Failure of Recombinant Vaccinia Viruses Expressing Plasmodium falciparum Antigens To Protect Saimiri Monkeys against Malaria
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INFECTION AND IMMUNITY, JUlY 1991, p. 2403-2411 Vol. 59, No. 7
0019-9567/91/072403-09$02.00/0
Copyright C 1991, American Society for Microbiology
Failure of Recombinant Vaccinia Viruses Expressing Plasmodium
falciparum Antigens To Protect Saimiri Monkeys against Malaria
D. PYE,l* S. J. EDWARDS,' R. F. ANDERS,2 C. M. O'BRIEN,2 P. FRANCHINA,1 L. N. CORCORAN,2t
C. MONGER,'t M. G. PETERSON,2§ K. L. VANDENBERG,' J. A. SMYTHE,211 S. R. WESTLEY,1
R. L. COPPEL,2 T. L. WEBSTER,' D. J. KEMP,2 A. W. HAMPSON,' AND C. J. LANGFORD2#
Commonwealth Serum Laboratories, 45 Poplar Road, Parkville, Victoria 3052,'
and Walter & Eliza Hall Institute, Parkville, Victoria 3050,2 Australia
Received 7 January 1991/Accepted 26 April 1991
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Saimiri sciurus monkeys were immunized at multiple sites with recombinant vaccinia viruses expressing
Plasmodium fakiparum antigen genes and boosted 4 weeks later. Control monkeys were immunized with a
thymidine kinase-negative vaccinia virus mutant. Two weeks later, all of the monkeys were challenged by
intravenous inoculation of P. falciparum (Indochina strain) parasites. A group of unimmunized monkeys was
challenged in parallel. All of the monkeys that received vaccinia virus recombinants or the control virus
produced good anti-vaccinia virus antibody responses. However, those that received a single construct
containing ring-infected erythrocyte surface antigen (RESA) given at eight sites did not produce significant
antibody to any of the three major RESA repeat epitopes after immunization but were primed for an enhanced
antibody response after challenge infection with P. falciparum. Most of the monkeys produced detectable
antibodies to the RESA epitopes after challenge infection. One group of monkeys was immunized with four
constructs (expressing RESA, two merozoite surface antigens [MSA-1 and MSA-2], and a rhoptry protein
[AMA-1]), each given at two sites. While these monkeys failed to produce significant antibody against MSA-2
or AMA-1 after immunization, they produced enhanced responses against these antigens after challenge
infection. Immunization involved an allelic form of MSA-2 different from that present in the parasite challenge
strain, so that the enhanced responses seen after challenge infection indicated the presence of T-cell epitopes
common to both allelic forms. No groups of monkeys showed any evidence of protection against challenge, as
determined by examination of the resulting parasitemias.
A number of Plasmodium falciparum proteins which cinia viruses expressing various viral antigens have been
might be suitable as components of a malaria vaccine have demonstrated to induce good antibody responses in immu-
been identified. While biological studies have suggested the nized animals and, more importantly, to protect such ani-
suitability of these protein antigens as vaccine components, mals from subsequent challenge infection with the viruses
ultimately their usefulness can be determined only by pro- from which the antigens were derived (11, 13, 14, 20).
tection studies in vivo, first in Aotus sp. or Saimiri sp. Successful demonstration of protection against malaria after
monkeys and then in human volunteers. recombinant vaccinia virus immunization would not only
A malaria vaccine requires development of delivery meth- contribute to acceptance of vaccinia virus as a delivery
ods suitable for developing countries. Vaccines must be system for use in humans but would also confirm the
easily stored, transported, and delivered. Because of the suitability of vaccinia virus as a delivery system for screen-
good record of smallpox (vaccinia virus) vaccines in the third ing of malaria antigens in monkeys.
world, the use of recombinant vaccinia viruses should over- A malaria antigen, ring-infected erythrocyte surface anti-
come these basic problems, while potentially offering other gen (RESA) (3, 15), has been shown to provide partial
advantages, such as broad and effective immune responses. protection in an Aotus sp. monkey trial (1), and antibody
In addition, the use of recombinant vaccinia virus could responses to epitopes encoded by sequence repeats in the
overcome many of the problems of antigen expression and RESA gene were correlated with protection. Therefore,
delivery associated with the preparation of recombinant RESA was a good antigen with which to evaluate delivery
proteins in bacterium or yeast systems. Recombinant vac- systems, and an immunization-and-challenge study based on
vaccinia virus constructs containing either RESA or its
epitopes was carried out in Saimiri sp. monkeys. Vaccinia
*
Corresponding author. virus constructs containing several other antigens were also
t Present address: Whitehead Institute for Biomedical Research, included in this study. Unfortunately, protection was not
Cambridge, MA 02142. demonstrated in this trial, but useful data in relation to
t Present address: Department of Molecular and Radiation Biol- immune responses obtained after immunization with recom-
ogy, Peter MacCallum Institute, Melbourne, Victoria 3001, Austra-
lia. binant vaccinia viruses were obtained. These results are
§ Present address: Tjian Laboratory, Howard Hughes Medical reported here.
