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Am. J. Trop. Med. Hyg., 74(3), 2006, pp. 422-424
Copyright © 2006 by The American Society of Tropical Medicine and Hygiene

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AOTUS NANCYMAAE AS A POTENTIAL MODEL FOR THE TESTING OF ANTI-SPOROZOITE AND LIVER STAGE VACCINES AGAINST PLASMODIUM FALCIPARUM

WILLIAM E. COLLINS*, JOANN S. SULLIVAN, ALLISON WILLIAMS, DOUGLAS NACE, TYRONE WILLIAMS, G. GALE GALLAND, AND JOHN W. BARNWELL
Division of Parasitic Diseases and Animal Resources Branch, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia; Atlanta Research and Education Foundation, Atlanta, Georgia


ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The Santa Lucia strain of Plasmodium falciparum was transmitted to Aotus lemurinus griseimembra, A. azarae boliviensis, A. vociferans, and A. nancymaae monkeys by bite and by intravenous inoculation of sporozoites dissected from Anopheles freeborni, An. stephensi, An. gambiae, An. albimanus, and An. maculatus mosquitoes. The data obtained from these infections indicate that A. nancymaae can be considered a suitable host model when combined with the Santa Lucia strain of P. falciparum for the testing of candidate anti-sporozoite and liver stage vaccines.


INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
One of the challenges of developing primate models for the testing of candidate malarial vaccines has been to develop one for sporozoite-induced Plasmodium falciparum malaria. Previously, we developed a reproducible model for Plasmodium vivax that combined the Salvador I strain of the parasite and Saimiri boliviensis boliviensis monkeys.1,2 This combination has been used successfully in several different trials to test candidate vaccines against P. vivax.3,4 Attempts are now being made to develop a similar model for P. falciparum.

Based on our experience, splenectomy appears to a critical function for high-density development of the erythrocytic stage after sporozoite challenge. However, with P. vivax, it was not necessary to splenectomize the animal prior to the challenge, nor prior to the completion of the exoerythrocytic development of the liver stage. Thus, immunization studies and assessments of anti-sporozoite and liver stage vaccines were not compromized by splenectomy in the P. vivax/S. boliviensis system. The splenectomy procedure was done at the end of the liver stage developmental cycle (6–7 days after sporozoite challenge).

Attempts to infect Saimiri boliviensis monkeys with sporozoites of P. falciparum have been discouraging, whereas different species of Aotus monkeys have been the standard hosts for these parasites for many years. Thus, an attempt was made to determine if the currently available Aotus species could be useful for the testing of anti-sporozoite and liver stage vaccines. Reported here are the results of our attempts to transmit the Santa Lucia strain of P. falciparum to different species of Aotus monkeys and the possible use of the most readily available species, A. nancymaae.


MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Aotus nancymaae and A. vociferans monkeys were wild-caught animals imported from Peru. The A. lemurinus griseimembra monkeys were imported from Colombia or laboratory-born animals. Similarly, A. azarae boliviensis were imported from Bolivia or were laboratory-born animals. Upon arrival at the facility, all animals were quarantined for a two-month conditioning period, weighed, and tested for tuberculosis. Parasitologic and serologic examination indicated that the animals were free of infection with malaria parasites before inoculation. All monkeys were splenectomized before exposure to infection. All surgeries were performed in an Association for the Assessment and Accreditation of Laboratory Animal Care, International, Inc. (Rockville, MD)–approved surgical suite appropriate for aseptic surgery. Protocols were reviewed and approved by the Centers for Disease Control and Prevention Institutional Animal Care and Use Committee, in accordance with procedures described in the U.S. Public Health Policy, 1986.

Animals were housed singly or doubly to avoid injuries caused by fighting with cage mates. Space recommendations for laboratory animals were followed as set forth in the Guide for the Care and use of Laboratory Animals (National Institutes of Health, Bethesda, MD). All animals were fed a diet that has been proven to provide adequate nutrition and calories in captive Aotus monkeys used in malaria-related research. Feed was free of contaminants and freshly prepared. Daily observations of the animals’ behavior, appetite, stool, and condition were recorded. All were treated as medical conditions arose by an attending veterinarian.

