after
the first antigen
a
injection
-
290 293 388
10 rabbits
thr-
thr-
0
11
17
f -
1500 870 880
-
+
21
71
116
324
4800 2930 3280
2400 2600 2300
1740 2720 1960
+
+
-
About 1000
a Antibody titers are expressed in micrograms per milliter of serum. For rabbits injected with TMVRP thr- only qualitative tests were made. The following notation is used: + = precipitation clearly visible, f = dubious precipitation, and - = no precipitation. Rabbits were injected with 20 mg antigen on days 1,4,9,16 and 18. No antibody against TMV thr- could be detected fifty days after the last injection of TMVRP thr-. TABLE DEPOSITION AND
Rabbit No.
W
2
TABLE
AND ELIMINATION OF 14C TMV TMVP IN THE NODES’
Time after injection (days)
201 202 206 204 205 203
Right popliteal node (% of injected 14C TMV)
Left popliteal node (% of injected ’ 4C TMVP)
7.38 6.70 4.13 1.69 0.54 0.32
1.05 0.28 0.11 0.06 0.022 0.007
1 2 4 8 15 34
Rabbit No.
14C TMV, (281,000
TABLE
Percent Rabbit No.
3
TRANSFER FROM ONE POPLITEAL LYMPH TO THE OTHER AND TO THE SPLEEN AFTER 24 HOURS
Antigen injected ‘:$n
of injected fixed by
antigen
Left popliteal node cells
Right popliteal node cells
9.65 0.45
0.0027 0.0036
~207 208
‘4C TMV ‘C TMVP
0.10 0.018
a Rabbits were injected only in the left hind foot pad with 73 pg (258,000 cpm) 14C TMV or with 74 pg (261,000 cpm) 14C TMVP. Radioactivities of the spleen and the two popliteal nodes were determined after 24 hours.
80 rg cpm)
14C TMVRP, Pg cm000 cpm)
ii
Percent of i njected antigen fixed by
Time after injection (hours)
Antigen injected
0 Each rabbit was injected with 85 pg (300,000 cpm) of 1% TMV into the right hind foot pad and with 74 ag (261,000 cpm) of 14C TMVP into the left hind foot pad. ANTIGEN NODE
4
DEPOSITION AND ELIMINATION OF ‘4C TMV AND OF 14C TMVRP IN LYMPHOID ORQANS~
74
I
Popliteal node cells
6 15.5 24 48 96 192 6 16 24 48 96 192
spleen cells
10.29 13.15 9.12 7.93 5.15 1.82
-
0.67 0.53 0.39 0.35 0.09 0.03$
0.127 0.018 0.029
-
A-
0.015 0.026 0.014
n Rabbits were injected into each hind foot pad. The two popliteal nodes were removed at different times after injection.
with the popliteal node cells was much greater than the radioactivity associated with the spleen cells; this suggests that antigen was mainly captured by lymphoid organs located near the injection site (Ada et al., 1964). DISCUSSION
My experiments show that intact TMV induces much greater amounts of anti-
IMMUNOGENICITIES
TMV
antibodies
in
rabbits
than
OF
does
TMVRP. My results are in good agreement with the observations of Marbrook and Matthews (1966) on TYMV immuno-
genicity in mice and rabbits, and confirm their few data on the immunogenicity of TMV in mice. These authors detected no circulating T&IV antibody 44 days after injection of TMVP or of TMVRP into mice, while intact TMV regularly stimulated the synthesis of such antibodies.
TMV
219
PROTEINS
The different degradation rates of TMVRP
and
of TMV
could
stem
from
their
different
st,ability. Indeed Caspar (1963) has pointed out that interaction between RNA and protein subunits stabilizes the assembly
of the intact
virus.
I propose the following hypothesis to explain antigen
how the influences
presence antibody
of RNA response.
