Failure of Recombinant Vaccinia Viruses Expressing Plasmodium ...

Vol. 59, No. 7

INFECTION AND IMMUNITY, JUlY 1991, p. 2403-2411 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

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 might be suitable as components of a malaria vaccine have been identified. While biological studies have suggested the suitability of these protein antigens as vaccine components, ultimately their usefulness can be determined only by protection studies in vivo, first in Aotus sp. or Saimiri sp. monkeys and then in human volunteers. A malaria vaccine requires development of delivery methods suitable for developing countries. Vaccines must be easily stored, transported, and delivered. Because of the good record of smallpox (vaccinia virus) vaccines in the third world, the use of recombinant vaccinia viruses should overcome these basic problems, while potentially offering other advantages, such as broad and effective immune responses. In addition, the use of recombinant vaccinia virus could overcome many of the problems of antigen expression and delivery associated with the preparation of recombinant proteins in bacterium or yeast systems. Recombinant vac*

cinia viruses expressing various viral antigens have been demonstrated to induce good antibody responses in immunized animals and, more importantly, to protect such animals from subsequent challenge infection with the viruses from which the antigens were derived (11, 13, 14, 20). Successful demonstration of protection against malaria after recombinant vaccinia virus immunization would not only contribute to acceptance of vaccinia virus as a delivery system for use in humans but would also confirm the suitability of vaccinia virus as a delivery system for screening of malaria antigens in monkeys. A malaria antigen, ring-infected erythrocyte surface antigen (RESA) (3, 15), has been shown to provide partial protection in an Aotus sp. monkey trial (1), and antibody responses to epitopes encoded by sequence repeats in the RESA gene were correlated with protection. Therefore, RESA was a good antigen with which to evaluate delivery 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 virus constructs containing several other antigens were also included in this study. Unfortunately, protection was not demonstrated in this trial, but useful data in relation to immune responses obtained after immunization with recombinant vaccinia viruses were obtained. These results are reported here.

Corresponding author.

t Present address: Whitehead Institute for Biomedical Research, Cambridge, MA 02142. t Present address: Department of Molecular and Radiation Biology, Peter MacCallum Institute, Melbourne, Victoria 3001, Australia.

§ Present address: Tjian Laboratory, Howard Hughes Medical Institute, Department of Biochemistry, University of California, Berkeley, CA 94720. 11 Present address: Laboratory of Tumor Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD

MATERIALS AND METHODS

Monkeys. Adult female squirrel monkeys (Saimiri sciurus) of Guyanan origin were obtained from Charles River Research Primates Corp. All had intact spleens, had weights of

20892. # Present address: Veterinary Institute of Animal Science, Attwood, Victoria 3049, Australia. 2403

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PYE ET AL.

TABLE 1. Immunization Group

Immunogen(s)

No.

of

monkeys

A None B TK- vaccinia virus C VRESA

6

D V5, V33 (RESA repeats)

6

5 5

TABLE 2. Antigens used in ELISA studies

groups

Comments

Vaccinia ......... Unvaccinated controls Vaccinated controls Eight dose sites; six animals immunized, with one death due to accident prior to challenge infection Four dose sites per virus