Institute, Department of Biochemistry, University of California,
Berkeley, CA 94720.
11 Present address: Laboratory of Tumor Cell Biology, National MATERIALS AND METHODS
Cancer Institute, National Institutes of Health, Bethesda, MD
20892. Monkeys. Adult female squirrel monkeys (Saimiri sciurus)
# Present address: Veterinary Institute of Animal Science, Att- of Guyanan origin were obtained from Charles River Re-
wood, Victoria 3049, Australia. search Primates Corp. All had intact spleens, had weights of
24032404 PYE ET AL. INFECT. IMMUN.
TABLE 1. Immunization groups TABLE 2. Antigens used in ELISA studies
Designation Description
Group Immunogen(s) No. of Comments
monkeys
Vaccinia ......... P-Propiolactone-inactivated purified TK-
A None 5 Unvaccinated controls vaccinia virus
B TK- vaccinia virus 5 Vaccinated controls RESA 8-mer ........ GST' fusion protein (19) containing 20 copies
C VRESA 6 Eight dose sites; six of the RESA 8-mer 3' repeat
animals immunized, RESA 11-mer ....... GST fusion protein containing six copies of the
with one death due RESA 11-mer 5' repeat
to accident prior to RESA 4-mer ........ GST fusion protein containing 15 copies of the
challenge infection RESA 4-mer 3' repeat
D V5, V33 (RESA repeats) 6 Four dose sites per MSA-1 ........ Affinity purified from strain FC27 parasites
virus MSA-2 ........ GST fusion protein containing the SspI
E VRESA, VMSA1, VAMA1, 6 Two dose sites per fragment of MSA-2 isolated from either the
VMSA2 virus FC27 or the IC1 strain of P. falciparum; full-
length antigen except for the terminal
hydrophobic amino acid domains
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AMA-1 ........ GST fusion protein containing the entire AMA-
1 antigen (FC27 origin) except for the
more than 600 g, and were preconditioned for several hydrophobic anchor region
months prior to use. They were free of tuberculosis infec- P513 ........ Peptide (CSQRSTNSAST) representing a
tion, not pregnant, and declared after thorough physical variable-region epitope of FC27 MSA-2
examination to be in general good health. Immunofluores- recognized by inhibitory monoclonal
cence testing for antibodies that cross-reacted with P. falci- antibodies; conjugated to BSAb by using
parum antigens failed to detect significant levels of such
glutaraldehyde
antibodies.
GGSA ........ Peptide repeat sequence from the variable
region of IC1 MSA-2 synthesized as a four-
Construction of recombinant vaccinia viruses. DNA frag- time tandem repeat and conjugated to BSA
ments encoding various malaria antigens and hybrid poly- by using glutaraldehyde
peptides from P. falciparum FC27 were inserted into the
multiple cloning site of vaccinia virus transfection plasmid
a
GST, glutathione S-transferase.
b BSA, bovine serum albumin.
pGS62 prior to transfection into the WR strain of vaccinia
virus (10).
Recombinant VRESA contained a DNA fragment encod-
ing the entire RESA antigen (4), derived from parasite strain 107 PFU in 0.1 ml. After healing (4 weeks), animals were
FC27. reimmunized with a dose of 108 PFU in 0.1 ml at each of four
Recombinant V5 contained a hybrid DNA fragment en- sites on the upper back and four sites on the lower abdomen.
coding the FC27 S antigen of P. falciparum which had been When animals received more than one virus strain, these
modified by addition of sequences encoding the hydrophobic were not mixed but a single strain was inoculated at each
transmembrane region and intracellular domain of the mem- site. For monkeys immunized with two strains, each virus
brane-bound form of the mouse immunoglobulin G, protein was inoculated at four sites, whereas monkeys immunized
(6) and deletion of the 12-amino-acid tandem repeat epitopes with four strains were inoculated with each virus at two
of the S antigen, which were replaced with sequences sites.
encoding six copies of the RESA undecamer repeat DDEH Parasite challenge. An S. sciurus-adapted P. falciparum
VEEPTVA. strain, Indochina 1/CDC (IC1), was obtained from W. Col-
Recombinant V33 was identical to V5 except that the lins (Centers for Disease Control, Atlanta, Ga.) and serially
S-antigen repeat sequences were replaced with sequences passaged in 10 further S. sciurus monkeys. When the para-
encoding 20 copies of the RESA octamer repeat EENVEH sitemia in the last of these monkeys first reached 3%
DA. Both the recombinant vaccinia viruses V5 and V33 have (subsequent peak of 10%), a stabilate was prepared and
been shown to be more immunogenic with respect to their stored in liquid nitrogen.
