THE JOURNAL OF INFECTIOUS DISEASES • VOL. 134, NO.1· © 1976 by the University of Chicago. All rights reserved.
JULY 1976
Host Immune Responses after Administration of Inactivated Venezuelan Equine Encephalomyelitis Virus Vaccines. II. Kinetics of Neutralizing Antibody Responses in Donors and Adoptively Immunized Recipients From the Infectious Diseases-Hypersensitivity Section, Department of Medicine, Veterans Administration Research Hospital, Northwestern University Medical Center, Chicago, Illinois
Stanley G. Rabinowitz
In a previous paper [1] the response of mice to immunization with various inactivated Venezuelan equine encephalomyelitis (VEE) virus vaccines was described and partially characterized. It appeared that the use of an adoptive transfer system provided an opportunity for viewing cellular events underlying responses to vaccination. As a result of our previous findings [1], we postulated
that responses of lymphoid spleen cells after immunization of donor mice with inactivated VEE vaccine and certain adjuvants were vigorous and long-lasting. We speculated on the possibility that these responses resulted from amplification of clones of VEE-reactive cells. This clonal amplification presumably resulted from adjuvant-mediated, intense, lymphocyte-antigen interaction enhancing by manyfold the usual response to vaccination with inactivated VEE vaccine. In contrast, immunization of mice with inactivated VEE vaccine alone did not result in substantial lymphoid spleen cell responses to VEE antigen. In support of the concept that immunopotentiation induced by adjuvants results from amplification of clones of VEE-reactive splenic lymphocytes, we proceeded to quantitate lymphoid spleen cell responses to inactivated VEE vaccines. We reasoned that, since neutralizing antibody was such a potent means of protecting mice against
Received for publication October 14, 1975, and in revised form January 30, 1976. This study was supported by grant no. MRS 7319 from the Veterans Administration Research Services and by contract no. DAMD 17-74-C-4112 from the U.S. Army Medical Research and Development Command. I gratefully acknowledge the expert technical assistance of Jayashree Huprikar and Mary Grover and the secretarial assistance of Judith Mann. Please address requests for reprints to Dr. Stanley G. Rabinowitz, Veterans Administration Lakeside Hospital, 333 East Huron Street, Chicago, Illinois 60611.
39
Downloaded from http://jid.oxfordjournals.org/ at University of Birmingham on August 21, 2015
Lymphoid cell responses to immunization with various formalin-inactivated Venezuelan equine encephalomyelitis (VEE) virus vaccines were monitored in mice by assessment of the development of both the neutralizing antibody response in sera of spleen cell donors and the adoptive neutralizing antibody response induced by spleen cell transfer in recipients. Donors immunized intraperitoneally with formalin-inactivated VEE vaccine (a single dose or a dose on three consecutive days) developed early serum neutralizing antibody responses (;::: 1:88-1 : 100) by seven days after immunization. Recipients of spleen cells from such mice were, however, incapable of eliciting a neutralizing antibody response (~1: 10). Only spleen cells from donors immunized with inactivated VEE vaccine plus adjuvants (particularly complete Freund's adjuvant and Bordetella pertussis) were consistently capable of producing early, high-titer serum neutralizing antibody responses in adoptively immunized recipients (;::: 1: 50-1 : 120 on day 4). The magnitude of neutralizing antibody responses of donors to inactivated VEE vaccines did not serve as a useful indicator of whether spleen cells from such mice could adoptively induce antibody responses in recipients. Finally, treatment of immune spleen cells with rabbit antiserum to mouse thymocytes, but not with rabbit antiserum to mouse y-globulin or normal rabbit serum, abolished the capacity of such cells to transfer an antibody response adoptively.