E

VRESA, VMSA1, VAMA1, VMSA2

6

Two dose sites virus

per

more than 600 g, and were preconditioned for several months prior to use. They were free of tuberculosis infection, not pregnant, and declared after thorough physical examination to be in general good health. Immunofluorescence testing for antibodies that cross-reacted with P. falciparum antigens failed to detect significant levels of such antibodies. Construction of recombinant vaccinia viruses. DNA fragments encoding various malaria antigens and hybrid polypeptides from P. falciparum FC27 were inserted into the multiple cloning site of vaccinia virus transfection plasmid pGS62 prior to transfection into the WR strain of vaccinia virus (10). Recombinant VRESA contained a DNA fragment encoding the entire RESA antigen (4), derived from parasite strain FC27. Recombinant V5 contained a hybrid DNA fragment encoding the FC27 S antigen of P. falciparum which had been modified by addition of sequences encoding the hydrophobic transmembrane region and intracellular domain of the membrane-bound form of the mouse immunoglobulin G, protein (6) and deletion of the 12-amino-acid tandem repeat epitopes of the S antigen, which were replaced with sequences encoding six copies of the RESA undecamer repeat DDEH VEEPTVA. Recombinant V33 was identical to V5 except that the S-antigen repeat sequences were replaced with sequences encoding 20 copies of the RESA octamer repeat EENVEH DA. Both the recombinant vaccinia viruses V5 and V33 have been shown to be more immunogenic with respect to their RESA repeat epitope than the VRESA recombinant virus when used to immunize mice and rabbits (8). Recombinant VMSA1 contained a DNA fragment encoding the entire MSA-1 antigen (17) from parasite strain FC27. Recombinant VAMAl contained a DNA fragment encoding the entire AMA-1 antigen (2, 18) from parasite strain FC27. Recombinant VMSA2 contained a DNA fragment encoding the entire MSA-2 antigen (21) from parasite strain FC27. Recombinant vaccinia virus transfection plasmids were used to construct recombinant vaccinia viruses, with bromodeoxyuridine selection of thymidine kinase-deficient (TK-) plaques as described previously (7). TK- nonrecombinant vaccinia virus was produced by growing wild-type virus in the presence of bromodeoxyuridine and selecting an individual plaque at random. Immunization of monkeys. Monkeys were randomly allocated into five groups (Table 1). Each monkey was immunized at each of eight shaved sites on the back with a dose of

Description

Designation

P-Propiolactone-inactivated purified TK-

vaccinia virus RESA 8-mer ........ GST' fusion protein (19) containing 20 copies of the RESA 8-mer 3' repeat RESA 11-mer ....... GST fusion protein containing six copies of the RESA 11-mer 5' repeat RESA 4-mer ........ GST fusion protein containing 15 copies of the RESA 4-mer 3' repeat MSA-1 ........ Affinity purified from strain FC27 parasites GST fusion protein containing the SspI MSA-2 ........ fragment of MSA-2 isolated from either the FC27 or the IC1 strain of P. falciparum; fulllength antigen except for the terminal hydrophobic amino acid domains GST fusion protein containing the entire AMAAMA-1 ........ 1 antigen (FC27 origin) except for the hydrophobic anchor region P513 ........ Peptide (CSQRSTNSAST) representing a variable-region epitope of FC27 MSA-2 recognized by inhibitory monoclonal antibodies; conjugated to BSAb by using glutaraldehyde GGSA ........ Peptide repeat sequence from the variable region of IC1 MSA-2 synthesized as a fourtime tandem repeat and conjugated to BSA by using glutaraldehyde a

GST, glutathione S-transferase.

b BSA, bovine serum albumin.

107 PFU in 0.1 ml. After healing (4 weeks), animals were reimmunized with a dose of 108 PFU in 0.1 ml at each of four sites on the upper back and four sites on the lower abdomen. When animals received more than one virus strain, these were not mixed but a single strain was inoculated at each site. For monkeys immunized with two strains, each virus was inoculated at four sites, whereas monkeys immunized with four strains were inoculated with each virus at two sites. Parasite challenge. An S. sciurus-adapted P. falciparum strain, Indochina 1/CDC (IC1), was obtained from W. Collins (Centers for Disease Control, Atlanta, Ga.) and serially passaged in 10 further S. sciurus monkeys. When the parasitemia in the last of these monkeys first reached 3% (subsequent peak of 10%), a stabilate was prepared and stored in liquid nitrogen. For challenge, an individual vial was thawed, passaged in a splenectomized monkey, and serially passaged further in intact monkeys until the time of challenge. At that time, blood was removed from the donor monkey (3% parasitemia) and the erythrocytes were suspended in saline to 3 x 107 parasites per ml. Monkeys were inoculated intravenously with 1 ml of parasites, and daily smears were prepared for parasitemia determination. Serological studies. Antibody responses were measured after dilution of serum (at 1:1,000, 1:3,000, 1:10,000, and 1:30,000 dilutions) and involved previously described enzyme-linked immunosorbent assay (ELISA) techniques (1). Antibody levels were determined on bleeds taken prior to immunization, at the time of challenge, and 2 weeks after challenge to measure responses to immunization and challenge. The antigens used are described in Table 2. The responses to MSA-2 were measured against two different allelic forms, from the FC27 and IC1 parasite strains. The forms differ in that while the 43 N-terminal and 74 C-terminal

