INFECTION
AND
IMMUNITY, OCt. 1991, p. 3498-3503
Vol. 59, No. 10
0019-9567/91/103498-06$02.00/0 Copyright © 1991, American Society for Microbiology
Ability of Recombinant or Native Proteins To Protect Monkeys against Heterologous Challenge with Plasmodium falciparum HOWARD M. ETLINGER,* PATRICK CASPERS, HUGUES MATILE, HANS-JOACHIM SCHOENFELD, DIETRICH STUEBER, AND BELA TAKACS Central Research Units, F. Hoffmann LaRoche Ltd., CH4002 Basel, Switzerland Received 18 December 1990/Accepted 10 July 1991
To circumvent problems associated with polymorphic vaccine candidates for Plasmodium falciparum malaria, we evaluated recombinant proteins representing sequences from relatively highly conserved regions of the precursor to the major merozoite surface proteins, gp190, for their ability to protect Saimiri monkeys against malaria challenge. Recombinant proteins represented amino acid residues 147 to 321 (p190-1) or 147 to 321 and 1060 to 1195 (p190-3), and their efficacy was compared with that of native gpl90 and its processed products. All antigens were derived from P. falciparum Ki, a Thai isolate, while the challenge strain was Palo Alto (from Uganda, Africa), which contains, with the exception of the N-terminal 375 amino acids, which are almost identical to the Kl sequence, essentially the MAD-20 allelic form of gpl90. By 12 days following challenge, each control monkey required drug treatment. Three monkeys injected with p190-3 required therapy, while one cleared the parasites without therapy. Two monkeys injected with p190-1 received therapy on day 14, while the remaining two cleared the parasites without therapy. Of four animals injected with native gpl90, because of health reasons unrelated to malaria, one was not challenged with parasites and one was removed from the study 8 days after challenge when its parasitemia was 1.1% (parasitemias in control animals ranged from 4.3 to 9%); the remaining two cleared the parasites after maximum parasitemias of 0.45 and 0.53%. The highest levels of antiparasite antibody were produced by animals immunized with native gpl90. There was a significant correlation between monkeys which did not require drug treatment and antiparasite antibody. These results may suggest that native gpl90 and/or its processed products can provide excellent protection against heterologous challenge and that antibody is important for protection. The challenge for vaccine development is to identify the protective sequence(s).
One of the difficulties associated with the development of
ideal vaccine will provide a broad degree of protection, we have evaluated the ability of recombinant proteins containing sequences from relatively highly conserved areas of isolates Ki and MAD-20 to protect against heterologous challenge. The efficacy of native gp190 to protect against heterologous challenge was also determined.
a successful malaria vaccine is the presence of parasite variability, which has been postulated to inhibit the appearance of effective immunity (1). For example, in the case of one group of vaccine candidates against the sporozoite stage
of the human malaria parasite, Plasmodium falciparum, the circumsporozoite proteins, the immunodominant sites for human T cell recognition, are polymorphic (11). A second group of vaccine candidates also characterized by polymorphism are the major merozoite surface proteins of P. falciparum. These are derived from a precursor protein of about 190 kDa, and the variability of gp190 reflects, in part, the presence of two prototypic forms of the gene, found in isolates Kl and MAD-20 (3, 6, 13, 15, 16, 18-21, 24-26, 28, 30, 35, 36). In addition to dimorphic segments, the protein contains relatively highly conserved as well as polymorphic regions. Purified gp190-derived proteins have been shown in some instances to eliminate the need for drug therapy in immunized monkeys challenged with P. falciparum (12, 29, 32). The monkey protection experiments which have been performed to date have utilized both heterologous and homologous parasite challenges. Since some areas of gp190 are relatively highly conserved, the term heterologous is used in the present study to indicate that the gp190-derived sequences used for immunization and those carried by the challenge parasite are not identical. The best protection was provided by gp190 and its processed products (native gpl90) when monkeys were challenged with parasites carrying the same gpl90 as that used for immunization (32). Since an *