Rabinowitz
40
challenge with VEE virus [2], it would be appropriate first to examine the development of neutralizing antibody in both donors and adoptively immunized recipients. We chose, therefore, in the present study to focus specifically on an analysis of the neutralizing antibody responses of both donor and adoptively immunized mice. Materials and Methods
Results
Effect of different immunization regimens on time of appearance of serum neutralizing antibody in donor and cell transfer recipients. Donor mice immunized with one dose of inactivated VEE vaccine (I-TC-83) developed serum neutralizing antibody detectable within five days of
Downloaded from http://jid.oxfordjournals.org/ at University of Birmingham on August 21, 2015
Animals, virus and vaccine preparations, and plaque assay have been described in detail [1]. The vaccines employed were: (1 ) formalininactivated TC-83 VEE virus given once (1-TC83 Xl), three times on consecutive days (1- TC83 X 3), or once weekly for three consecutive weeks (I-TC-83 X 3 wk); (2) I-TC-83 combined with Bordetella pertussis (2 X 1010 heatkilled organisms/ml; lot no. 985739; Parke, Davis, Detroit, Mich.); (3) 1-TC-83 combined with complete Freund's adjuvant (CFA; 4 mg/rnl of heat-killed, lyophilized bacillus CalmetteGuerin); (4) 1-TC-83 combined with incomplete Freund's adjuvant (Difco Laboratories, Detroit, Mich.). Vaccine was administered ip in a dose of 2.4 X 108 pfu/0.3 ml, unless otherwise indicated. When 1-TC-83 was combined with adjuvant, 0.2 ml of 1-TC-83 was added to 0.1 ml of adjuvant. Booster injections of 1-TC-83 alone were given for the next two days after vaccine and adjuvant were administered (0.3 ml per mouse per day). Adoptive transfer protocol. Donor mice were immunized; at given intervals after immunization, blood was collected aseptically, and spleens were harvested and processed as previously described [1]. Viable spleen cells (1.2-1.6 X 10 8 ) were transferred ip to normal, adult, syngeneic recipients; groups of these animals were sacrificed by exsanguination two, four or five, and six or seven days after cell transfer. At least five donors and three adoptively immunized recipients were bled at each interval studied. Individually collected samples of sera were heat-inactivated at 56 C for 30 min and then stored at -20 C for subsequent antibody determination. Plaque reduction neutralization test. Equal volumes of heat-inactivated mouse serum and neurovirulent TC-83 strain VEE virus, adjusted to yield 100 pfu of virus/O.! ml, were allowed
to stand at room temperature (about 24 C) for 60 min, and 0.1 ml of the mixture was added to chick embryo fibroblast (CEF) monolayers in 35-mm tissue culture plates (FB-6TC, Linbro Co., New Haven, Conn.). Plaque overlays were done as described previously [1], and the highest dilution resulting in a 50% reduction in plaques was taken as the end point. For each experiment involving determination of neutralizing antibody titer, aliquots of known negative and positive control sera were included. All sera were tested in triplicate. Treatment with cytotoxic antisera. Normal rabbit serum, rabbit antiserum to mouse y-globulin, and rabbit antiserum to mouse thymocytes were prepared, absorbed, and tested for specificity as described previously [3]. In brief, treatment of spleen cells with rabbit antiserum to mouse y-globulin plus complement was shown to abolish lipopolysaccharide reactivity while leaving phytohemagglutinin reactivity intact. Conversely, treatment of spleen cells with rabbit antiserum to mouse thymocytes plus complement abolished phytohemagglutinin reactivity while leaving lipopolysaccharide responsiveness intact. Lyophilized rabbit complement (Grand Island Biological Co., Grand Island, N.Y.) was reconstituted with buffer and absorbed twice with C57B16/J spleen cells at 4 C before use for removal of nonspecific lymphocytotoxicity. Spleen cells (1.2-1.4 X 109 ) were suspended in 4.5 ml of RPMI-1640 with 1.0 ml of absorbed antisera or control sera and 4.5 ml of a 1: 3 dilution of absorbed rabbit complement. After the mixture was incubated at 37 C for 30 min, treated cells were washed twice with RPMI-1640 and counted, and 1.4-1.5 X 108 viable (trypan blue, 0.5% dye exclusion) spleen cells were injected ip into each recipient. Amounts and dilutions of antisera and complement used were based on previously described data [3].
41
Host Immune Responses to VEE Vaccines II
donor spleen cells
6
7 day 14 day _._._._ 21 day" ..
40 I
i •
•
I•
•
100
!
••
7
20
14
21
day after immunization
2
4
6
8
day after cell transfer
Figure 1. Left, development of serum neutralizing antibody response in mice immunized with one ip dose of formalin-inactivated Venezuelan equine encephalomyelitis virus vaccine (I-TC-83; 2.4 X 108 pfu/0.3 ml), Values shown represent reciprocal of the mean (- SE) of five determinations for each day studied. Right, development of serum neutralizing antibody response in adoptively immunized recipients of spleen cells seven, 14, and 21 days after immunization of donors with one ip dose of I-TC-83. Values shown represent reciprocal of the mean (- SE) of three determinations for each day studied (two, four, and seven days) after cell transfer.