VACCINATION OF MONKEYS AGAINST MALARIA

VOL. 59, 1991

2405

O.D. at 1:30,000 dilution 3 _

2.5

M -E3

Prebleed 4 wks post 1st dose 2 wks post 2nd dose

2

1.5 _

I

0.5 _-

0

I

I. I JI LA

db , . 20 42 66 38 78

74 75 81 82 88

A

B

ti.

+. I --I

I]

1]

14 15 31 48 85 87

ii.

28 35 40 41 79 80

D

C

J

1

AA

03 29 47 77 83 88

E

Immunisation groups (monkey nos.) 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 are major differences between the alleles (22). In the FC27 strain, there are two copies of a 32-amino-acid repeat sequence. In contrast, the IC1 strain contains 12 copies of the four-amino-acid repeat sequence Gly-Gly-Ser-Ala (GGSA). The sequences flanking the repeats also differ between the alleles. RESULTS Responses to immunization. Primary immunization with vaccinia virus produced lesions in all immunized monkeys. Lesions appeared within 5 days as red, swollen areas approximately 8 mm in diameter which developed into crusty scabs during the next few days. Within 12 days of vaccination, these scabs were lifting to reveal healed tissue beneath. Healing was complete within 19 days. No secondary lesions developed, and no difference in lesions was observed between animals immunized with recombinant vaccinia virus and those immunized with nonrecombinant vaccinia virus. Lesions developed after secondary immunizations but were smaller and resolved quickly (10 to 11 days). All vaccinated monkeys produced antibody to vaccinia virus, with boosted responses after the second immunization (Fig. 1). Responses to challenge with P. falciparum. After challenge with the IC1 strain of P. falciparum, all monkeys developed

patent infections which subsequently cleared without drug treatment. In general, parasitemias were detected within 2 days of inoculation and reached maximum levels at around 7 or 9 days. Parasites grew synchronously and presumably cytoadhered, as evidenced by the 48-h interval between peaks of parasitemia. Only ring forms were seen in the peripheral blood until a day or two prior to the fall in parasite levels. At that time, mature forms (late trophozoites and schizonts) became evident, as did crisis forms. The duration of detectable parasitemia was generally around 2 weeks. Within each group, there was considerable variation in peak parasitemias (Table 3), with a maximum of 6% and a minimum of 0.2%. No significant differences were found between groups in the maximum parasitemia or the time taken to reach maximum parasitemia. Thus, by using these criteria, we found no evidence that any of the P. falciparum

antigens expressed in recombinant vaccinia virus could protect S. sciurus monkeys against challenge with P. falciparum.

Antibody responses to RESA epitopes. Antibody responses to the three major RESA repeat epitopes were determined by using sera collected prior to the primary immunization, at the times of the second immunization and the challenge, and 2 weeks after the challenge (Fig. 2). Of the 18 animals that received recombinant vaccinia virus containing the RESA 11-mer repeat, none produced detectable responses to immunization (Fig. 2a). However, a num-

PYE ET AL.

2406

a.

INFECT. IMMUN.

RESA llmer

TABLE 3. Responses to challenge parasite infection Mon-

key

Peak parasitemia (%

no.

erythrocytes)

A, unvaccinated controls

20 42 66 38 78

1.25 0.55 0.15 3.31 0.17

parasitemia (days) 16 7 7 13 13

B, vaccinated controls (TK- vaccinia virus)

74 75 81 82 86

2.9 2.53 1.06 0.61 2.0

15 15 15 14 19

C, VRESA

14 15 31 48 85 87

2.16 4.5 6.75 2.0 0.6 3.0

13 15 15 14 12 10

D, V5-V33

26 35 40 41 79 80

1.46 6.0 0.9 2.1 1.6 0.27

14 14 15 10 14 8

E, VRESA-VMSA1VMSA2-VAMA

3 29 47 77 83 88

1.6 1.6a 1.1 0.5 1.85 2.16

15

O.D. at 113000 dilution

so 42 186 4T.5 1 .66

A

a

Group

14 1. 614 66667

lmmunlbatlom

C

16"4641 700 D

groups (monkey no.)