MATERIALS AND METHODS Immunogens. (i) Recombinant proteins. DNAs coding for p190-1 (amino acid residues 147 to 321) and p190-3 (amino acid residues 147 to 321 and 1060 to 1195), representing highly conserved regions of the Kl isolate, were cloned and expressed in Escherichia coli as previously described (9). p190-1 is similar to 190 L (33) and each recombinant protein described in this report had an additional 4 to 6 amino acids resulting from the cloning. p190-1 contained 6 histidine (hexa-His) residues at the amino end, while p190-3 had a hexa-His sequence at the carboxy end. The recombinant proteins were purified to homogeneity from E. coli lysates by a combination of chromatofocusing and Ni2'-chelate affinity chromatography as described previously (33) or by successive hydrophobic interaction chromatography, Ni2+-chelate chromatography, ion-exchange chromatography, and gel filtration chromatography. Each recombinant protein was judged to be at least 95% pure by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. (ii) Native gp190. Native gpl90 was purified by immunoaffinity chromatography from a lysate of P. falciparum Ki cultured in human A' erythrocytes as described previously (27). Nonsynchronous cultures were harvested when parasitemia reached 10 to 15%, and parasites were isolated after
Corresponding author. 3498
VOL. 59, 1991
IMMUNIZATION AGAINST MALARIA WITH HETEROLOGOUS PROTEIN
saponin lysis of erythrocytes (10). Parasite pellets were extracted with 10 volumes of 1% Nonidet P-40 in 5 mM Tris-Cl (pH 7.2)-50 mM NaCl-5 mM EDTA-5 mM ethylene glycol-bis(P-aminoethyl ether)-N,N,N',N'-tetraacetic acid-1 mM phenylmethylsulfonyl fluoride-100 U of aprotinin (Trasylol) per ml-10 ,ug of leupeptin per ml for 60 min on ice. Lysates were centrifuged at 48,000 x g for 30 min, and the supernatant fractions were passed through a column containing 2 ml of Sepharose 4B coupled to protein A-purified rabbit immunoglobulin. This immunoglobulin was from an animal injected with a Ki-derived recombinant protein containing p190-3 amino acid residues 1060 to 1195 and 147 to 321 (190 N) (33). To remove nonspecifically bound proteins, we washed the column with 50 volumes of 50 mM Tris-Cl (pH 7.4) containing 0.3 M NaCl, 0.5% Nonidet P-40, and 100 U of aprotinin per ml. Bound proteins were eluted with 0.2 M glycine-HCl (pH 2.8) containing 0.5% Nonidet P-40 and 0.3 M NaCl. Fractions containing gp190, as determined by dot blotting, were pooled and concentrated in collodion bags (Sartorius) with N2 pressure. Immunization. Four Saimiri monkeys each received subcutaneously 100 ,ug of p190-1 or p190-3 in Freund's complete adjuvant on day 1 and 100 jig of the same antigen in Freund's incomplete adjuvant on days 24 and 48. Monkeys in a third group received approximately 8 ,ug of native gpl90 in Freund's complete adjuvant on day 1 and the same amount of native gpi90 in Freund's incomplete adjuvant on days 24 and 48. Control animals received 0.9% NaCl in the adjuvants on the same days. Parasite challenge. On day 60, each monkey received intraveneously 3.5 x 107 P. falciparum (Palo Alto)-infected erythrocytes from a donor monkey. Serological typing results, obtained with blood smears containing erythrocytes infected with our parasite challenge strain and a panel of 18 monoclonal antibodies, were consistent with the gpl90 sequence of Palo Alto published by Chang et al., who found that, except for a 36-bp deletion in the repeat region, the N-terminal 375 amino acids of the Kl and Palo Alto proteins are 99.9% identical; the remainder of the Palo Alto protein is very similar to the MAD-20 protein (4). There was no evidence for parasite heterogeneity on the basis of reactivity of the monoclonal antibodies, since each monoclonal antibody stained either all or none of the parasites. Following challenge, blood smears obtained