Downloaded from http://jid.oxfordjournals.org/ at University of Birmingham on August 21, 2015
tion of donors (figure 2). Splenocytes transferred 14 days after immunization, however, did elicit a small (titer, approximately 1:20) antibody response by two to five days after transfer. This response was short-lived. Furthermore, inability to immunize recipients adoptively against challenge with VEE virus appeared to correlate with a lack of or a minimal neutralizing antibody response in recipients [1]. Mice immunized with 1-TC-83 once weekly for three weeks behaved differently. Serum neutralizing antibody responses of donors were already well developed by seven days after the last immunization (figure 3). In fact, antibody responses had already reached a plateau by that time and were on the decline by 21 days after the last immunization. In general, weekly immunization with 1-TC-83 seemed to produce the highest neutralizing antibody response in the groups of donor mice immunized with I-TC-83 alone. In contrast to previously immunized groups, mice adoptively immunized with spleen cells harvested seven days
vaccine administration (figure 1). After five days, the level of serum antibody rose rapidly and began to level off by 21 days after immunization. No serum neutralizing antibody was apparent in adoptively immunized recipients receiving spleen cells seven, 14, or 21 days after vaccination (figure 1). Furthermore, lack of antibody in adoptively immunized recipients correlated with susceptibility to challenge with VEE virus [1]. Donors immunized with I-TC-83 daily for three days also developed early serum neutralizing antibody titers (figure 2). Between seven and 14 days, antibody titers increased linearly and began to level off by 21 days. The kinetics of antibody production resembles that seen for the group given only one dose of 1-TC-83. The only difference readily appreciated between donors receiving one or three doses of 1-TC-83 is that antibody titers are higher in the latter group. Adoptively immunized recipients failed again to demonstrate serum neutralizing antibody when spleen cells were transferred seven or 21 days after vaccina-
42
Rabinowitz
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too
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14
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Figure 2. Left, development of serum neutralizing antibody response in mice immunized with three ip doses (on consecutive days) of formalin-inactivated Venezuelan equine encephalomyelitis virus vaccine (I-TC-83; 2.4 X 108 pfu/0.3 mI). Values shown represent reciprocal of the mean (- SE) of five determinations for each day studied. Right, development of serum neutralizing antibody response in adoptively immunized recipients of spleen cells seven, 14, and 21 days after immunization of donors as described above. Values shown represent reciprocal of the mean (- SE) of three determinations for each day studied (two, five, and six days) after cell transfer.
after the last weekly immunization developed serum neutralizing antibody (titer, 1:40) by four days after cell transfer. By seven days after cell transfer, recipients had serum neutralization titers of > 1: 80. A similar pattern appeared when spleen cells were transferred two weeks after the last injection of inactivated VEE vaccine. By 21 days after immunization, spleen cells appeared to lose their ability to transfer the capacity for antibody production. Appearance of neutralizing antibody responses in adoptively immunized recipients seemed to correlate with the ability of the mice to resist challenge with VEE virus [1]. Resistance to challenge, however, was not as complete in this group as one would have expected, based on appearance of serum neutralizing antibody in recipients [1]. This lower resistance may be explained by the fact that these adoptively immunized recipients develop late antibody responses (four to seven days). Donor mice immunized ·with I-TC-83 combined with CFA demonstrated high titers of serum
neutralizing antibody (about 1:240) by five to seven days after immunization, with substantial increases by 14 days (figure 4). In contrast to responses in the previously discussed groups, however, antibody responses of donors were extremely high by 21 days. Adoptively immunized recipients also showed clear-cut evidence of serum neutralizing antibody between two and four days after cell transfer, with peak serum titers four days after cell transfer. Although the peak titer of antibody in sera of adoptively immunized recipients was not higher than that in animals given weekly doses of 1-TC-83 vaccine, the response occurred earlier. Early appearance of a substantial level (~1 :40) of serum neutralizing antibody in adoptively immunized recipients seemed to correlate with successful transfer of adoptive immunity to challenge with VEE virus [1]. However, although mice were protected as early as seven days after immunization with 1-TC-83 and CFA (sc ) , no antibody was detected in such adoptively immunized recipients.
Downloaded from http://jid.oxfordjournals.org/ at University of Birmingham on August 21, 2015
(l C
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Host Immune Responses to VEE Vaccines 11
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7
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4
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Figure 3. Lett, development of serum neutralizing antibody response in mice immunized with three ip doses (given weekly for three weeks) of formalin-inactivated Venezuelan equine encephalomyelitis virus vaccine (I-TC-83; 2.4 X 108 pfu/0.3 ml). Values shown represent reciprocal of the mean (- SE) of five determinations for each day studied. Right, development of serum neutralizing antibody response in adoptively immunized recipients of spleen cells seven, 14, and 21 days after immunization of donors with I-TC-83 vaccine in three doses described above. Values shown represent reciprocal of the mean (- SE) of three determinations for each day studied (two, four, and seven days) after cell transfer.