0616477768o" E

b. RESA 8mer

Duration of

12 11 14 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

A

*

C

0

lmmunlmfion groups (monkey nos.)

06647 776

E

c. RESA 4mer

1041666T6 A

74766161S6

14 If 146667 666440417660 C D B lmmumlatlon group. (monley no.)

061647776866 E

FIG. 2. Responses to RESA. Sera prepared from bleeds taken prior to immunization (prebleed), after 2 immunizing doses (postimmunization), and 2 weeks postchallenge were diluted 1:3,000 and assayed by ELISA against the RESA 5' 11-mer repeat (a), the RESA 3' 8-mer repeat (b), and the RESA 3' 4-mer repeat (c). Each cluster of bars represents the optical densities (O.D.) after ELISA of the

ber of these monkeys had detectable responses after challenge. Particularly enhanced responses after challenge were 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 all six animals that received VRESA alone but in only one of

three sera obtained from an individual animal. The immunization groups were unvaccinated controls (A), vaccinated controls (B), VRESA (C), V5-V33 (D), and VRESA-VMSA1-VAMA1-VMSA2

(E).

VACCINATION OF MONKEYS AGAINST MALARIA

VOL. 59, 1991

FC27 MSA2

a.

Peptide 513

a.

O.D. at 1:3000 dllutlon.

O.D. at 1:3000 dilution

PrObl sod M post Immunleatton EJ poet 1Challenge _

2.0

2407

Prebleed

_

M post Immunlletlon 2

_CE

poet Challenge

2-

1.6 _ I

0.6 _ 0.6 _ n

Hj 20

-n

42 663 6

A

14

163 1 46 667

0

22

47

77

W., &A Jl

20 426

36

Immunisation groups (monkey nos.)

b. IC1 MSA2

14 16

n

31 486

C Immunisation groups

A

E

c

.-n -wi

36 7

_rl

67

tfl

-

tfl fl J--ni

03 29 4

(monkey nos.)

77

66

E

b. GGSA O.D. at 1:1000 dilution. 3

Prebleed poet Immunheatlon post Challenge

1

2.6

2

I1.6

3.1

c

20

42

66

A

36

78

14

16

31

48

66

67

03

C Immunisation groups (monkey nos.)

29

47

77

63 66

E

6367

6 87 14 16 31 4 03 20 47 77 63 86 C E Immunisation groups (monkey nos.) FIG. 4. Responses to MSA-2 variable-region repeats. Sera prepared from bleeds taken prior to immunization (prebleed), after 2

20

42

A

FIG. 3. Responses to the FC27 and IC1 forms of MSA-2. Sera prepared from bleeds taken prior to immunization (prebleed), after 2 immunizing doses (postimmunization), and 2 weeks postchallenge were diluted and assayed by ELISA against recombinant FC27 strain MSA-2 protein (1:3,000 dilution; a) and recombinant IC1 strain MSA-2 protein (1:3,000 dilution, b). Each cluster of bars represents the optical densities (O.D.) after ELISA of the three sera obtained from an individual animal. The immunization groups were unvaccinated controls (A), VRESA (C), and VRESA-VMSA1VAMA1-VMSA2 (E).

immunizing doses (postimmunization), and 2 weeks postchallenge diluted and assayed by ELISA against FC27 strain variableregion repeat epitope P513 (1:3,000 dilution; a) and IC1 strain variable-region repeat epitope GGSA (1:1,000 dilution; b). Each cluster of bars represents the optical densities (O.D.) after ELISA of the three sera obtained from an individual animal. The immunization groups were unvaccinated controls (A), VRESA (C), and VRESAVMSA1-VAMA1-VMSA2 (E).

the six animals that received VRESA together with other recombinants. In general, more monkeys produced strong responses to the 8-mer and 4-mer repeats than to the 11-mer repeat. A small number of monkeys that responded well to the 8-mer repeat after challenge infection failed to respond to the l1-mer repeat (e.g., monkeys 31 and 82). Antibody responses to MSA-2. Monkeys were assayed for responses to the FC27 and IC1 forms of MSA-2, a variableregion epitope (P513) found in the FC27 strain of P. falciparum involved in immunization but not in the IC1 strain used for challenge infection, and a variable-region epitope (GGSA) found in the IC1 strain but not in the FC27 strain.