RESA repeat epitope than the VRESA recombinant virus For challenge, an individual vial was thawed, passaged in
when used to immunize mice and rabbits (8). a splenectomized monkey, and serially passaged further in
Recombinant VMSA1 contained a DNA fragment encod- intact monkeys until the time of challenge. At that time,
ing the entire MSA-1 antigen (17) from parasite strain FC27. blood was removed from the donor monkey (3% para-
Recombinant VAMAl contained a DNA fragment encod- sitemia) and the erythrocytes were suspended in saline to 3
ing the entire AMA-1 antigen (2, 18) from parasite strain x 107 parasites per ml. Monkeys were inoculated intrave-
FC27. nously with 1 ml of parasites, and daily smears were pre-
Recombinant VMSA2 contained a DNA fragment encod- pared for parasitemia determination.
ing the entire MSA-2 antigen (21) from parasite strain FC27. Serological studies. Antibody responses were measured
Recombinant vaccinia virus transfection plasmids were after dilution of serum (at 1:1,000, 1:3,000, 1:10,000, and
used to construct recombinant vaccinia viruses, with bro- 1:30,000 dilutions) and involved previously described en-
modeoxyuridine selection of thymidine kinase-deficient zyme-linked immunosorbent assay (ELISA) techniques (1).
(TK-) plaques as described previously (7). Antibody levels were determined on bleeds taken prior to
TK- nonrecombinant vaccinia virus was produced by immunization, at the time of challenge, and 2 weeks after
growing wild-type virus in the presence of bromodeoxyuri- challenge to measure responses to immunization and chal-
dine and selecting an individual plaque at random. lenge. The antigens used are described in Table 2. The
Immunization of monkeys. Monkeys were randomly allo- responses to MSA-2 were measured against two different
cated into five groups (Table 1). Each monkey was immu- allelic forms, from the FC27 and IC1 parasite strains. The
nized at each of eight shaved sites on the back with a dose of forms differ in that while the 43 N-terminal and 74 C-terminalVOL. 59, 1991 VACCINATION OF MONKEYS AGAINST MALARIA 2405
O.D. at 1:30,000 dilution
3
_ Prebleed
M 4 wks post 1st dose
2.5 -E3 2 wks post 2nd dose
2
1.5 _
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I
0.5 _-
0 , . db
20 42 66 38 78
A
I I. I JI
LA
74 75 81 82 88
B
ti.
+.
I --I
14 15 31 48 85 87
C
Immunisation groups (monkey nos.)
I] 1] ii. J
28 35 40 41 79 80
D
1 AA
03 29 47 77 83 88
E
FIG. 1. Response of monkeys to vaccinia virus. Sera prepared from bleeds taken prior to the first immunization (prebleeds), 4 weeks later,
and 2 weeks after the second immunization were diluted 1:30,000 in phosphate-buffered saline diluent and assayed by ELISA against
inactivated vaccinia virus (TK- strain). Each cluster of bars represents the optical densities (O.D.) after ELISA of the three sera obtained
from an individual monkey. The immunization groups were unvaccinated controls (A), vaccinated controls (B), VRESA (C), V5-V33 (D), and
VRESA-VMSA1-VAMA1-VMSA2 (E).
residues are highly conserved, between these regions there patent infections which subsequently cleared without drug
are major differences between the alleles (22). In the FC27 treatment. In general, parasitemias were detected within 2
strain, there are two copies of a 32-amino-acid repeat se- days of inoculation and reached maximum levels at around 7
quence. In contrast, the IC1 strain contains 12 copies of the or 9 days. Parasites grew synchronously and presumably
four-amino-acid repeat sequence Gly-Gly-Ser-Ala (GGSA). cytoadhered, as evidenced by the 48-h interval between
The sequences flanking the repeats also differ between the peaks of parasitemia. Only ring forms were seen in the
alleles. peripheral blood until a day or two prior to the fall in parasite
levels. At that time, mature forms (late trophozoites and
RESULTS schizonts) became evident, as did crisis forms. The duration
of detectable parasitemia was generally around 2 weeks.
Responses to immunization. Primary immunization with Within each group, there was considerable variation in
vaccinia virus produced lesions in all immunized monkeys. peak parasitemias (Table 3), with a maximum of 6% and a
Lesions appeared within 5 days as red, swollen areas ap- minimum of 0.2%. No significant differences were found
proximately 8 mm in diameter which developed into crusty between groups in the maximum parasitemia or the time
scabs during the next few days. Within 12 days of vaccina- taken to reach maximum parasitemia. Thus, by using these
tion, these scabs were lifting to reveal healed tissue beneath. criteria, we found no evidence that any of the P. falciparum
Healing was complete within 19 days. No secondary lesions antigens expressed in recombinant vaccinia virus could
developed, and no difference in lesions was observed be- protect S. sciurus monkeys against challenge with P. falci-
tween animals immunized with recombinant vaccinia virus parum.
and those immunized with nonrecombinant vaccinia virus. Antibody responses to RESA epitopes. Antibody responses
Lesions developed after secondary immunizations but to the three major RESA repeat epitopes were determined
were smaller and resolved quickly (10 to 11 days). All by using sera collected prior to the primary immunization, at
vaccinated monkeys produced antibody to vaccinia virus, the times of the second immunization and the challenge, and
with boosted responses after the second immunization (Fig. 2 weeks after the challenge (Fig. 2).