Anopheles freeborni (F-1 strain originally from California), An. gambiae (originally from The Gambia), An. albimanus (originally from Central America), An. maculatus (originally from Malaysia), and An. stephensi (originally from Delhi, India), were laboratory-reared and maintained at the Division of Parasitic Diseases/Centers for Disease Control and Prevention insectaries. During periods when gametocytes were present, mosquitoes were allowed to feed on tranquilized monkeys as previously described.5 After feeding, mosquitoes were held in an incubator at 25°C until examined one week later for the presence of oocysts on their midguts. If shown to be positive, mosquitoes were then held for either feeding on the tranquilized recipient monkey or dissected to provide sporozoites for intravenous challenge.

For intravenous challenge with sporozoites, infected mosquitoes were dissected in 20% fetal bovine serum/phosphate-buffered saline. The salivary glands were removed and crushed under a cover slip. The sporozoites were washed into a vial, counted using a Neubauer cell counting chamber, and injected into the femoral vein of the recipient monkey.

Blood-stage parasitemia was monitored by the daily examination of thick and thin blood films by the method of Earle and Perez.6 Infections were terminated by treatment with 30 mg chloroquine given over a three-day period by oral intubation.


RESULTS
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The initial studies conducted over a number of years with A. lemurinusn griseimembra monkeys resulted in 39 successful transmissions by the bites of Anopheles freeborni, An. stephensi, An. albimanus, and An. maculatus mosquitoes (Table 1Go). Prepatent periods ranged from 10 to 46 days with a median of 21 days. There was no relationship between the number of bites (intensity of salivary gland ratings) and the prepatent period, nor the species of mosquito. However, the percentage success that was obtained via mosquito bite was only 61.9% (39 of 63 attempts).


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TABLE 1
Infection of Aotus lemurinus griseimembra monkeys with the Santa Lucia strain of Plasmodium falciparum by the feeding of Anopheles freeborni, An. stephensi, An. albimanus, and An. maculatus mosquitoes*
 
Attempts were made to improve the transmission rate by the intravenous injection of sporozoites dissected from the salivary glands (Table 2Go). There were 61 successful transmissions obtained by the intravenous injection of sporozoites. Of 51 A. l. griseimembra that were inoculated, only 22 (41.5%) developed detectable infections. Of the 11 A. azarae boliviensis that were challenged with sporozoites, 8 (72.7%) developed detectable infections. Twelve A. vociferans were also challenged and 10 (83.3%) developed detectable infections.


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TABLE 2
Infection of Aotus lemurinus griseimembra, A. azarae boliviensis, A. nancymaae, and A. vociferans monkeys with the Santa Lucia strain of Plasmodium falciparum by the intravenous injection of sporozoites*
 
In addition to the above species, 33 A. nancymaae were challenged intravenously and 21 (63.6%) developed detectable infections. One that failed to develop a detectable infection was not splenectomized. Six of the animals that did not develop detectable parasitemia were not splenectomized until seven days after challenge. The remaining 21 animals were splenectomized before challenge. Thus, 21 (77.8%) of 27 monkeys that had been splenectomized before challenge were infected.

Of the 61 animals that developed detectable parasitemia after intravenous injection of sporozoites, the prepatent periods ranged from 15 to 50 days with a median of 21 days. There was little difference between the different monkey species in regards to the median day of patency, and there was no apparent relationship between the number of sporozoites injected and the prepatent period.


DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
An examination of the data indicated that the source of the sporozoites did not appear to influence the prepatent period. More surprisingly, the number of sporozoites injected, whether by bite or by intravenous injection, also did not greatly influence the prepatent period.

Currently, the species of animals that are available for vaccine trials are A. nancymaae, and to a lesser extent, A. vociferans. The rate of success when compared with the existing preferred model species A. l. griseimembra suggests that splenectomized A. nancymaae may also be a useful model for the testing of anti-sporozoite and anti-liver stage vaccines against the Santa Lucia strain of P. falciparum. The data suggests that A. nancymaae monkeys be challenged intravenously with 20,000–30,000 sporozoites from whatever species of mosquito has been infected. Prior splenectomy of the animals is preferred and appears to be required for reproducible appearance of parasitemia. However, deferment to completion of the liver stage development may be necessary for the assessment of the effect of liver stage vaccines. Polymerase chain reaction techniques in conjunction with suitable controls could be used to confirm the presence of erythrocytic stages developing after parasites have emerged from the liver and for parasite counts that never rise to levels detectable by blood film examination, but more desirable is actual microscopic detection.