Donors immunized with inactivated VEE virus vaccine and incomplete Freund's adjuvant developed substantial titers of neutralizing antibody seven days (~1 :80) and 14 days (~1 :360) after immunization. By 21 days after immunization, titers of neutralizing antibody in sera of donors approached 1: 1,000 (figure 5). When adoptively immunized recipients were studied, it was apparent that only recipients immunized 14 days after vaccination of donors developed significant serum antibody titers (~1 :50). These results were similar to antibody responses noted in recipients receiving spleen cells from donors immunized weekly with inactivated VEE virus vaccine (figure 3). Mice adoptively immunized seven and 21 days after vaccination of donors with 1-TC-83 and incomplete Freund's adjuvant developed peak serum antibody titers of only ~ 1: 20 (figure 5). Thus, immunization with 1-TC-83 and incomplete Freund's adjuvant produces a less vigorous adoptive immune response in recipients
than immunization with I-TC-83 and CFA (figures 4 and 5). Donors immunized with I-TC-83 and B. pertussis developed prompt neutralizing antibody responses by seven to 14 days (figure 6). Serum neutralizing antibody titers at 21 days, however, were considerably lower than those resulting from administration of 1-TC-83 for three days. Finally, adoptively immunized recipients receiving splenocytes seven, 14, and 21 days after immunization of donors developed comparable early, high-titer serum neutralizing antibody. Furthermore, as seen in most groups, the ability to transfer protection adop.tively against challenge with VEE virus correlated with the capacity to produce an early serum neutralizing antibody response in immunized recipients [1]. We next examined whether the enhanced humoral antibody responses seen in adoptively immunized recipients depended on the presence of either thymus (T-) cells or bone marrow-derived
Downloaded from http://jid.oxfordjournals.org/ at University of Birmingham on August 21, 2015
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44
Rabinowitz
donor spleen cells
120
7 day 14 day 21 day normal
80
2 800
40
Q) III
400
7 dev
14
4
21
6
doy oFter cell tronsFer
ftfter immunizotion
Figure 4. Left, development of serum neutralizing antibody response in mice immunized with an sc dose of formalin-inactivated Venezuelan equine encephalomyelitis virus vaccine (I-TC-83; 1.6 X 108 pfu/0.2 ml) plus complete Freund's adjuvant, followed by daily administration of vaccine ip for two days. Values shown represent reciprocal of the mean (- SE) of five determinations for each day studied. Right, development of serum neutralizing antibody response in adoptively immunized recipients of spleen cells seven, 14, and 21 days after immunization of donors as described above. Values shown represent reciprocal of the mean (- SE) of three antibody determinations for each day studied (two, four, and six days) after cell transfer.
(B-) cells in the spleen cell transfer. We selectively depleted the spleen cell preparation of either T-cells or B-cells by using rabbit antiserum to mouse thymocytes or rabbit antiserum to mouse y-globulin plus complement. For spleen cell donors we chose mice immunized 14 days previously with 1-TC-8 3 and CFA. This group of vaccinees was chosen because their spleen cells uniformly protected recipients against challenge with VEE virus and induced vigorous antibody responses in recipients. Recipients receiving spleen cells after in vitro treatment with rabbit antiserum to mouse thymocytes plus complement demonstrated a markedly blunted antibody response (figure 7). On the other hand, recipients who received spleen cells after in vitro treatment with rabbit antiserum to mouse y-globulin or normal rabbit serum produced substantial titers of neutralizing antibody. Thus depletion of T-cells rather than B-cells from donor spleen cell suspensions resulted in a markedly inhibited antibody response in recipients.
