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

INTRODUCTION

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

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

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 1). 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).

Attempts were made to improve the transmission rate by the intravenous injection of sporozoites dissected from the salivary glands (Table 2). 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.

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

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.