1). Of the 18 animals that received recombinant vaccinia virus
Responses to challenge with P. falciparum. After challenge containing the RESA 11-mer repeat, none produced detect-
with the IC1 strain of P. falciparum, all monkeys developed able responses to immunization (Fig. 2a). However, a num-2406 PYE ET AL. INFECT. IMMUN.
a. RESA llmer TABLE 3. Responses to challenge parasite infection
O.D. at 113000 dilution Mon- Peak para- Duration of
Group key sitemia (% parasitemia
no. erythrocytes) (days)
A, unvaccinated controls 20 1.25 16
42 0.55 7
66 0.15 7
38 3.31 13
78 0.17 13
B, vaccinated controls 74 2.9 15
(TK- vaccinia virus) 75 2.53 15
81 1.06 15
82 0.61 14
86 2.0 19
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C, VRESA 14 2.16 13
15 4.5 15
31 6.75 15
so 42 4T.5 1 .66
186 14 1. 614 66667 16"4641 700 0616477768o" 48 2.0 14
A a C D E
lmmunlbatlom groups (monkey no.) 85 0.6 12
b. RESA 8mer 87 3.0 10
D, V5-V33 26 1.46 14
35 6.0 14
40 0.9 15
41 2.1 10
79 1.6 14
80 0.27 8
E, VRESA-VMSA1- 3 1.6 15
VMSA2-VAMA 29 1.6a
47 1.1 12
77 0.5 11
83 1.85 14
88 2.16 14
a This animal died as a result of an accident, and its parasitemia may not
have achieved its potential peak by the time of death.
20416676 74766161 o 14 16146667 16 6O 40 41 7660 06647 776
ber of these monkeys had detectable responses after chal-
A * C 0 E
lmmunlmfion groups (monkey nos.) lenge. Particularly enhanced responses after challenge were
c. RESA 4mer seen in four of the six monkeys immunized with VRESA
alone.
Of the 18 monkeys that received recombinant vaccinia
virus containing the RESA 8-mer repeat in the form of
VRESA or V33, most failed to produce detectable antibody
responses to immunization (Fig. 2b). In contrast, a majority
of monkeys produced detectable responses after challenge.
Again, it was clear that immunization with VRESA alone
primed for an enhanced response to the 8-mer repeat as a
result of the challenge infection.
Of the 12 animals that received VRESA alone or in
conjunction with recombinant vaccinia virus expressing
other antigens, most failed to produce detectable antibody
responses to immunization with the RESA 4-mer repeat
(Fig. 2c). As was observed with responses to the other two
major RESA repeat epitopes, immunization with VRESA
primed for an enhanced antibody response to the 4-mer
repeat after challenge. This enhanced response was seen in
1041666T6
A
74766161S6
B
14 If 146667 666440417660
C D
061647776866
E
all six animals that received VRESA alone but in only one of
lmmumlatlon group. (monley no.)
FIG. 2. Responses to RESA. Sera prepared from bleeds taken
prior to immunization (prebleed), after 2 immunizing doses (postim-
munization), and 2 weeks postchallenge were diluted 1:3,000 and three sera obtained from an individual animal. The immunization
assayed by ELISA against the RESA 5' 11-mer repeat (a), the RESA groups were unvaccinated controls (A), vaccinated controls (B),
3' 8-mer repeat (b), and the RESA 3' 4-mer repeat (c). Each cluster VRESA (C), V5-V33 (D), and VRESA-VMSA1-VAMA1-VMSA2
of bars represents the optical densities (O.D.) after ELISA of the (E).VOL. 59, 1991 VACCINATION OF MONKEYS AGAINST MALARIA 2407
a. FC27 MSA2 a. Peptide 513
O.D. at 1:3000 dllutlon. O.D. at 1:3000 dilution
_ PrObl sod _ Prebleed
M post Immunleatton M post Immunlletlon
2.0 EJ poet 1Challenge
2 _CE poet Challenge
2-
1.6 _
I
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0.6 _
0.6 _
n
Hj
20 42 663 6 14 163 1 46 667 0 22 47 77 36
-n
20 426
W., &A Jl
36 7
.-n -wi
14 16
n
31 486
_rl
67
tfl -
03 29 4
tfl fl J--ni
77 66
A c E A C E
Immunisation groups (monkey nos.) Immunisation groups (monkey nos.)
b. IC1 MSA2 b. GGSA
O.D. at 1:1000 dilution.
3
Prebleed
1 poet Immunheatlon
2.6 post Challenge
2
I1.6
20 42 66
A
36 78 14 16 31
C
48 66
Immunisation groups (monkey nos.)
FIG. 3. Responses to the FC27 and IC1 forms of MSA-2. Sera
67 03 29 47 77
E
63 66
3.1
c
20 42
A
6367
14 16 31 4
C
6 87 03 20 47 77 63 86
Immunisation groups (monkey nos.)