daily were stained with Giemsa stain and the percentage of parasitemia was determined. Animals which had 20% parasitemia received drug therapy consisting of quinine and pyrimethamine-sulfadoxine (Fansidar). The experiment was stopped 50 days after parasite challenge, at which time all remaining monkeys received the antimalarial drugs. Antibody analyses. Sera or plasma were collected prior to immunizations and parasite challenge. Reactivity was evaluated by immunofluorescence (27), enzyme-linked immunosorbent assay (ELISA) (5), and immunoblotting (34) and with a Bio-Rad Mini-Protean II dual-slab-cell unit (used as described by the manufacturer) with the gel and buffer system described by Laemmli (23). Other p190 recombinant proteins. DNAs coding for recombinant proteins from the MAD-20 gpl90 gene were cloned, expressed, and purified from E. coli cell lysates (9). These proteins contained amino acid residues 123 to 302, 384 to 595, 595 to 897, 1078 to 1251, 1250 to 1398, and 1397 to 1563; each construct also contained hexa-His residues on either the amino- or the carboxy-terminal end. Four additional recombinant proteins contained amino acid residues 34 to 595, 1250 to 1563, 1250 to 1679, and 1397 to 1679; they also
3499
200 94 67
'
-
-
*
CY)
43
, *
0
I-a
30 *
a-1
14
a
b
c
d
e
FIG. 1. Immunoblot analysis of native gpl90. Total P. falciparum detergent extracts (lanes b, d, and e) and immunoaffinitypurified gpl90 (lane c) were probed with either endemic-area plasma (lanes b and c), monkey immunoaffinity-purified gpl90 antiserum obtained prior to challenge with parasites (lane d), or rabbit 190 N antiserum (lane e). Lane a contains '4C-labeled molecular weight standards. Polypeptides marked with an asterisk in lanes c and d represent immunoglobulin heavy and light chains that were eluted from the immunoaffinity column and elicited the production of antibodies.
had hexa-His residues at both the amino- and carboxyterminal ends. Plasma collected 4 days prior to parasite challenge was tested by ELISA and immunoblotting for reactivity with these proteins. Nucleotide sequence accession number. The EMBL accession number for P. falciparum Kl gpl90 is X03371. There are no other gene accession numbers applicable for any of the recombinant proteins used in this report.
RESULTS Immunoblot analysis of native gpl90. The apparent molecular weights of the polypeptides in the affinity-purified native gpl90 immunogen, as revealed by reactivity with endemicarea plasma, corresponded to those previously published (13, 15, 16, 30) for the gpl90 precursor and its processed products (Fig. 1). When reacted with a total parasite extract, the rabbit antiserum used to purify native gpl90 as well as the monkey antisera obtained prior to challenge from monkeys immunized with native gpl90 revealed polypeptides with molecular weights expected for native gpl90 (Fig. 1). Monkey protection studies. The results of monkey protection studies are shown in Fig. 2. By 12 days after receiving parasites, the four control monkeys required drug treatment. Three of four monkeys which had been immunized with p190-3 also required drug treatment by 12 days, while the remaining animal cleared the parasites after having maximum parasitemia of 10%. Two of four monkeys which had been injected with p190-1 required drug treatment on day 14, while one cleared the parasites after 16 days, with maximum parasitemia of 4%, and one still had 1% parasitemia by day 21, with maximum parasitemia of 13% over this time period. The group of animals that received native gpl90 had the lowest levels of parasitemia. Because of a neck infection, which was discovered following immunization, one animal in this group did not receive the parasite challenge. Of the
3500
INFECT. IMMUN.
ETLINGER ET AL.
Control
T
p1 90-1
T
20
20
10
10
-
0^
12
0
0 5
10
15
20
25
C
15
10
5
20
25
'Eu
pl 90-3
native gpl90 20-
rT
10
10
0
o
5
10
15
20
25
1
0
Time after challenge
A
&MM
.-
--.f
- -.0.
5
.
15
10
20
25
(days)
FIG. 2. Effect of immunization with recombinant (p190-1 or p190-3) or native (native gpl90) polypeptides P. falciparum. Control monkeys received only Freund's complete adjuvant. T, drug therapy.