Discussion
Donor lymphoid cell responses to immunization with inactivated VEE virus vaccine are meager (figure 1). Thus spleen cells prepared from such donors are incapable of transferring the capacity to produce neutralizing antibody to adoptively immunized recipients at any time after donor immunization. It is curious that this finding is valid at the same time that donor humoral antibody responses to immunization are proceeding rapidly. One cannot, therefore, ascribe the failure of adoptive antibody transfer either to a generalized inability of the host to respond to 1-TC-83 or to any lack of immunogenicity associated with 1-TC-83 itself. Donors immunized on three consecutive days with inactivated VEE virus vaccine also fail to demonstrate any capacity to transfer an adoptive antibody response (figure 2). In contrast, immunization with I-TC-83 once weekly for three weeks produces both a vigorous neu-
Downloaded from http://jid.oxfordjournals.org/ at University of Birmingham on August 21, 2015
E
Host Immune Responses to VEE Vaccines II
45
1000
800
80
~ 600
60
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~
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JULY 1976
Host Immune Responses after Administration of Inactivated Venezuelan Equine Encephalomyelitis Virus Vaccines. II. Kinetics of Neutralizing Antibody Responses in Donors and Adoptively Immunized Recipients From the Infectious Diseases-Hypersensitivity Section, Department of Medicine, Veterans Administration Research Hospital, Northwestern University Medical Center, Chicago, Illinois
Stanley G. Rabinowitz
In a previous paper [1] the response of mice to immunization with various inactivated Venezuelan equine encephalomyelitis (VEE) virus vaccines was described and partially characterized. It appeared that the use of an adoptive transfer system provided an opportunity for viewing cellular events underlying responses to vaccination. As a result of our previous findings [1], we postulated
that responses of lymphoid spleen cells after immunization of donor mice with inactivated VEE vaccine and certain adjuvants were vigorous and long-lasting. We speculated on the possibility that these responses resulted from amplification of clones of VEE-reactive cells. This clonal amplification presumably resulted from adjuvant-mediated, intense, lymphocyte-antigen interaction enhancing by manyfold the usual response to vaccination with inactivated VEE vaccine. In contrast, immunization of mice with inactivated VEE vaccine alone did not result in substantial lymphoid spleen cell responses to VEE antigen. In support of the concept that immunopotentiation induced by adjuvants results from amplification of clones of VEE-reactive splenic lymphocytes, we proceeded to quantitate lymphoid spleen cell responses to inactivated VEE vaccines. We reasoned that, since neutralizing antibody was such a potent means of protecting mice against
Received for publication October 14, 1975, and in revised form January 30, 1976. This study was supported by grant no. MRS 7319 from the Veterans Administration Research Services and by contract no. DAMD 17-74-C-4112 from the U.S. Army Medical Research and Development Command. I gratefully acknowledge the expert technical assistance of Jayashree Huprikar and Mary Grover and the secretarial assistance of Judith Mann. Please address requests for reprints to Dr. Stanley G. Rabinowitz, Veterans Administration Lakeside Hospital, 333 East Huron Street, Chicago, Illinois 60611.
39
Downloaded from http://jid.oxfordjournals.org/ at University of Birmingham on August 21, 2015
Lymphoid cell responses to immunization with various formalin-inactivated Venezuelan equine encephalomyelitis (VEE) virus vaccines were monitored in mice by assessment of the development of both the neutralizing antibody response in sera of spleen cell donors and the adoptive neutralizing antibody response induced by spleen cell transfer in recipients. Donors immunized intraperitoneally with formalin-inactivated VEE vaccine (a single dose or a dose on three consecutive days) developed early serum neutralizing antibody responses (;::: 1:88-1 : 100) by seven days after immunization. Recipients of spleen cells from such mice were, however, incapable of eliciting a neutralizing antibody response (~1: 10). Only spleen cells from donors immunized with inactivated VEE vaccine plus adjuvants (particularly complete Freund's adjuvant and Bordetella pertussis) were consistently capable of producing early, high-titer serum neutralizing antibody responses in adoptively immunized recipients (;::: 1: 50-1 : 120 on day 4). The magnitude of neutralizing antibody responses of donors to inactivated VEE vaccines did not serve as a useful indicator of whether spleen cells from such mice could adoptively induce antibody responses in recipients. Finally, treatment of immune spleen cells with rabbit antiserum to mouse thymocytes, but not with rabbit antiserum to mouse y-globulin or normal rabbit serum, abolished the capacity of such cells to transfer an antibody response adoptively.