Responses were determined in three of the five monkey (A, unvaccinated controls; C, VRESA alone; and E, VMSA2, VMSA1, VRESA, and VAMA1). Immunization with VMSA2 (FC27 MSA-2) produced weak responses to both the FC27 and IC1 forms of MSA-2 (Fig. 3). After challenge (IC1 MSA-2), responses to both the FC27 and IC1 forms of MSA-2 were detected in all monkeys (Fig. 3). Unvaccinated monkeys and those that received VRESA mounted antibody responses which preferentially reacted with the homologous IC1 form of MSA-2. Several of the monkeys which received VMSA2 made antibody responses that reacted almost as well with FC27 MSA-2 as with the homologous IC1 MSA-2. Thus, immunization with the one

were

groups

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PYE ET AL.

INFECT. IMMUN.

O.D. at 1:10,000 dilution 2.5

2

1.5

0.5

0

20

42

66 A

38 78

14

15

31

48 85 87

C Immunisation groups (monkey nos.)

03

29

47

77

83

88

E

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 primed animals for enhanced responses to both forms of MSA-2 following infection. Responses to the epitope from the FC27 variable region were not detected after immunization (Fig. 4a), although after challenge with the IC1 strain, some animals showed evidence of weak responses to P513. A few animals produced detectable responses to the IC1 variable-region repeat epitope GGSA after challenge infection (Fig. 4b), with no evidence that the GGSA response was enhanced because of prior immunization with VMSA2. Antibody responses to AMA-1. Monkeys that received recombinant vaccinia virus expressing AMA-1 (VAMA1) failed to mount significant responses to AMA-1 after immunization (Fig. 5). However, all of the monkeys tested (groups A, C, and E) produced good antibody responses after challenge infection (detected at a 1:10,000 or greater dilution), and those immunized with VAMAl were primed for an enhanced response. Antibody responses to MSA-1. Only a small amount of affinity-purified MSA-1 was available, and therefore the analysis of antibody responses to MSA-1 was limited. However, the results (Fig. 6) indicated that immunization failed to produce a detectable response in the six animals that received the relevant vaccinia virus recombinant. All of the monkeys tested produced good responses after challenge infection, which were detectable at a 1:10,000 dilution, but

there was no evidence of priming for an enhanced response in monkeys immunized with the VMSA1 recombinant. DISCUSSION The purpose of this study was to determine whether immunization with recombinant vaccinia viruses expressing selected malaria antigens could protect S. sciurus monkeys against challenge with blood stages of P. falciparum. RESA was the major antigen under study, but monkeys were also immunized with recombinant vaccinia virus expressing merozoite surface antigens MSA-1 and MSA-2 and rhoptry protein AMA-1. Although immunization resulted in high vaccinia virus antibody responses in all of the animals, there was no evidence of protection induced by any of the malaria antigens delivered in this way. Vaccinia virus infections were similar in all of the monkeys, and relatively small lesions were produced. The infection took a mild course, with no indication of clinical illness like that frequently seen in humans who receive smallpox vaccine. While the mildness of the infections may have been due to the reduced virulence of the recombinant viruses, it is also likely that S. sciurus monkeys are less susceptible than humans to vaccinia virus infection. In a comparative study of responses to recombinant vaccinia viruses in Saimiri sp., Aotus sp., Macaca sp., Cercopithecus sp., and Erythrocebus sp. monkeys (12), Saimiri sp. monkeys produced the