remaining three, one was removed from the experiment 8 days after challenge because of a blood infection and fever; during this time, its maximum parasitemia was 1.1%, whereas parasitemias in controls varied between 4.3 and 9%. The effect of the infection on susceptibility to parasite challenge is unknown. The other two monkeys had maximum parasitemias of 0.53 and 0.45% during the course of the experiment. Monkey antibody responses. (i) Reactivity with recombinant proteins. Prechallenge antibody responses in ELISA to both p190-1 and p190-3 were high in animals receiving either of these recombinant proteins (Table 1). Monkeys receiving native gp190 also produced high antibody titers to p190-3 but somewhat lower antibody titers to p190-1. There was no obvious relationship between ELISA titer and protection. An attempt was made to more precisely localize the areas of gp190 which were recognized by prechallenge sera. The various p190 recombinant proteins were tested in ELISA and immunoblotting and yielded, in general, equivalent
---
on
challenge with blood-stage
results (data not shown). Native gp190 elicited antibody reactive with residues 123 to 302 in the three monkeys. In addition, serum from one monkey (Milo) reacted with residues 384 to 595, while that from another (Patrik) reacted with residues 595 to 897 and 1078 to 1251. Sera from the three gpl90 monkeys reacted with residues 34 to 595 and 1397 to 1679. Each monkey receiving p190-3 or p190-1 produced antibody against residues 123 to 302, a sequence present in the original antigen. In addition, each monkey injected with p190-3 produced antibody reactive with residues 1078 to 1251, a sequence present in the original antigen. It should be stressed that almost all of the antibody (>99%) elicited by p190-3 was specific for this parasite sequence. Nevertheless, three of the four monkeys immunized with p190-3 produced antibody reactive with the hexa-His sequence (data not shown). (ii) Reactivity with parasites. In the p190-1 group, the two monkeys with the highest titers against Palo Alto did not
VOL. 59, 1991
IMMUNIZATION AGAINST MALARIA WITH HETEROLOGOUS PROTEIN
TABLE 1. Reactivity of monkey sera with p190-1 or p190-3 Elisa titer (103)
Antigen
None
Monkey
Uno Vasco Walo Xeno
Preimmune
Prechallenge
p190-1 p190-3 p190-1 p190-3 2.3 0.9 5.8 2.3 0.9 2.3 0.9 2.3 2.3 2.3 2.3 0.9 2.3 5.8 5.8 5.8
% Maximum parasitemiaa
3501
TABLE 2. Reactivity of monkey sera with parasites Fluorescence titer
Antigen
Monkey
Preimmune Kl
T T T T
None
Palo Alto
Prechallenge Kl
Palo Alto
% Maximum parasitemiaa
Uno
AND
IMMUNITY, OCt. 1991, p. 3498-3503
Vol. 59, No. 10
0019-9567/91/103498-06$02.00/0 Copyright © 1991, American Society for Microbiology
Ability of Recombinant or Native Proteins To Protect Monkeys against Heterologous Challenge with Plasmodium falciparum HOWARD M. ETLINGER,* PATRICK CASPERS, HUGUES MATILE, HANS-JOACHIM SCHOENFELD, DIETRICH STUEBER, AND BELA TAKACS Central Research Units, F. Hoffmann LaRoche Ltd., CH4002 Basel, Switzerland Received 18 December 1990/Accepted 10 July 1991
To circumvent problems associated with polymorphic vaccine candidates for Plasmodium falciparum malaria, we evaluated recombinant proteins representing sequences from relatively highly conserved regions of the precursor to the major merozoite surface proteins, gp190, for their ability to protect Saimiri monkeys against malaria challenge. Recombinant proteins represented amino acid residues 147 to 321 (p190-1) or 147 to 321 and 1060 to 1195 (p190-3), and their efficacy was compared with that of native gpl90 and its processed products. All antigens were derived from P. falciparum Ki, a Thai isolate, while the challenge strain was Palo Alto (from Uganda, Africa), which contains, with the exception of the N-terminal 375 amino acids, which are almost identical to the Kl sequence, essentially the MAD-20 allelic form of gpl90. By 12 days following challenge, each control monkey required drug treatment. Three monkeys injected with p190-3 required therapy, while one cleared the parasites without therapy. Two monkeys injected with p190-1 received therapy on day 14, while the remaining two cleared the parasites without therapy. Of four animals injected with native gpl90, because of health reasons unrelated to malaria, one was not challenged with parasites and one was removed from the study 8 days after challenge when its parasitemia was 1.1% (parasitemias in control animals ranged from 4.3 to 9%); the remaining two cleared the parasites after maximum parasitemias of 0.45 and 0.53%. The highest levels of antiparasite antibody were produced by animals immunized with native gpl90. There was a significant correlation between monkeys which did not require drug treatment and antiparasite antibody. These results may suggest that native gpl90 and/or its processed products can provide excellent protection against heterologous challenge and that antibody is important for protection. The challenge for vaccine development is to identify the protective sequence(s).