Rabinowitz
40
challenge with VEE virus [2], it would be appropriate first to examine the development of neutralizing antibody in both donors and adoptively immunized recipients. We chose, therefore, in the present study to focus specifically on an analysis of the neutralizing antibody responses of both donor and adoptively immunized mice. Materials and Methods
Results
Effect of different immunization regimens on time of appearance of serum neutralizing antibody in donor and cell transfer recipients. Donor mice immunized with one dose of inactivated VEE vaccine (I-TC-83) developed serum neutralizing antibody detectable within five days of
Downloaded from http://jid.oxfordjournals.org/ at University of Birmingham on August 21, 2015
Animals, virus and vaccine preparations, and plaque assay have been described in detail [1]. The vaccines employed were: (1 ) formalininactivated TC-83 VEE virus given once (1-TC83 Xl), three times on consecutive days (1- TC83 X 3), or once weekly for three consecutive weeks (I-TC-83 X 3 wk); (2) I-TC-83 combined with Bordetella pertussis (2 X 1010 heatkilled organisms/ml; lot no. 985739; Parke, Davis, Detroit, Mich.); (3) 1-TC-83 combined with complete Freund's adjuvant (CFA; 4 mg/rnl of heat-killed, lyophilized bacillus CalmetteGuerin); (4) 1-TC-83 combined with incomplete Freund's adjuvant (Difco Laboratories, Detroit, Mich.). Vaccine was administered ip in a dose of 2.4 X 108 pfu/0.3 ml, unless otherwise indicated. When 1-TC-83 was combined with adjuvant, 0.2 ml of 1-TC-83 was added to 0.1 ml of adjuvant. Booster injections of 1-TC-83 alone were given for the next two days after vaccine and adjuvant were administered (0.3 ml per mouse per day). Adoptive transfer protocol. Donor mice were immunized; at given intervals after immunization, blood was collected aseptically, and spleens were harvested and processed as previously described [1]. Viable spleen cells (1.2-1.6 X 10 8 ) were transferred ip to normal, adult, syngeneic recipients; groups of these animals were sacrificed by exsanguination two, four or five, and six or seven days after cell transfer. At least five donors and three adoptively immunized recipients were bled at each interval studied. Individually collected samples of sera were heat-inactivated at 56 C for 30 min and then stored at -20 C for subsequent antibody determination. Plaque reduction neutralization test. Equal volumes of heat-inactivated mouse serum and neurovirulent TC-83 strain VEE virus, adjusted to yield 100 pfu of virus/O.! ml, were allowed
to stand at room temperature (about 24 C) for 60 min, and 0.1 ml of the mixture was added to chick embryo fibroblast (CEF) monolayers in 35-mm tissue culture plates (FB-6TC, Linbro Co., New Haven, Conn.). Plaque overlays were done as described previously [1], and the highest dilution resulting in a 50% reduction in plaques was taken as the end point. For each experiment involving determination of neutralizing antibody titer, aliquots of known negative and positive control sera were included. All sera were tested in triplicate. Treatment with cytotoxic antisera. Normal rabbit serum, rabbit antiserum to mouse y-globulin, and rabbit antiserum to mouse thymocytes were prepared, absorbed, and tested for specificity as described previously [3]. In brief, treatment of spleen cells with rabbit antiserum to mouse y-globulin plus complement was shown to abolish lipopolysaccharide reactivity while leaving phytohemagglutinin reactivity intact. Conversely, treatment of spleen cells with rabbit antiserum to mouse thymocytes plus complement abolished phytohemagglutinin reactivity while leaving lipopolysaccharide responsiveness intact. Lyophilized rabbit complement (Grand Island Biological Co., Grand Island, N.Y.) was reconstituted with buffer and absorbed twice with C57B16/J spleen cells at 4 C before use for removal of nonspecific lymphocytotoxicity. Spleen cells (1.2-1.4 X 109 ) were suspended in 4.5 ml of RPMI-1640 with 1.0 ml of absorbed antisera or control sera and 4.5 ml of a 1: 3 dilution of absorbed rabbit complement. After the mixture was incubated at 37 C for 30 min, treated cells were washed twice with RPMI-1640 and counted, and 1.4-1.5 X 108 viable (trypan blue, 0.5% dye exclusion) spleen cells were injected ip into each recipient. Amounts and dilutions of antisera and complement used were based on previously described data [3].
41
Host Immune Responses to VEE Vaccines II
donor spleen cells
6
7 day 14 day _._._._ 21 day" ..
40 I
i •
•
I•
•
100
!
••
7
20
14
21
day after immunization
2
4
6
8
day after cell transfer
Figure 1. Left, development of serum neutralizing antibody response in mice immunized with one ip dose of formalin-inactivated Venezuelan equine encephalomyelitis virus vaccine (I-TC-83; 2.4 X 108 pfu/0.3 ml), Values shown represent reciprocal of the mean (- SE) of five determinations for each day studied. Right, development of serum neutralizing antibody response in adoptively immunized recipients of spleen cells seven, 14, and 21 days after immunization of donors with one ip dose of I-TC-83. Values shown represent reciprocal of the mean (- SE) of three determinations for each day studied (two, four, and seven days) after cell transfer.