E
FIG. 4. Responses to MSA-2 variable-region repeats. Sera pre-
prepared from bleeds taken prior to immunization (prebleed), after 2 pared from bleeds taken prior to immunization (prebleed), after 2
immunizing doses (postimmunization), and 2 weeks postchallenge immunizing doses (postimmunization), and 2 weeks postchallenge
were diluted and assayed by ELISA against recombinant FC27 were diluted and assayed by ELISA against FC27 strain variable-
strain MSA-2 protein (1:3,000 dilution; a) and recombinant IC1 region repeat epitope P513 (1:3,000 dilution; a) and IC1 strain
strain MSA-2 protein (1:3,000 dilution, b). Each cluster of bars variable-region repeat epitope GGSA (1:1,000 dilution; b). Each
represents the optical densities (O.D.) after ELISA of the three sera cluster of bars represents the optical densities (O.D.) after ELISA of
obtained from an individual animal. The immunization groups were the three sera obtained from an individual animal. The immunization
unvaccinated controls (A), VRESA (C), and VRESA-VMSA1- groups were unvaccinated controls (A), VRESA (C), and VRESA-
VAMA1-VMSA2 (E). VMSA1-VAMA1-VMSA2 (E).
the six animals that received VRESA together with other Responses were determined in three of the five monkey
recombinants. groups (A, unvaccinated controls; C, VRESA alone; and E,
In general, more monkeys produced strong responses to VMSA2, VMSA1, VRESA, and VAMA1). Immunization
the 8-mer and 4-mer repeats than to the 11-mer repeat. A with VMSA2 (FC27 MSA-2) produced weak responses to
small number of monkeys that responded well to the 8-mer both the FC27 and IC1 forms of MSA-2 (Fig. 3). After
repeat after challenge infection failed to respond to the challenge (IC1 MSA-2), responses to both the FC27 and IC1
l1-mer repeat (e.g., monkeys 31 and 82). forms of MSA-2 were detected in all monkeys (Fig. 3).
Antibody responses to MSA-2. Monkeys were assayed for Unvaccinated monkeys and those that received VRESA
responses to the FC27 and IC1 forms of MSA-2, a variable- mounted antibody responses which preferentially reacted
region epitope (P513) found in the FC27 strain of P. falci- with the homologous IC1 form of MSA-2. Several of the
parum involved in immunization but not in the IC1 strain monkeys which received VMSA2 made antibody responses
used for challenge infection, and a variable-region epitope that reacted almost as well with FC27 MSA-2 as with the
(GGSA) found in the IC1 strain but not in the FC27 strain. homologous IC1 MSA-2. Thus, immunization with the one2408 PYE ET AL. INFECT. IMMUN.
O.D. at 1:10,000 dilution
2.5
2
1.5
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0.5
0
20 42 66 38 78 14 15 31 48 85 87 03 29 47 77 83 88
A C E
Immunisation groups (monkey nos.)
FIG. 5. Responses to AMA-1. Sera prepared from bleeds taken prior to immunization (prebleeds), after two immunizing doses, and 2
weeks after challenge were diluted 1:10,000 in phosphate-buffered saline diluent and assayed by ELISA against fusion protein GST352. Each
cluster of bars represents the optical density (O.D.) after ELISA of the three sera obtained from an individual monkey. The immunization
groups were unvaccinated controls (A), VRESA (C), and VRESA-VMSA1-VAMA1-VMSA2 (E).
allelic form of MSA-2 using recombinant vaccinia virus there was no evidence of priming for an enhanced response
primed animals for enhanced responses to both forms of in monkeys immunized with the VMSA1 recombinant.
MSA-2 following infection.
Responses to the epitope from the FC27 variable region DISCUSSION
were not detected after immunization (Fig. 4a), although
after challenge with the IC1 strain, some animals showed The purpose of this study was to determine whether
evidence of weak responses to P513. A few animals pro- immunization with recombinant vaccinia viruses expressing
duced detectable responses to the IC1 variable-region repeat selected malaria antigens could protect S. sciurus monkeys
epitope GGSA after challenge infection (Fig. 4b), with no against challenge with blood stages of P. falciparum. RESA
evidence that the GGSA response was enhanced because of was the major antigen under study, but monkeys were also
prior immunization with VMSA2. immunized with recombinant vaccinia virus expressing
Antibody responses to AMA-1. Monkeys that received merozoite surface antigens MSA-1 and MSA-2 and rhoptry
recombinant vaccinia virus expressing AMA-1 (VAMA1) protein AMA-1. Although immunization resulted in high
failed to mount significant responses to AMA-1 after immu- vaccinia virus antibody responses in all of the animals, there
nization (Fig. 5). However, all of the monkeys tested (groups was no evidence of protection induced by any of the malaria
A, C, and E) produced good antibody responses after antigens delivered in this way.