in an Two
factors related to the presence of RNA lead to persistence of the antigen in lymphoid organs: (1) the greater deposition
The similar immunogenicity, deposition, of 14C TMV (probably due to its “particle” and persistence of TMV subunits in lymph nodes of injected animals, whether they character), and (2) its slower degradation are completely dissociated or reassociated by proteolytic enzymes. It is well known that the persistence of into protein rods, may be due to the great antigen plays an important role in antibody dissociability of reaggregated TMV protein at the physiological pH (Caspar, 1963). production. As observed by Richter et al. It is reasonable to assume that TMVRP (1965), the decrease in antibody titer several months after injection of antigen dissociates shortly after injection. It seems likely that lymph node cells remarkably parallels that of antigen content. capture greater amounts of intact TMV than TMV subunits, because particular antigens are more actively incorporated
than soluble antigens. Indeed Ada et al. (1964) have shown that the content of flagella of Salmonella adelaide in rat lymph nodes is about 10 times greater than that of flagellin, a soluble protein prepared by acid dissociation of the flagella. Possibly, cells use distinct mechanisms of
different efficiency to capture each of the two types of antigens. The antigen content of cells decreases more rapidly after injection of 14C TMVP or 14C TMVRP than of intact 14C TMV. Prelimina,ry experiments (Loor, in preparation) show that after injection of native virus, TMV protein is split into amino acids. Thus, the degradation products may be rapidly eliminated from the lymphoid
organs. Since TMVP
and TMV have similar
amino acid sequences, the question arises why t’hese antigens have different rates of degradation. It is well known that TMV is highly resistant to in vitro proteolysis and that the isolated virus protein can be hydrolyzed under conditions that do not
affect the intact virus (Anderer, 1963). Experiments of Kleczkowski and van Kammen (1961) suggest that virus dissociation at~tack.
is a necessary
step for enzymatic
I
may
thus
postulate
that
a low
sistence of antigen (e.g., TMVP) lymphoid
organs
becomes
rapidly
per-
in t.he a limiting
factor for multiplication of immunocompetent cells, and thus for antibody production. ACKNOWLEDGMENTS
The authors wishes to express his gratitude to Dr. R. Jeener for many useful suggestions and counsel throughollt this work. REFEREKCES
G. L., NOSSAL, G. J. V., and PYE, J. (1964). Antigens in immunity. III. Distribution of iodinated antigens following injection into rats via the hind foot pats. Australian J. Exptl. Biol.
ADA,
Med. Sci. ANDERER,
42, 295-310.
F. A. (1959). Reversible Denaturierung des Proteins aus Tabakmosaikvirus. 2. Natur-
forsch. ANDERER,
14b,
642-647.
F. A. (1963). Recent studies structure of tobacco mosaic virus.
on the
Advan. Protein Chem. 18, 15-19. CASPAR, D. L. D. (1963). Assembly and stability of the tobacco mosaic virus. Advan. Protein Chem. 18, 37-121. COCHRAN,G. W., CHIDESTER, J.L.,~~~STOCKS,D.
L. (1957). Chromatography of tobacco mosaic virus on a cellulose cat,ion exchange adsorbent. Nature 180, 1281-1282. FRAENKEL-CONRAT, H. (1957). Degradation tobacco mosaic virus with acetic acid. Viro/ogy 4, l-4. HARRIS, J. I., and KNIGHT, C. A. (1955). Studies
of
on the action of carboxypeptidase on J. Biol. Chem. 214, 215-230. JEENER, R. (1965). Effects on the antigenic minants of tobacco mosaic virus of a small ber of base replacements in the ribonucleic Virology 26, 10-15. KLECZKOWSKI, A., and VAN KAMMEN, A. Protection from proteolysis by aggregating protein isolated from tobacco mosaic Biochim. Biophys. Acta 53, 181.
TMV. deternumacid. (1961). the virus.
LOOR, F. (1966). Recherches sur la synthbse d’anticorps anti-TMV non prkipitables par l’antig&e inject&. Arch. Intern. Physiol. Biochim. 74, 929-930. MARBROOK, J., and MATTHEWS, R. E. F. (1966). The differential immunogenicity of plant viral protein and nucleoproteins. Virology 28, 219-228. RICHTER, M., ZIMMERMAN, S., and HAUROWITZ, F. (1965). Relation of antibody titer to persistence of antigen. J. Immunol. 94, 938-941.
33, 215-220
Comparative
(1967)
lmmunogenicities Subunits,
and
of Tobacco
Reaggregated
Mosaic
Protein
Virus,
Protein
Subunits’
F. LOOR* Laboratory
of Animal
Physiology, Accepted
When TMV protein, jected into rabbits, the virus is %8 times smaller are injected. Duration month instead of more deposition and rate of organs.