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O.D. at 1:1000 dilution 3

2.5

2

1.5

1

0.5

0

20

42

66

38

78

74

81

86

75

82

03

47

77

83

88

E

B

A

29

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) and the lowest resulting antibody responses. (Alternatively, a cellular immune response of greater potency might also result in smaller lesions.) However, in our study, the high vaccinia virus antibody responses induced by the primary immunizations and the boosting of these after the secondary immunization indicated good virus replication and, therefore, potentially good expression of inserted antigens. After the challenge infection with P. falciparum, the resulting parasitemias achieved peaks between 0.15 and 6.75%, with the unvaccinated control group showing the greatest range of parasitemias seen in any group (0.15 to 3.3%) and the lowest median peak (0.55%). Therefore, protection could not be demonstrated in any test group, and the model was less than ideal, since protection would be detected only if a majority of monkeys within a group showed nearly complete absence of parasites (nearly sterile immunity). While these results cast some doubt on the value of the S. sciurus protection model, more recent studies by us (unpublished data) have demonstrated that nearly sterile immunity can be obtained as a consequence of immunization. Thus, factors other than suitability of the monkey model should be considered among the reasons for failure to demonstrate protection in this trial. In contrast to the high induced antibody responses to vaccinia virus, immunization with the recombinant viruses

failed to induce significant antibody responses to the various parasite antigens. This contrasted with previous studies with rabbits and mice in which the RESA constructs given singly had stimulated satisfactory antibody responses (7-9). In addition, Lew and coworkers (9) had demonstrated proliferative T-cell responses in lymphocytes taken from mice previously immunized with the RESA constructs. Three different groups of monkeys were immunized with vaccinia virus constructs expressing RESA either as the whole antigen alone or in combination with other antigens or as fragments containing repetitive epitopes previously implicated in protective immune responses (1). Despite the poor antibody responses to RESA induced by immunization, all of the monkeys that were immunized with VRESA alone and one of the monkeys that received VRESA in combination with other antigens were primed for a markedly enhanced antibody to RESA following challenge. The failure of immunization with V5 and V33 to prime in an equivalent way to VRESA suggests that the RESA 8-mer and 11-mer repeats expressed by these two recombinant viruses lack the T-cell epitopes relevant to secondary antibody responses to RESA. This contrasts with other studies which indicated that the three different repeats of RESA all contain T-cell epitopes (9) and that when various strains of mice were immunized with VRESA, T-cell epitopes encoded by the repeats were involved in the response. The four-

2410

PYE ET AL.

amino-acid repeats in RESA have been shown to encode T-cell epitopes recognized by infected humans (5). We did not immunize monkeys with a recombinant vaccinia virus expressing these repeats alone, and it is possible that the priming effect of full-length RESA involved recognition of T-cell epitopes encoded by the 4-mer repeats. However, the uniformly good responses in all of these outbred animals suggest that other T-cell epitopes are involved. Alternatively, the failure of VS and V33 to prime for an enhanced response may have been caused by insufficient antigen to sensitize immunized animals, but this is unlikely, since antigen production in vitro by these constructs is at least as good as with VRESA (unpublished data) and their ability to induce immune responses in rabbits and mice is at least equivalent (8, 9). Merozoite surface antigens MSA-1 and MSA-2 are of particular interest as potential components of a malaria vaccine. The failure to achieve protection with recombinant vaccinia virus expressing these antigens should not lessen interest in these antigens because immunization failed to induce significant antibody responses and, for both antigens, the MSA allele expressed by the challenge strain (IC1) differed from that used to immunize the monkeys (FC27). While it would have been desirable to immunize the monkeys with the MSA allele present in the challenge parasite strain, constructs containing these alleles were not available. In the case of MSA-2, only one allele (21) was evident at the time of immunization of the monkeys. The subsequent discovery of different alleles (22) came too late to permit their inclusion in this experiment but did allow their involvement in the serological studies. Unlike the constructs expressing RESA, MSA-2, or AMA-1, the construct expressing MSA-1 failed to prime for an enhanced response. Since the level of expression of malaria antigen by that construct had been demonstrated to be low compared with the other constructs (unpublished data), it is likely that immunized animals received a dose insufficient for sensitization. As was observed in the case of immunization with the VRESA construct, immunization with recombinant vaccinia virus expressing MSA-2 (VMSA2), although failing to induce a good antibody response, primed for an enhanced antibody response following challenge. In addition to the enhanced response, prior immunization with VMSA2 appeared to alter the specificity of the MSA-2 response following challenge infection in that antibodies in the immunized group were not as specific for the homologous MSA-2 as those induced by infection alone. Structural studies on MSA-2 genes have shown that they fall into two major allelic families (22). The FC27 and IC1 genes are representatives of the two different family types of MSA-2, and it is therefore of considerable importance that immunization with the FC27 molecule primes for an enhanced immune response when animals are infected with IC1. This indicates that the epitopes seen by helper T cells are common to the two different forms of MSA-2 and suggests that the effects of a vaccine containing one or a small number of MSA-2 molecules would be boosted by natural exposure to P. falciparum infections. Recombinant VAMAl was included in this trial because of the evidence that molecules located in the rhoptries induce active antiparasitic immune responses (16). As observed with the other antigens, this antigen failed to protect and significant antibody responses were seen only after challenge. In a previous trial, partial protection of Aotus sp. monkeys