challenge infection (detected at a 1:10,000 or greater dilu- Vaccinia virus infections were similar in all of the mon-
tion), and those immunized with VAMAl were primed for an keys, and relatively small lesions were produced. The infec-
enhanced response. tion took a mild course, with no indication of clinical illness
Antibody responses to MSA-1. Only a small amount of like that frequently seen in humans who receive smallpox
affinity-purified MSA-1 was available, and therefore the vaccine. While the mildness of the infections may have been
analysis of antibody responses to MSA-1 was limited. How- due to the reduced virulence of the recombinant viruses, it is
ever, the results (Fig. 6) indicated that immunization failed also likely that S. sciurus monkeys are less susceptible than
to produce a detectable response in the six animals that humans to vaccinia virus infection. In a comparative study
received the relevant vaccinia virus recombinant. All of the of responses to recombinant vaccinia viruses in Saimiri sp.,
monkeys tested produced good responses after challenge Aotus sp., Macaca sp., Cercopithecus sp., and Erythroce-
infection, which were detectable at a 1:10,000 dilution, but bus sp. monkeys (12), Saimiri sp. monkeys produced theVOL. 59, 1991 VACCINATION OF MONKEYS AGAINST MALARIA 2409
O.D. at 1:1000 dilution
3
2.5
2
1.5
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1
0.5
0
20 42 66 38 78 74 81 86 75 82 03 29 47 77 83 88
A B E
Immunisation groups (monkey nos.)
FIG. 6. Responses to MSA-1. Sera prepared from bleeds taken prior to the first immunization (prebleeds), after two immunizing doses,
and 2 weeks after challenge were diluted 1:1,000 in phosphate-buffered saline diluent and assayed by ELISA against FC27 strain MSA-1
protein purified from whole parasites by affinity chromatography. Limited availability of the antigen permitted assay of all sera from one group
only. For the two control groups, only postchallenge data were obtained. Each cluster of bars represents the optical density (O.D.) after
ELISA of the three sera obtained from an individual monkey. The immunization groups were unvaccinated controls (A), vaccinated controls
(B), and VRESA-VMSA1-VAMA1-VMSA2 (E).
smallest lesions (suggesting a reduced ability to replicate) failed to induce significant antibody responses to the various
and the lowest resulting antibody responses. (Alternatively, parasite antigens. This contrasted with previous studies with
a cellular immune response of greater potency might also rabbits and mice in which the RESA constructs given singly
result in smaller lesions.) However, in our study, the high had stimulated satisfactory antibody responses (7-9). In
vaccinia virus antibody responses induced by the primary addition, Lew and coworkers (9) had demonstrated prolifer-
immunizations and the boosting of these after the secondary ative T-cell responses in lymphocytes taken from mice
immunization indicated good virus replication and, there- previously immunized with the RESA constructs.
fore, potentially good expression of inserted antigens. Three different groups of monkeys were immunized with
After the challenge infection with P. falciparum, the vaccinia virus constructs expressing RESA either as the
resulting parasitemias achieved peaks between 0.15 and whole antigen alone or in combination with other antigens or
6.75%, with the unvaccinated control group showing the as fragments containing repetitive epitopes previously impli-
greatest range of parasitemias seen in any group (0.15 to cated in protective immune responses (1). Despite the poor
3.3%) and the lowest median peak (0.55%). Therefore, antibody responses to RESA induced by immunization, all
protection could not be demonstrated in any test group, and of the monkeys that were immunized with VRESA alone and
the model was less than ideal, since protection would be one of the monkeys that received VRESA in combination
detected only if a majority of monkeys within a group with other antigens were primed for a markedly enhanced
showed nearly complete absence of parasites (nearly sterile antibody to RESA following challenge.
immunity). While these results cast some doubt on the value The failure of immunization with V5 and V33 to prime in
of the S. sciurus protection model, more recent studies by us an equivalent way to VRESA suggests that the RESA 8-mer
(unpublished data) have demonstrated that nearly sterile and 11-mer repeats expressed by these two recombinant
immunity can be obtained as a consequence of immuniza- viruses lack the T-cell epitopes relevant to secondary anti-
tion. Thus, factors other than suitability of the monkey body responses to RESA. This contrasts with other studies
model should be considered among the reasons for failure to which indicated that the three different repeats of RESA all
demonstrate protection in this trial. contain T-cell epitopes (9) and that when various strains of
In contrast to the high induced antibody responses to mice were immunized with VRESA, T-cell epitopes encoded
vaccinia virus, immunization with the recombinant viruses by the repeats were involved in the response. The four-2410 PYE ET AL. INFECT. IMMUN.
amino-acid repeats in RESA have been shown to encode was obtained with RESA fusion proteins (1). These monkeys
T-cell epitopes recognized by infected humans (5). We did had high antibody titers induced by immunization at the time
not immunize monkeys with a recombinant vaccinia virus of challenge. If the immunogenicity of vaccinia virus recom-
expressing these repeats alone, and it is possible that the binants could be improved so that similarly high antibody
priming effect of full-length RESA involved recognition of responses were induced, then such recombinants could be
T-cell epitopes encoded by the 4-mer repeats. However, the expected to provide protective immunity.
uniformly good responses in all of these outbred animals
suggest that other T-cell epitopes are involved. Alterna- ACKNOWLEDGMENTS
tively, the failure of VS and V33 to prime for an enhanced
response may have been caused by insufficient antigen to We thank Bill Collins for advice and for supplying the IC1 parasite
sensitize immunized animals, but this is unlikely, since strain. We thank David Irving and Graeme Jones for supplying some
antigen production in vitro by these constructs is at least as of the ELISA antigens.