One of the difficulties associated with the development of
ideal vaccine will provide a broad degree of protection, we have evaluated the ability of recombinant proteins containing sequences from relatively highly conserved areas of isolates Ki and MAD-20 to protect against heterologous challenge. The efficacy of native gp190 to protect against heterologous challenge was also determined.
a successful malaria vaccine is the presence of parasite variability, which has been postulated to inhibit the appearance of effective immunity (1). For example, in the case of one group of vaccine candidates against the sporozoite stage
of the human malaria parasite, Plasmodium falciparum, the circumsporozoite proteins, the immunodominant sites for human T cell recognition, are polymorphic (11). A second group of vaccine candidates also characterized by polymorphism are the major merozoite surface proteins of P. falciparum. These are derived from a precursor protein of about 190 kDa, and the variability of gp190 reflects, in part, the presence of two prototypic forms of the gene, found in isolates Kl and MAD-20 (3, 6, 13, 15, 16, 18-21, 24-26, 28, 30, 35, 36). In addition to dimorphic segments, the protein contains relatively highly conserved as well as polymorphic regions. Purified gp190-derived proteins have been shown in some instances to eliminate the need for drug therapy in immunized monkeys challenged with P. falciparum (12, 29, 32). The monkey protection experiments which have been performed to date have utilized both heterologous and homologous parasite challenges. Since some areas of gp190 are relatively highly conserved, the term heterologous is used in the present study to indicate that the gp190-derived sequences used for immunization and those carried by the challenge parasite are not identical. The best protection was provided by gp190 and its processed products (native gpl90) when monkeys were challenged with parasites carrying the same gpl90 as that used for immunization (32). Since an *
MATERIALS AND METHODS Immunogens. (i) Recombinant proteins. DNAs coding for p190-1 (amino acid residues 147 to 321) and p190-3 (amino acid residues 147 to 321 and 1060 to 1195), representing highly conserved regions of the Kl isolate, were cloned and expressed in Escherichia coli as previously described (9). p190-1 is similar to 190 L (33) and each recombinant protein described in this report had an additional 4 to 6 amino acids resulting from the cloning. p190-1 contained 6 histidine (hexa-His) residues at the amino end, while p190-3 had a hexa-His sequence at the carboxy end. The recombinant proteins were purified to homogeneity from E. coli lysates by a combination of chromatofocusing and Ni2'-chelate affinity chromatography as described previously (33) or by successive hydrophobic interaction chromatography, Ni2+-chelate chromatography, ion-exchange chromatography, and gel filtration chromatography. Each recombinant protein was judged to be at least 95% pure by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. (ii) Native gp190. Native gpl90 was purified by immunoaffinity chromatography from a lysate of P. falciparum Ki cultured in human A' erythrocytes as described previously (27). Nonsynchronous cultures were harvested when parasitemia reached 10 to 15%, and parasites were isolated after
Corresponding author. 3498
VOL. 59, 1991
IMMUNIZATION AGAINST MALARIA WITH HETEROLOGOUS PROTEIN
saponin lysis of erythrocytes (10). Parasite pellets were extracted with 10 volumes of 1% Nonidet P-40 in 5 mM Tris-Cl (pH 7.2)-50 mM NaCl-5 mM EDTA-5 mM ethylene glycol-bis(P-aminoethyl ether)-N,N,N',N'-tetraacetic acid-1 mM phenylmethylsulfonyl fluoride-100 U of aprotinin (Trasylol) per ml-10 ,ug of leupeptin per ml for 60 min on ice. Lysates were centrifuged at 48,000 x g for 30 min, and the supernatant fractions were passed through a column containing 2 ml of Sepharose 4B coupled to protein A-purified rabbit immunoglobulin. This immunoglobulin was from an animal injected with a Ki-derived recombinant protein containing p190-3 amino acid