Downloaded from http://jid.oxfordjournals.org/ at University of Birmingham on August 21, 2015
tion of donors (figure 2). Splenocytes transferred 14 days after immunization, however, did elicit a small (titer, approximately 1:20) antibody response by two to five days after transfer. This response was short-lived. Furthermore, inability to immunize recipients adoptively against challenge with VEE virus appeared to correlate with a lack of or a minimal neutralizing antibody response in recipients [1]. Mice immunized with 1-TC-83 once weekly for three weeks behaved differently. Serum neutralizing antibody responses of donors were already well developed by seven days after the last immunization (figure 3). In fact, antibody responses had already reached a plateau by that time and were on the decline by 21 days after the last immunization. In general, weekly immunization with 1-TC-83 seemed to produce the highest neutralizing antibody response in the groups of donor mice immunized with I-TC-83 alone. In contrast to previously immunized groups, mice adoptively immunized with spleen cells harvested seven days
vaccine administration (figure 1). After five days, the level of serum antibody rose rapidly and began to level off by 21 days after immunization. No serum neutralizing antibody was apparent in adoptively immunized recipients receiving spleen cells seven, 14, or 21 days after vaccination (figure 1). Furthermore, lack of antibody in adoptively immunized recipients correlated with susceptibility to challenge with VEE virus [1]. Donors immunized with I-TC-83 daily for three days also developed early serum neutralizing antibody titers (figure 2). Between seven and 14 days, antibody titers increased linearly and began to level off by 21 days. The kinetics of antibody production resembles that seen for the group given only one dose of 1-TC-83. The only difference readily appreciated between donors receiving one or three doses of 1-TC-83 is that antibody titers are higher in the latter group. Adoptively immunized recipients failed again to demonstrate serum neutralizing antibody when spleen cells were transferred seven or 21 days after vaccina-
42
Rabinowitz
50
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40
.!::!
~
7 dey 14 dey 21 dey
6
normal
I I I
, ,
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""
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300
...
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I I I
20
!
too
7
14
~
doy oFter imrnunizetion
2
4
6
doy ofter cell tronsFer
Figure 2. Left, development of serum neutralizing antibody response in mice immunized with three ip doses (on consecutive days) of formalin-inactivated Venezuelan equine encephalomyelitis virus vaccine (I-TC-83; 2.4 X 108 pfu/0.3 mI). Values shown represent reciprocal of the mean (- SE) of five determinations for each day studied. Right, development of serum neutralizing antibody response in adoptively immunized recipients of spleen cells seven, 14, and 21 days after immunization of donors as described above. Values shown represent reciprocal of the mean (- SE) of three determinations for each day studied (two, five, and six days) after cell transfer.
after the last weekly immunization developed serum neutralizing antibody (titer, 1:40) by four days after cell transfer. By seven days after cell transfer, recipients had serum neutralization titers of > 1: 80. A similar pattern appeared when spleen cells were transferred two weeks after the last injection of inactivated VEE vaccine. By 21 days after immunization, spleen cells appeared to lose their ability to transfer the capacity for antibody production. Appearance of neutralizing antibody responses in adoptively immunized recipients seemed to correlate with the ability of the mice to resist challenge with VEE virus [1]. Resistance to challenge, however, was not as complete in this group as one would have expected, based on appearance of serum neutralizing antibody in recipients [1]. This lower resistance may be explained by the fact that these adoptively immunized recipients develop late antibody responses (four to seven days). Donor mice immunized ·with I-TC-83 combined with CFA demonstrated high titers of serum
neutralizing antibody (about 1:240) by five to seven days after immunization, with substantial increases by 14 days (figure 4). In contrast to responses in the previously discussed groups, however, antibody responses of donors were extremely high by 21 days. Adoptively immunized recipients also showed clear-cut evidence of serum neutralizing antibody between two and four days after cell transfer, with peak serum titers four days after cell transfer. Although the peak titer of antibody in sera of adoptively immunized recipients was not higher than that in animals given weekly doses of 1-TC-83 vaccine, the response occurred earlier. Early appearance of a substantial level (~1 :40) of serum neutralizing antibody in adoptively immunized recipients seemed to correlate with successful transfer of adoptive immunity to challenge with VEE virus [1]. However, although mice were protected as early as seven days after immunization with 1-TC-83 and CFA (sc ) , no antibody was detected in such adoptively immunized recipients.