good as with VRESA (unpublished data) and their ability to This work was supported by the Australian Malaria Vaccine Joint
Venture (Saramane Pty. Ltd.).
induce immune responses in rabbits and mice is at least
equivalent (8, 9). REFERENCES
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Merozoite surface antigens MSA-1 and MSA-2 are of
particular interest as potential components of a malaria 1. Collins, W. E., R. F. Anders, M. Pappaioanou, G. H. Campbell,
G. V. Brown, D. J. Kemp, R. L. Coppel, J. C. Skinner, P. M.
vaccine. The failure to achieve protection with recombinant Andrysiak, J. M. Favaloro, L. M. Corcoran, J. R. Broderson,
vaccinia virus expressing these antigens should not lessen G. F. Mitchell, and C. C. Campbell. 1986. Immunization of
interest in these antigens because immunization failed to Aotus monkeys with recombinant proteins of an erythrocyte
induce significant antibody responses and, for both antigens, surface antigen of Plasmodium falciparum. Nature (London)
the MSA allele expressed by the challenge strain (IC1) 323:259-262.
differed from that used to immunize the monkeys (FC27). 2. Coppel, R. L., A. E. Bianco, J. G. Culvenor, P. E. Crewther,
While it would have been desirable to immunize the mon- G. V. Brown, R. F. Anders, and D. J. Kemp. 1987. Identification
keys with the MSA allele present in the challenge parasite of a cDNA clone expressing a rhoptry protein of Plasmodium
strain, constructs containing these alleles were not available. falciparum. Mol. Biochem. Parasitol. 25:73-81.
3. Coppel, R. L., A. F. Cowman, R. F. Anders, A. E. Bianco, R. B.
In the case of MSA-2, only one allele (21) was evident at the Saint, K. R. Lingelbach, D. J. Kemp, and G. V. Brown. 1984.
time of immunization of the monkeys. The subsequent Immune sera recognize on erythrocytes a Plasmodium falci-
discovery of different alleles (22) came too late to permit parum antigen composed of repeated amino acid sequences.
their inclusion in this experiment but did allow their involve- Nature (London) 310:789-791.
ment in the serological studies. 4. Favaloro, J. M., R. L. Coppel, L. M. Corcoran, S. J. Foote,
Unlike the constructs expressing RESA, MSA-2, or G. V. Brown, R. F. Anders, and D. J. Kemp. 1986. Structure of
AMA-1, the construct expressing MSA-1 failed to prime for the RESA gene of Plasmodium falciparum. Nucleic Acids Res.
an enhanced response. Since the level of expression of 14:8265-8277.
malaria antigen by that construct had been demonstrated to 5. Kabilan, L., M. Troye-Blomberg, H. Perlmann, G. Anderson, B.
Hogh, E. Petersen, A. Bjorkman, and P. Perlmann. 1988. T-cell
be low compared with the other constructs (unpublished epitopes in P155/RESA, a major candidate for a Plasmodium
data), it is likely that immunized animals received a dose falciparum malaria vaccine. Proc. Natl. Acad. Sci. USA 85:
insufficient for sensitization. 5659-5663.
As was observed in the case of immunization with the 6. Langford, C. J., S. J. Edwards, G. L. Smith, G. F. Mitchell, B.
VRESA construct, immunization with recombinant vaccinia Moss, D. J. Kemp, and R. F. Anders. 1986. Anchoring a secreted
virus expressing MSA-2 (VMSA2), although failing to induce plasmodium antigen on the surface of recombinant vaccinia
a good antibody response, primed for an enhanced antibody virus-infected cells increases its immunogenicity. Mol. Cell.
response following challenge. In addition to the enhanced Biol. 6:3191-3199.
response, prior immunization with VMSA2 appeared to alter 7. Langford, C. J., D. J. Kemp, R. F. Anders, G. F. Mitchell, S. J.
Edwards, G. L. Smith, and B. Moss. 1986. Antigens of the
the specificity of the MSA-2 response following challenge asexual blood stages of Plasmodium falciparum and their
infection in that antibodies in the immunized group were not expression in recombinant vaccinia virus, p. 145-148. In F.
as specific for the homologous MSA-2 as those induced by Brown, R. M. Chanock, and R. A. Lerner (ed.), Vaccines
infection alone. 86-new approaches to immunization. Cold Spring Harbor
Structural studies on MSA-2 genes have shown that they Laboratory, Cold Spring Harbor, N.Y.
fall into two major allelic families (22). The FC27 and IC1 8. Langford, C. J., D. B. Smith, S. J. Edwards, L. Keam, L. M.
genes are representatives of the two different family types of Corcoran, G. Peterson, P. McIntyre, D. Pye, D. J. Kemp, and
MSA-2, and it is therefore of considerable importance that R. F. Anders. 1988. "Cocktail" vaccines against falciparum
immunization with the FC27 molecule primes for an en- malaria, p. 89-94. In R. M. Chanock, R. A. Lerner, F. Brown,
and H. Ginsberg (ed.), Vaccines 88-modem approaches to
hanced immune response when animals are infected with vaccines including the prevention of AIDS. Cold Spring Harbor
IC1. This indicates that the epitopes seen by helper T cells Laboratory, Cold Spring Harbor, N.Y.
are common to the two different forms of MSA-2 and 9. Lew, A. M., C. J. Langford, D. Pye, S. J. Edwards, L. M.
suggests that the effects of a vaccine containing one or a Corcoran, and R. F. Anders. 1989. Class II restriction in mice to
small number of MSA-2 molecules would be boosted by the malaria candidate vaccine antigen, RESA: as synthetic
natural exposure to P. falciparum infections. peptides or as expressed in recombinant vaccinia. J. Immunol.