INFECT. IMMUN.

was obtained with RESA fusion proteins (1). These monkeys had high antibody titers induced by immunization at the time of challenge. If the immunogenicity of vaccinia virus recombinants could be improved so that similarly high antibody responses were induced, then such recombinants could be expected to provide protective immunity. ACKNOWLEDGMENTS We thank Bill Collins for advice and for supplying the IC1 parasite strain. We thank David Irving and Graeme Jones for supplying some of the ELISA antigens. This work was supported by the Australian Malaria Vaccine Joint Venture (Saramane Pty. Ltd.). REFERENCES 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. Andrysiak, J. M. Favaloro, L. M. Corcoran, J. R. Broderson, G. F. Mitchell, and C. C. Campbell. 1986. Immunization of Aotus monkeys with recombinant proteins of an erythrocyte surface antigen of Plasmodium falciparum. Nature (London) 323:259-262. 2. Coppel, R. L., A. E. Bianco, J. G. Culvenor, P. E. Crewther, G. V. Brown, R. F. Anders, and D. J. Kemp. 1987. Identification of a cDNA clone expressing a rhoptry protein of Plasmodium falciparum. Mol. Biochem. Parasitol. 25:73-81. 3. Coppel, R. L., A. F. Cowman, R. F. Anders, A. E. Bianco, R. B. Saint, K. R. Lingelbach, D. J. Kemp, and G. V. Brown. 1984. Immune sera recognize on erythrocytes a Plasmodium falciparum antigen composed of repeated amino acid sequences. Nature (London) 310:789-791. 4. Favaloro, J. M., R. L. Coppel, L. M. Corcoran, S. J. Foote, G. V. Brown, R. F. Anders, and D. J. Kemp. 1986. Structure of the RESA gene of Plasmodium falciparum. Nucleic Acids Res.

14:8265-8277. 5. Kabilan, L., M. Troye-Blomberg, H. Perlmann, G. Anderson, B. Hogh, E. Petersen, A. Bjorkman, and P. Perlmann. 1988. T-cell epitopes in P155/RESA, a major candidate for a Plasmodium falciparum malaria vaccine. Proc. Natl. Acad. Sci. USA 85: 5659-5663. 6. Langford, C. J., S. J. Edwards, G. L. Smith, G. F. Mitchell, B. Moss, D. J. Kemp, and R. F. Anders. 1986. Anchoring a secreted plasmodium antigen on the surface of recombinant vaccinia virus-infected cells increases its immunogenicity. Mol. Cell. Biol. 6:3191-3199. 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 asexual blood stages of Plasmodium falciparum and their expression in recombinant vaccinia virus, p. 145-148. In F. Brown, R. M. Chanock, and R. A. Lerner (ed.), Vaccines 86-new approaches to immunization. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 8. Langford, C. J., D. B. Smith, S. J. Edwards, L. Keam, L. M. Corcoran, G. Peterson, P. McIntyre, D. Pye, D. J. Kemp, and R. F. Anders. 1988. "Cocktail" vaccines against falciparum malaria, p. 89-94. In R. M. Chanock, R. A. Lerner, F. Brown, and H. Ginsberg (ed.), Vaccines 88-modem approaches to vaccines including the prevention of AIDS. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 9. Lew, A. M., C. J. Langford, D. Pye, S. J. Edwards, L. M. Corcoran, and R. F. Anders. 1989. Class II restriction in mice to the malaria candidate vaccine antigen, RESA: as synthetic peptides or as expressed in recombinant vaccinia. J. Immunol.