University June
The immunogenicities of intact tobacco mosaic virus (TMV), of dissociated protein subunits (TMVP), and of reconstituted protein rods (TMVRP) were studied by Marbrook and Matthews (1966) in mice and independently by me in rabbits. My results were published as a prereport
(Loor,
1966)
and
are
detailed here. In contrast, to Marbrook and Matthews, I have followed antibody production during a period ranging from a few days to many months and have attempted to correlate the differential immunogenicity observed with the fate of injected antigens. MATERIALS
AND
Belgium
either dissociated subunits or reconstituted protein rods, is inamount of precipitating antibodies directed against the intact at the peak of synthesis than when similar amounts of TMV of the synthesis of these TMV antibodies is also shorter (one than one year). These differences seem to be related to unequal degradation of the three antigens in the antibody-producing
INTRODUCTION
liminary
of Brussels,
6, 1967
METHODS
Antigens. TMV was isolated from an extract, of infected leaves of Nicotianiu tabacum, by the method of Jeener (1965) using several cycles of isoelectric precipitation and fractionation by UItracentrifugation. The virus was further purified by CM-cellulose chromatography (Cochran et al., 1957). The purity of such TMV 1 Project carried out under a Euratom contract the University of Brussels (007-61-10 ABIB). 2 Aspirant de recherches du Fonds National de la Recherche Scientifique. with
215
preparations was checked by electron microscopy. Uniformly and highly 14C-labeled TMV was obtained by a new method (to be published in detail elsewhere). TMV was grown in tobacco plants that had incorporated W02 by photosynthesis. Young tobacco plants, deprived of C02, were grown for 5 weeks at 26-28” in the presence of ‘*CO2 in an air-tight Lucite box. They were infected with TMV after the first, week. A concentration of 0.2% v/v of 14C02 was obtained and maintained in the air-tight box by the reaction of lactic acid on Ba14C03. After purification of the virus, 3-4 mg of 14C TMV per gram of infected leaves was obtained. About 5% of the 14C used was incorporated in the virus. The specific radioactivity of 14C TMV used in the following experiments was 5.25 pC/mg, corresponding to about 3500 cpm per microgram of virus. TMV protein subunits (TMVP) were prepared by the acetic acid method (Fraenkel-Conrat, 1957). Reaggregated TMV protein (TMVRP) was obtained in the following way. A purified TMVP solution was first, treated with ribonuclease (RNase) to ensure the complete removal of viral RNA. TMVP was then reassociated into protein rods devoid
216
LOOR
of RNA by dialysis for 2 hours against ammonium acetat’e buffer (5 X low3 ill, 19.59). TMVRP was pH 5.6) (Anderer, finally washed several times by differential ultracentrifugation. In some experiments, TMV and TMVRP were first treated with carboxypeptidase A (Worthington CoA DFP) (Harris and Knight, 1955) to remove the C-terminal threoninc before injection. Extent of Cterminal threonine removal was checked by t’he method of Jeener (1965). These carboxypept,idase-treated antigens are designated thr-, and the others are designated thr+. Immunization 0s rabbits. To study the immunogenicity of the different antigens, rabbits were subjected to five injections of 20 mg of purified ant,igen (10 mg intravenously and 5 mg in each hind food pad) (see Results). To study the persistence of antigens in lymphoid organs, rabbit,s received one or two injections of about 75 pg of 14Clabeled antigen in the hind foot pad (see Resu1t.s). Antibody content. The antibodies were precipitated by successive additions to the antiserum of small amounts of 14C-labeled antigen until no more precipitation occurred. The successive precipitates were pooled, washed 3 times with 0.15 A4 NaCI, and dissolved in 0.1 N NaOH. The amount of antibody in the final precipitate was calculated from the difference between the total amount of protein and the amount of viral protein. The total amount’ of protein was determined by Folin-Ciocalteau measurements performed with an Auto-Analyzer Technicon apparatus. The amount of viral protein was determined from the specific radioactivity of 1% TMV and the radioactivity of the final precipitate. Radioactivities were measured with a lowbackground Nuclear detector. ilssays for antigens in tissues. 14C-labeled antigens were injected into rabbits, and the radioactivity fixed by the cells of the lymphoid organs was determined as follows. Different organs were cut in small several Gmes with 1% pieces, washed ammonium acetate buffer (pH 7.5), and
homogenized at 0°C in a Virtis enizer. RESULTS Comparatioe Immunogenicity VP, and TMVRP
“45” homog-
of TM V, TM-
Antibodies synthesized after injection of antigen thr+ or thr were precipitated with TMV thr+ or TMV thr-, respectively. Results of qualitative and yuant)itative tests are summarized in Fig. 1 and in Table 1. After injection of TMV thrf or TMV thr-, high titers of TMV antibodies persisted in the serum for many months. For example, in one rabbit injected with TMV thr+, antibody content reached 7.5 mg per milliliter of serum, and was still 1 mg/ml 400 days after the last injection of antigen. In contrast, after injection of TMVP or of TMVRP, the TMV antibody titer was smaller (at most 1 mg/ml), and antibody synthesis seemed to have stopped a few days after the last injection of antigen. One month after the last injection of t’hese could be found. antigens, no antibody At the peak of synthesis, the antibody titer aft’er inject’ion of TMV was 2-8 times greater than after injecbion of TMVP or TMVRP. However, the difference in immunogenicity is still more evident, when the amount of antibodies synthesized during the total period of antibody production is compared; indeed t,he antibody synthesis is much longer after injection of TMV than of TMVP 01’ TMVRP (Fig. 1). The removal of C-terminal threonine did not affect the immunogenicity of TMV. In fact, no significant difference in t,he antibody titer appeared after injection of TMV t,hr+ and TMV thr-. Until now, differences in immunogenicit)y were observed by studying all antibodies that reacted with TMV. I have attempted to determine whether the amounts of some antibodies of very restricted specificity (Loor, 1966) were also different after injection of the different antigens (with or without RNA4). Preliminary experiments agree wit’h the results described.