Downloaded from http://jid.oxfordjournals.org/ at University of Birmingham on August 21, 2015
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Host Immune Responses to VEE Vaccines 11
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Figure 3. Lett, development of serum neutralizing antibody response in mice immunized with three ip doses (given weekly for three weeks) of formalin-inactivated Venezuelan equine encephalomyelitis virus vaccine (I-TC-83; 2.4 X 108 pfu/0.3 ml). Values shown represent reciprocal of the mean (- SE) of five determinations for each day studied. Right, development of serum neutralizing antibody response in adoptively immunized recipients of spleen cells seven, 14, and 21 days after immunization of donors with I-TC-83 vaccine in three doses described above. Values shown represent reciprocal of the mean (- SE) of three determinations for each day studied (two, four, and seven days) after cell transfer.
Donors immunized with inactivated VEE virus vaccine and incomplete Freund's adjuvant developed substantial titers of neutralizing antibody seven days (~1 :80) and 14 days (~1 :360) after immunization. By 21 days after immunization, titers of neutralizing antibody in sera of donors approached 1: 1,000 (figure 5). When adoptively immunized recipients were studied, it was apparent that only recipients immunized 14 days after vaccination of donors developed significant serum antibody titers (~1 :50). These results were similar to antibody responses noted in recipients receiving spleen cells from donors immunized weekly with inactivated VEE virus vaccine (figure 3). Mice adoptively immunized seven and 21 days after vaccination of donors with 1-TC-83 and incomplete Freund's adjuvant developed peak serum antibody titers of only ~ 1: 20 (figure 5). Thus, immunization with 1-TC-83 and incomplete Freund's adjuvant produces a less vigorous adoptive immune response in recipients
than immunization with I-TC-83 and CFA (figures 4 and 5). Donors immunized with I-TC-83 and B. pertussis developed prompt neutralizing antibody responses by seven to 14 days (figure 6). Serum neutralizing antibody titers at 21 days, however, were considerably lower than those resulting from administration of 1-TC-83 for three days. Finally, adoptively immunized recipients receiving splenocytes seven, 14, and 21 days after immunization of donors developed comparable early, high-titer serum neutralizing antibody. Furthermore, as seen in most groups, the ability to transfer protection adop.tively against challenge with VEE virus correlated with the capacity to produce an early serum neutralizing antibody response in immunized recipients [1]. We next examined whether the enhanced humoral antibody responses seen in adoptively immunized recipients depended on the presence of either thymus (T-) cells or bone marrow-derived
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Figure 4. Left, development of serum neutralizing antibody response in mice immunized with an sc dose of formalin-inactivated Venezuelan equine encephalomyelitis virus vaccine (I-TC-83; 1.6 X 108 pfu/0.2 ml) plus complete Freund's adjuvant, followed by daily administration of vaccine ip for two days. Values shown represent reciprocal of the mean (- SE) of five determinations for each day studied. Right, development of serum neutralizing antibody response in adoptively immunized recipients of spleen cells seven, 14, and 21 days after immunization of donors as described above. Values shown represent reciprocal of the mean (- SE) of three antibody determinations for each day studied (two, four, and six days) after cell transfer.
(B-) cells in the spleen cell transfer. We selectively depleted the spleen cell preparation of either T-cells or B-cells by using rabbit antiserum to mouse thymocytes or rabbit antiserum to mouse y-globulin plus complement. For spleen cell donors we chose mice immunized 14 days previously with 1-TC-8 3 and CFA. This group of vaccinees was chosen because their spleen cells uniformly protected recipients against challenge with VEE virus and induced vigorous antibody responses in recipients. Recipients receiving spleen cells after in vitro treatment with rabbit antiserum to mouse thymocytes plus complement demonstrated a markedly blunted antibody response (figure 7). On the other hand, recipients who received spleen cells after in vitro treatment with rabbit antiserum to mouse y-globulin or normal rabbit serum produced substantial titers of neutralizing antibody. Thus depletion of T-cells rather than B-cells from donor spleen cell suspensions resulted in a markedly inhibited antibody response in recipients.
Discussion
Donor lymphoid cell responses to immunization with inactivated VEE virus vaccine are meager (figure 1). Thus spleen cells prepared from such donors are incapable of transferring the capacity to produce neutralizing antibody to adoptively immunized recipients at any time after donor immunization. It is curious that this finding is valid at the same time that donor humoral antibody responses to immunization are proceeding rapidly. One cannot, therefore, ascribe the failure of adoptive antibody transfer either to a generalized inability of the host to respond to 1-TC-83 or to any lack of immunogenicity associated with 1-TC-83 itself. Donors immunized on three consecutive days with inactivated VEE virus vaccine also fail to demonstrate any capacity to transfer an adoptive antibody response (figure 2). In contrast, immunization with I-TC-83 once weekly for three weeks produces both a vigorous neu-
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