At the present time, this appears to be one of the few models available as an in vivo model for the testing of anti-sporozoite and anti-liver stage vaccines against P. falciparum prior to initiating human studies. With a predicted transmission rate of approximately 80% (excluding the intact animal and the six animals in which splenectomy was delayed), A. nancymaae and the Santa Lucia strain of P. falciparum appears to be a usable model for preliminary trials of candidate vaccines. However, statistics would indicate the need for larger groups of animals than with the Salvador I strain of P. vivax and the Saimiri boliviensis monkey model where greater than 95% successful transmission would be expected after challenge with 10,000 sporozoites.

Received September 2, 2005. Accepted for publication November 6, 2005.

Financial support: This work was supported in part by an interagency agreement between the United States Agency for International Development, Malaria Vaccine Development Program, and the Centers for Disease Control and Prevention, Project # 936-6001, and the Atlanta Research and Education Foundation of the Veterans Affairs Medical Center of Atlanta.

* Address correspondence to William E. Collins, Division of Parasitic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Mailstop F-36, 4770 Buford Highway, Atlanta, GA 30341. E-mail: wec1{at}cdc.gov Back

Authors’ addresses: William E. Collins, JoAnn S. Sullivan, Douglas Nace, and John H. Barnwell, Division of Parasitic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Mailstop F-36, 4770 Buford Highway, Atlanta, GA 30341, E-mail: wec1{at}cdc.gov. Allison Williams and G. Gale Galland, Animal Resources Branch, Centers for Disease Control and Prevention, Atlanta, GA 30333. Tyrone Williams, Atlanta Research and Education Foundation, VA Medical Center, Atlanta, GA 30033.


REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Collins WE, Skinner JC, Pappaioanou M, Broderson JR, Filipski VK, McClure HM, Strobert E, Sutton BB, Stanfill PS, Huong AY, 1988. Sporozoite-induced infections of the Salvador I strain of Plasmodium vivax in Saimiri sciureus boliviensis monkeys. J Parasitol 74: 582–585.[Medline]
  2. Collins WE, Morris CL, Richardson BB, Sullivan JS, Galland GG, 1994. Further studies on the sporozoite transmission of the Salvador I strain of Plasmodium vivax. J Parasitol 80: 512–517.[Medline]
  3. Collins WE, Nussenzweig RS, Ballou WR, Ruebush TK II, Nardin EH, Chulay JD, Majarian WR, Young JF, Wasserman GF, Bathurst I, Gibson HL, Barr PJ, Hoffman SL, Wasserman SS, Broderson JR, Skinner JC, Procell PM, Filipski VK, Wilson CL, 1989. Immunization of Saimiri sciureus boliviensis monkeys with recombinant vaccines based on the circumsporozoite protein of Plasmodium vivax. Am J Trop Med Hyg 40: 455–464.[Abstract/Free Full Text]
  4. Collins WE, Nussenzweig RS, Ruebush TK II, Bathurst I, Nardin EH, Gibson HL, Campbell GH, Barr PJ, Broderson JR, Skinner JC, Filipski VK, Stanfill PS, Roberts JM, Wilson CL, 1990. Further studies on the immunization of Saimiri sciureus boliviensis monkeys with recombinant vaccines based on the circumsporozoite protein of Plasmodium vivax. Am J Trop Med Hyg 43: 576–583.[Abstract/Free Full Text]
  5. Collins WE, Contacos PG, Guinn EG, Held JR, 1966. Studies on the transmission of simian malarias. I. Transmission of two strains of Plasmodium inui by Anopheles maculatus and A. stephensi. J Parasitol 52: 664–668.[Medline]
  6. Earle WC, Perez M, 1932. Enumeration of parasites in the blood of malarial patients. J Lab Clin Med 17: 1124–1130.[Web of Science]



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This Article
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