IMMUNOGENICITIES
OF
TMV
injected
x-x
FIG. 1. TMV antibody synthesis jected with 20 mg antigen on days after the last injection of TMVkP
in rabbits injected with TMV, TMVP, 1, 3, 8, 12, and 15. No TMV antibody or of TMVP.
Comparison of the Deposition and the Persistence of Di$erent Antigens in Popliteal Nodes
217
PROTEINS
antigen
I
TMVP
or TMVRP. Rabbits were incould be detected one month
after immunization 14C subunits were eliminated faster than 14CTMV. l*C TMV and 14CTMVRP. Each rabbit received either 14C TMV or an identical 14CTMV and 14C TMVP. Rabbits were quantity of 14C TMVRP into the hind injected with identical quantities of 14C TMVP in the left hind foot pad and of foot pads. After a few hours, one of the intact 14C TMV in the right one. Some popliteal nodes was aseptically removed, rabbits received only one of these injections. later the second node and the spleen were At different intervals after injection, radio- removed; the radioactivity associated with activity associated with the cells of the their cells was then determined. Results popliteal lymph nodes and of the spleenwas are summarized in Table 4. Radioactivity determined. Results are summarized in fixed by the lymph node cells was at least 20 times greater and decreased two times Table 2. The control experiment (rabbits 207 slower after injection of intact 14C TMV and 208) shows that transfer of the antigen than of 14CTMVRP. For example (Table from one foot to the other, at least during 4), in 1 week the radioactivity decreased the first 24 hours following injection, may by a factor of 5 after injection of 14CTMV be neglected (Table 3). Much more 14C (rabbit 3) and by a factor of 10 after inTMV was deposited in the nodes than jection of 14C TMVRP (rabbit’ 6). In all 14C TMV subunits. During the first week experiments, the radioactivity associated
218
LOOR TABLE COMPARATIVE
IMMUNOGENICITY
1
OF TMV Days
Antigen
TMV
injected
Rabbit
thr-
TMVRP
No.
AND
TMVRP
thr-
after
the first antigen
a
injection
-
290 293 388
10 rabbits
thr-
thr-
0
11
17
f -
1500 870 880
-
+
21
71
116
324
4800 2930 3280
2400 2600 2300
1740 2720 1960
+
+
-
About 1000
a Antibody titers are expressed in micrograms per milliter of serum. For rabbits injected with TMVRP thr- only qualitative tests were made. The following notation is used: + = precipitation clearly visible, f = dubious precipitation, and - = no precipitation. Rabbits were injected with 20 mg antigen on days 1,4,9,16 and 18. No antibody against TMV thr- could be detected fifty days after the last injection of TMVRP thr-. TABLE DEPOSITION AND
Rabbit No.
W
2
TABLE
AND ELIMINATION OF 14C TMV TMVP IN THE NODES’
Time after injection (days)
201 202 206 204 205 203
Right popliteal node (% of injected 14C TMV)
Left popliteal node (% of injected ’ 4C TMVP)
7.38 6.70 4.13 1.69 0.54 0.32
1.05 0.28 0.11 0.06 0.022 0.007
1 2 4 8 15 34
Rabbit No.
14C TMV, (281,000
TABLE
Percent Rabbit No.