residues 1060 to 1195 and 147 to 321 (190 N) (33). To remove nonspecifically bound proteins, we washed the column with 50 volumes of 50 mM Tris-Cl (pH 7.4) containing 0.3 M NaCl, 0.5% Nonidet P-40, and 100 U of aprotinin per ml. Bound proteins were eluted with 0.2 M glycine-HCl (pH 2.8) containing 0.5% Nonidet P-40 and 0.3 M NaCl. Fractions containing gp190, as determined by dot blotting, were pooled and concentrated in collodion bags (Sartorius) with N2 pressure. Immunization. Four Saimiri monkeys each received subcutaneously 100 ,ug of p190-1 or p190-3 in Freund's complete adjuvant on day 1 and 100 jig of the same antigen in Freund's incomplete adjuvant on days 24 and 48. Monkeys in a third group received approximately 8 ,ug of native gpl90 in Freund's complete adjuvant on day 1 and the same amount of native gpi90 in Freund's incomplete adjuvant on days 24 and 48. Control animals received 0.9% NaCl in the adjuvants on the same days. Parasite challenge. On day 60, each monkey received intraveneously 3.5 x 107 P. falciparum (Palo Alto)-infected erythrocytes from a donor monkey. Serological typing results, obtained with blood smears containing erythrocytes infected with our parasite challenge strain and a panel of 18 monoclonal antibodies, were consistent with the gpl90 sequence of Palo Alto published by Chang et al., who found that, except for a 36-bp deletion in the repeat region, the N-terminal 375 amino acids of the Kl and Palo Alto proteins are 99.9% identical; the remainder of the Palo Alto protein is very similar to the MAD-20 protein (4). There was no evidence for parasite heterogeneity on the basis of reactivity of the monoclonal antibodies, since each monoclonal antibody stained either all or none of the parasites. Following challenge, blood smears obtained daily were stained with Giemsa stain and the percentage of parasitemia was determined. Animals which had 20% parasitemia received drug therapy consisting of quinine and pyrimethamine-sulfadoxine (Fansidar). The experiment was stopped 50 days after parasite challenge, at which time all remaining monkeys received the antimalarial drugs. Antibody analyses. Sera or plasma were collected prior to immunizations and parasite challenge. Reactivity was evaluated by immunofluorescence (27), enzyme-linked immunosorbent assay (ELISA) (5), and immunoblotting (34) and with a Bio-Rad Mini-Protean II dual-slab-cell unit (used as described by the manufacturer) with the gel and buffer system described by Laemmli (23). Other p190 recombinant proteins. DNAs coding for recombinant proteins from the MAD-20 gpl90 gene were cloned, expressed, and purified from E. coli cell lysates (9). These proteins contained amino acid residues 123 to 302, 384 to 595, 595 to 897, 1078 to 1251, 1250 to 1398, and 1397 to 1563; each construct also contained hexa-His residues on either the amino- or the carboxy-terminal end. Four additional recombinant proteins contained amino acid residues 34 to 595, 1250 to 1563, 1250 to 1679, and 1397 to 1679; they also
3499
200 94 67
'
-
-
*
CY)
43
, *
0
I-a
30 *
a-1
14
a
b
c
d
e
FIG. 1. Immunoblot analysis of native gpl90. Total P. falciparum detergent extracts (lanes b, d, and e) and immunoaffinitypurified gpl90 (lane c) were probed with either endemic-area plasma (lanes b and c), monkey immunoaffinity-purified gpl90 antiserum obtained prior to challenge with parasites (lane d), or rabbit 190 N antiserum (lane e). Lane a contains '4C-labeled molecular weight standards. Polypeptides marked with an asterisk in lanes c and d represent immunoglobulin heavy and light chains that were eluted from the immunoaffinity column and elicited the production of antibodies.
had hexa-His residues at both the amino- and carboxyterminal ends. Plasma collected 4 days prior to parasite challenge was tested by ELISA and immunoblotting for reactivity with these proteins. Nucleotide sequence accession number. The EMBL accession number for P. falciparum Kl gpl90 is X03371. There are no other gene accession numbers applicable for any of the recombinant proteins used in this report.