142:4012-4016. 10. Mackett, M., G. L. Smith, and B. Moss. 1984. General method for production and selection of infectious vaccinia virus recombinants expressing foreign genes. J. Virol. 49:857-864. 11. Moss, B., G. L. Smith, J. L. Gerin, and R. H. Purcell. 1984. Live recombinant vaccinia virus protects chimpanzees against hepatitis B. Nature (London) 311:67-69. 12. Olmsted, R. A., R. M. L. Buller, P. L. Collins, W. T. London,

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J. A. Beeler, G. A. Prince, R. M. Chanock, and B. R. Murphy. 1988. Evaluation in non-human primates of the safety, immunogenicity and efficacy of recombinant vaccinia viruses expressing the F or G glycoprotein of respiratory syncytial virus. Vaccine 6:519-524. Panicali, D., S. W. Davis, R. L. Weinberg, and E. Paoletti. 1983. Construction of live vaccines using genetically engineered poxviruses: biological activity of vaccinia virus recombinants expressing influenza virus hemagglutinin. Proc. Natl. Acad. Sci. USA 80:5364-5368. Paoletti, E., B. R. Lipinskas, C. Samsonoff, S. Mercer, and D. Panicali. 1983. Construction of live vaccines using genetically engineered poxviruses: biological activity of vaccinia virus recombinants expressing the hepatitis B virus surface antigen and the herpes simplex virus glycoprotein D. Proc. Natl. Acad. Sci. USA 81:193-197. Perlmann, H., K. Berzins, M. Wahlgren, J. Carlsson, A. Bjorkman, M. E. Patarroyo, and P. Perlmann. 1984. Antibodies in malarial sera to parasite antigens in the membrane of erythrocytes infected with early asexual stages of Plasmodium falciparum. J. Exp. Med. 159:1686-1704. Perrin, L. H., B. Merkli, M. S. Gabra, J. W. Stocker, C. Chizolini, and R. Richle. 1985. Immunisation with a Plasmodium falciparum merozoite surface antigen induces a partial immunity in monkeys. J. Clin. Invest. 75:1718-1721.

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17. Peterson, M. G., R. L. Coppel, P. McIntyre, C. J. Langford, G. Woodrow, G. V. Brown, R. F. Anders, and D. J. Kemp. 1988. Variations in the precursor to the major merozoite surface antigens of Plasmodium falciparum. Mol. Biochem. Parasitol. 27:291-302. 18. Peterson, M. G., V. M. Marshall, J. A. Smythe, P. E. Crewther, A. Lew, A. Sylva, R. F. Anders, and D. J. Kemp. 1989. Integral membrane protein located in the apical complex of Plasmodium falciparum. Mol. Cell. Biol. 9:3584-3587. 19. Smith, D. B., and K. S. Johnson. 1988. Single step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 67:31-40. 20. Smith, G. L., B. R. Murphy, and B. Moss. 1983. Construction and characterization of an infectious vaccinia virus recombinant that expresses the influenza virus hemagglutinin gene and induces resistance to influenza infection in hamsters. Proc. Natl. Acad. Sci. USA 80:7155-7159. 21. Smythe, J. A., R. L. Coppel, G. V. Brown, R. Ramasamy, D. J. Kemp, and R. F. Anders. 1988. Identification of two integral membrane proteins of Plasmodium falciparum. Proc. Natl. Acad. Sci. USA 85:5195-5199. 22. Smythe, J. A., M. G. Peterson, R. L. Coppel, A. J. Saul, D. J. Kemp, and R. F. Anders. 1990. Structural diversity in the 45-kilodalton merozoite surface antigen of Plasmodium falciparum. Mol. Biochem. Parasitol. 39:227-234.