Recombinant VAMAl was included in this trial because of 142:4012-4016.
the evidence that molecules located in the rhoptries induce 10. Mackett, M., G. L. Smith, and B. Moss. 1984. General method
active antiparasitic immune responses (16). As observed for production and selection of infectious vaccinia virus recom-
binants expressing foreign genes. J. Virol. 49:857-864.
with the other antigens, this antigen failed to protect and 11. Moss, B., G. L. Smith, J. L. Gerin, and R. H. Purcell. 1984. Live
significant antibody responses were seen only after chal- recombinant vaccinia virus protects chimpanzees against hepa-
lenge. titis B. Nature (London) 311:67-69.
In a previous trial, partial protection of Aotus sp. monkeys 12. Olmsted, R. A., R. M. L. Buller, P. L. Collins, W. T. London,VOL. 59, 1991 VACCINATION OF MONKEYS AGAINST MALARIA 2411
J. A. Beeler, G. A. Prince, R. M. Chanock, and B. R. Murphy. 17. Peterson, M. G., R. L. Coppel, P. McIntyre, C. J. Langford, G.
1988. Evaluation in non-human primates of the safety, immuno- Woodrow, G. V. Brown, R. F. Anders, and D. J. Kemp. 1988.
genicity and efficacy of recombinant vaccinia viruses expressing Variations in the precursor to the major merozoite surface
the F or G glycoprotein of respiratory syncytial virus. Vaccine antigens of Plasmodium falciparum. Mol. Biochem. Parasitol.
6:519-524. 27:291-302.
13. Panicali, D., S. W. Davis, R. L. Weinberg, and E. Paoletti. 1983. 18. Peterson, M. G., V. M. Marshall, J. A. Smythe, P. E. Crewther,
Construction of live vaccines using genetically engineered pox- A. Lew, A. Sylva, R. F. Anders, and D. J. Kemp. 1989. Integral
viruses: biological activity of vaccinia virus recombinants ex- membrane protein located in the apical complex of Plasmodium
pressing influenza virus hemagglutinin. Proc. Natl. Acad. Sci. falciparum. Mol. Cell. Biol. 9:3584-3587.
USA 80:5364-5368. 19. Smith, D. B., and K. S. Johnson. 1988. Single step purification of
14. Paoletti, E., B. R. Lipinskas, C. Samsonoff, S. Mercer, and D. polypeptides expressed in Escherichia coli as fusions with
Panicali. 1983. Construction of live vaccines using genetically glutathione S-transferase. Gene 67:31-40.
engineered poxviruses: biological activity of vaccinia virus 20. Smith, G. L., B. R. Murphy, and B. Moss. 1983. Construction
recombinants expressing the hepatitis B virus surface antigen and characterization of an infectious vaccinia virus recombinant
and the herpes simplex virus glycoprotein D. Proc. Natl. Acad. that expresses the influenza virus hemagglutinin gene and in-
Sci. USA 81:193-197. duces resistance to influenza infection in hamsters. Proc. Natl.
15. Perlmann, H., K. Berzins, M. Wahlgren, J. Carlsson, A. Bjork-
Downloaded from http://iai.asm.org/ on February 28, 2021 by guest
Acad. Sci. USA 80:7155-7159.
man, M. E. Patarroyo, and P. Perlmann. 1984. Antibodies in 21. Smythe, J. A., R. L. Coppel, G. V. Brown, R. Ramasamy, D. J.
malarial sera to parasite antigens in the membrane of erythro- Kemp, and R. F. Anders. 1988. Identification of two integral
cytes infected with early asexual stages of Plasmodium falci- membrane proteins of Plasmodium falciparum. Proc. Natl.
parum. J. Exp. Med. 159:1686-1704. Acad. Sci. USA 85:5195-5199.
16. Perrin, L. H., B. Merkli, M. S. Gabra, J. W. Stocker, C. 22. Smythe, J. A., M. G. Peterson, R. L. Coppel, A. J. Saul, D. J.
Chizolini, and R. Richle. 1985. Immunisation with a Plasmodium Kemp, and R. F. Anders. 1990. Structural diversity in the
falciparum merozoite surface antigen induces a partial immu- 45-kilodalton merozoite surface antigen of Plasmodium falci-
nity in monkeys. J. Clin. Invest. 75:1718-1721. parum. Mol. Biochem. Parasitol. 39:227-234.You can also read