RESULTS Immunoblot analysis of native gpl90. The apparent molecular weights of the polypeptides in the affinity-purified native gpl90 immunogen, as revealed by reactivity with endemicarea plasma, corresponded to those previously published (13, 15, 16, 30) for the gpl90 precursor and its processed products (Fig. 1). When reacted with a total parasite extract, the rabbit antiserum used to purify native gpl90 as well as the monkey antisera obtained prior to challenge from monkeys immunized with native gpl90 revealed polypeptides with molecular weights expected for native gpl90 (Fig. 1). Monkey protection studies. The results of monkey protection studies are shown in Fig. 2. By 12 days after receiving parasites, the four control monkeys required drug treatment. Three of four monkeys which had been immunized with p190-3 also required drug treatment by 12 days, while the remaining animal cleared the parasites after having maximum parasitemia of 10%. Two of four monkeys which had been injected with p190-1 required drug treatment on day 14, while one cleared the parasites after 16 days, with maximum parasitemia of 4%, and one still had 1% parasitemia by day 21, with maximum parasitemia of 13% over this time period. The group of animals that received native gpl90 had the lowest levels of parasitemia. Because of a neck infection, which was discovered following immunization, one animal in this group did not receive the parasite challenge. Of the
3500
INFECT. IMMUN.
ETLINGER ET AL.
Control
T
p1 90-1
T
20
20
10
10
-
0^
12
0
0 5
10
15
20
25
C
15
10
5
20
25
'Eu
pl 90-3
native gpl90 20-
rT
10
10
0
o
5
10
15
20
25
1
0
Time after challenge
A
&MM
.-
--.f
- -.0.
5
.
15
10
20
25
(days)
FIG. 2. Effect of immunization with recombinant (p190-1 or p190-3) or native (native gpl90) polypeptides P. falciparum. Control monkeys received only Freund's complete adjuvant. T, drug therapy.
remaining three, one was removed from the experiment 8 days after challenge because of a blood infection and fever; during this time, its maximum parasitemia was 1.1%, whereas parasitemias in controls varied between 4.3 and 9%. The effect of the infection on susceptibility to parasite challenge is unknown. The other two monkeys had maximum parasitemias of 0.53 and 0.45% during the course of the experiment. Monkey antibody responses. (i) Reactivity with recombinant proteins. Prechallenge antibody responses in ELISA to both p190-1 and p190-3 were high in animals receiving either of these recombinant proteins (Table 1). Monkeys receiving native gp190 also produced high antibody titers to p190-3 but somewhat lower antibody titers to p190-1. There was no obvious relationship between ELISA titer and protection. An attempt was made to more precisely localize the areas of gp190 which were recognized by prechallenge sera. The various p190 recombinant proteins were tested in ELISA and immunoblotting and yielded, in general, equivalent
---
on
challenge with blood-stage
results (data not shown). Native gp190 elicited antibody reactive with residues 123 to 302 in the three monkeys. In addition, serum from one monkey (Milo) reacted with residues 384 to 595, while that from another (Patrik) reacted with residues 595 to 897 and 1078 to 1251. Sera from the three gpl90 monkeys reacted with residues 34 to 595 and 1397 to 1679. Each monkey receiving p190-3 or p190-1 produced antibody against residues 123 to 302, a sequence present in the original antigen. In addition, each monkey injected with p190-3 produced antibody reactive with residues 1078 to 1251, a sequence present in the original antigen. It should be stressed that almost all of the antibody (>99%) elicited by p190-3 was specific for this parasite sequence. Nevertheless, three of the four monkeys immunized with p190-3 produced antibody reactive with the hexa-His sequence (data not shown). (ii) Reactivity with parasites. In the p190-1 group, the two monkeys with the highest titers against Palo Alto did not
VOL. 59, 1991
IMMUNIZATION AGAINST MALARIA WITH HETEROLOGOUS PROTEIN
TABLE 1. Reactivity of monkey sera with p190-1 or p190-3 Elisa titer (103)
Antigen
None
Monkey
Uno Vasco Walo Xeno
Preimmune
Prechallenge
p190-1 p190-3 p190-1 p190-3 2.3 0.9 5.8 2.3 0.9 2.3 0.9 2.3 2.3 2.3 2.3 0.9 2.3 5.8 5.8 5.8
% Maximum parasitemiaa
3501
TABLE 2. Reactivity of monkey sera with parasites Fluorescence titer
Antigen
Monkey
Preimmune Kl
T T T T
None
Palo Alto
Prechallenge Kl
Palo Alto
% Maximum parasitemiaa
Uno