3
TRANSFER FROM ONE POPLITEAL LYMPH TO THE OTHER AND TO THE SPLEEN AFTER 24 HOURS
Antigen injected ‘:$n
of injected fixed by
antigen
Left popliteal node cells
Right popliteal node cells
9.65 0.45
0.0027 0.0036
~207 208
‘4C TMV ‘C TMVP
0.10 0.018
a Rabbits were injected only in the left hind foot pad with 73 pg (258,000 cpm) 14C TMV or with 74 pg (261,000 cpm) 14C TMVP. Radioactivities of the spleen and the two popliteal nodes were determined after 24 hours.
80 rg cpm)
14C TMVRP, Pg cm000 cpm)
ii
Percent of i njected antigen fixed by
Time after injection (hours)
Antigen injected
0 Each rabbit was injected with 85 pg (300,000 cpm) of 1% TMV into the right hind foot pad and with 74 ag (261,000 cpm) of 14C TMVP into the left hind foot pad. ANTIGEN NODE
4
DEPOSITION AND ELIMINATION OF ‘4C TMV AND OF 14C TMVRP IN LYMPHOID ORQANS~
74
I
Popliteal node cells
6 15.5 24 48 96 192 6 16 24 48 96 192
spleen cells
10.29 13.15 9.12 7.93 5.15 1.82
-
0.67 0.53 0.39 0.35 0.09 0.03$
0.127 0.018 0.029
-
A-
0.015 0.026 0.014
n Rabbits were injected into each hind foot pad. The two popliteal nodes were removed at different times after injection.
with the popliteal node cells was much greater than the radioactivity associated with the spleen cells; this suggests that antigen was mainly captured by lymphoid organs located near the injection site (Ada et al., 1964). DISCUSSION
My experiments show that intact TMV induces much greater amounts of anti-
IMMUNOGENICITIES
TMV
antibodies
in
rabbits
than
OF
does
TMVRP. My results are in good agreement with the observations of Marbrook and Matthews (1966) on TYMV immuno-
genicity in mice and rabbits, and confirm their few data on the immunogenicity of TMV in mice. These authors detected no circulating T&IV antibody 44 days after injection of TMVP or of TMVRP into mice, while intact TMV regularly stimulated the synthesis of such antibodies.
TMV
219
PROTEINS
The different degradation rates of TMVRP
and
of TMV
could
stem
from
their
different
st,ability. Indeed Caspar (1963) has pointed out that interaction between RNA and protein subunits stabilizes the assembly
of the intact
virus.
I propose the following hypothesis to explain antigen
how the influences
presence antibody
of RNA response.
in an Two
factors related to the presence of RNA lead to persistence of the antigen in lymphoid organs: (1) the greater deposition
The similar immunogenicity, deposition, of 14C TMV (probably due to its “particle” and persistence of TMV subunits in lymph nodes of injected animals, whether they character), and (2) its slower degradation are completely dissociated or reassociated by proteolytic enzymes. It is well known that the persistence of into protein rods, may be due to the great antigen plays an important role in antibody dissociability of reaggregated TMV protein at the physiological pH (Caspar, 1963). production. As observed by Richter et al. It is reasonable to assume that TMVRP (1965), the decrease in antibody titer several months after injection of antigen dissociates shortly after injection. It seems likely that lymph node cells remarkably parallels that of antigen content. capture greater amounts of intact TMV than TMV subunits, because particular antigens are more actively incorporated
than soluble antigens. Indeed Ada et al. (1964) have shown that the content of flagella of Salmonella adelaide in rat lymph nodes is about 10 times greater than that of flagellin, a soluble protein prepared by acid dissociation of the flagella. Possibly, cells use distinct mechanisms of
different efficiency to capture each of the two types of antigens. The antigen content of cells decreases more rapidly after injection of 14C TMVP or 14C TMVRP than of intact 14C TMV. Prelimina,ry experiments (Loor, in preparation) show that after injection of native virus, TMV protein is split into amino acids. Thus, the degradation products may be rapidly eliminated from the lymphoid
organs. Since TMVP
and TMV have similar
amino acid sequences, the question arises why t’hese antigens have different rates of degradation. It is well known that TMV is highly resistant to in vitro proteolysis and that the isolated virus protein can be hydrolyzed under conditions that do not
affect the intact virus (Anderer, 1963). Experiments of Kleczkowski and van Kammen (1961) suggest that virus dissociation at~tack.
is a necessary
step for enzymatic
I
may
thus
postulate
that
a low
sistence of antigen (e.g., TMVP) lymphoid
organs
becomes
rapidly
per-
in t.he a limiting
factor for multiplication of immunocompetent cells, and thus for antibody production. ACKNOWLEDGMENTS
The authors wishes to express his gratitude to Dr. R. Jeener for many useful suggestions and counsel throughollt this work. REFEREKCES
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