Protective efficacy of a Plasmodium vivax circumsporozoite protein-based vaccine in Aotus nancymaae is associated with antibodies to the repeat region.

We have previously reported that Vivax Malaria Protein 001 (VMP001), a vaccine candidate based on the circumsporozoite protein of Plasmodium vivax, is immunogenic in mice and rhesus monkeys in the presence of various adjuvants. In the present study, we evaluated the immunogenicity and efficacy of VMP001 formulated with a TLR9 agonist in a water-in-oil emulsion. Following immunization, the vaccine efficacy was assessed by challenging Aotus nancymaae monkeys with P. vivax sporozoites. Monkeys from both the low- and high-dose vaccine groups generated strong humoral immune responses to the vaccine (peak median titers of 291,622), and its subunits (peak median titers to the N-term, central repeat and C-term regions of 22,188; 66,120 and 179,947, respectively). 66.7% of vaccinated monkeys demonstrated sterile protection following challenge. Protection was associated with antibodies directed against the central repeat region. The protected monkeys had a median anti-repeat titer of 97,841 compared to 14,822 in the non-protected monkeys. This is the first report demonstrating P. vivax CSP vaccine-induced protection of Aotus monkeys challenged with P. vivax sporozoites.

Mosquito infection studies with Aotus monkeys and humans infected with the Chesson strain of Plasmodiun vivax.

Oocyst counts were compared between mosquitoes that fed on humans versus mosquitoes that fed on Aotus monkeys, both of which were infected with the Chesson strain of Plasmodium vivax. Oocyst counts obtained from mosquitoes fed on humans were almost 10-fold higher in number. Mosquitoes were more likely to be infected and with a higher rate of infection when they fed on monkeys before the peak in the asexual parasite count. Mosquitoes that fed on humans were more likely to be more heavily infected when fed after the peak in the asexual count. Of several species of owl monkeys, Aotus vociferans was infected at a higher frequency. On the basis of oocyst counts, Anopheles dirus were the most susceptible and An. maculatus were the least susceptible of the mosquito species tested.

Observations on the Uganda I strain of Plasmodium malariae and Plasmodium brasilianum in Aotus and Saimiri monkeys and Anopheles mosquitoes.

Splenectomized Aotus lemurinus griseimembra, A. azarae boliviensis, A. nancymaae, A. vociferans, and Saimiri boliviensis monkeys were infected with the Uganda I/CDC strain of Plasmodium malariae. The maximum parasite counts were lower if the animals had been previously infected with Plasmodium vivax. Mosquito infection was concentrated in the 12 days following the rise in count above 1,000/microl. Mosquito infection and parasite counts were highest with A. l. griseimembra. Anopheles freeborni was more readily infected than An. gambiae, which was more readily infected than An. stephensi. Parasite counts and mosquito infection with P. brasilianum were much higher in S. boliviensis monkeys than with the Uganda I strain of P. malariae in this host, suggesting marked differences between the host-parasite-vector relationships and indicating that P. brasilianum in S. boliviensis monkeys may be a better reflection of the relationship of P. malariae in the human host.

Infection of mosquitoes with Plasmodium falciparum by feeding on humans and on Aotus monkeys.

Of 1,004 positive lots of mosquitoes fed on 229 humans infected with Plasmodium falciparum, 46.2% had 1-10 oocysts/(+)gut, 21.2% had 10-30 oocysts/(+)gut, 22.2% had 30-100 oocysts/(+)gut, and 10.4% had > 100 oocysts/(+) gut. The highest levels of infection occurred between 6 and 15 days after the peak in the asexual parasite count. Of 2,281 lots of Anopheles freeborni mosquitoes fed on splenectomized Aotus monkeys infected with the Santa Lucia strain of P. falciparum, 1,191 were infected (52.2%). The highest intensity infections ranged from 2.78 oocysts per positive gut in mosquitoes fed on Aotus vociferans to 6.08 oocysts per positive gut for those fed on A. lemurinus griseimembra to 10.4 oocysts per positive gut for those fed on A. nancymaae. The pattern of infection for mosquitoes fed on splenectomized Aotus monkeys was similar to that obtained by feeding on humans, but the intensity, based on oocyst/(+)gut, was much lower.

The Santa Lucia strain of Plasmodium falciparum in Aotus monkeys.

The Santa Lucia strain of Plasmodium falciparum was studied in 150 Aotus lemurinus griseimembra, 30 A. azarae boliviensis, 103 A. nancymaae, and 121 A. vociferans monkeys. All four of these splenectomized hosts supported the production of gametocytes infective to Anopheles freeborni mosquitoes. Transmission through sporozoites from An. freeborni, An. stephensi, An. maculatus, and An. albimanus mosquitoes was successful to all four species of Aotus on a total of 100 occasions with a median pre-patent period of 21 days. For the production of infective mosquitoes for vaccine challenge studies, A. l. griseimembra and A. vociferans were the most predictable hosts.

Studies on the Salvador I strain of Plasmodium vivax in non-human primates and anopheline mosquitoes.

A review is presented on studies conducted in New World monkeys and chimpanzees with the Salvador I strain of Plasmodium vivax. This isolate has been adapted to Aotus and Saimiri (squirrel) monkeys and developed as a model for the testing of antimalarial vaccines. After the injection of 10,000 sporozoites, the median prepatent period in S. boliviensis monkeys was 21.5 days. In 103 sporozoite-induced infections in splenectomized monkeys, the median maximum parasite count ranged from 2,139 to 202,368/microL, with a median maximum parasite count of 48,174/microL. Median maximum parasite counts in Aotus lemurinus griseimembra, A. nancymaae, A. azarae boliviensis, and A. vociferans monkeys were 19,902, 18,390, 21,420, and 18,210/microL, respectively and ranged from 124 to 156,000/microL. Mosquito infections were readily obtained in different species of Anopheles mosquitoes. The S. boliviensis monkey and Salvador I strain seems suitable for the testing of sporozoite and liver stage vaccines but not for blood-stage vaccines against P. vivax unless adapted further in spleen-intact Saimiri boliviensis monkeys.

The Chesson strain of plasmodium vivax in humans and different species of Aotus monkeys.

Comparison was made between the parasitemia of Chesson strain Plasmodium vivax in humans and in splenectomized Aotus lemurinus griseimembra, A. nancymaae, A. vociferans, and A. azarae boliviensis monkeys. In the monkeys, 56.3% of the animals had maximum counts > 25,000/muL and in humans 59.6% were above this peak parasitemia. In humans, it took an average of 9.3 days to reach the maximum parasite count. In monkeys with no previous infections, it took an average of 18.9 days to reach the maximum parasite count; for those with previous infections, it took an average of 15 days. Human and nonhuman primate data on this parasite suggest that splenectomized Aotus monkeys, particularly A. lemurinus griseimembra, and to a somewhat lesser extent A. vociferans, can mimic the course of Chesson malaria in humans regarding parasitemia and mosquito infection.

Transmission of different strains of Plasmodium cynomolgi to Aotus nancymaae monkeys and relapse.

Forty-four splenectomized Aotus nancymaae monkeys were infected with 6 different strains of Plasmodium cynomolgi, 11 via trophozoites and 33 via sporozoites. Sporozoites from Anopheles dirus, Anopheles freeborni, Anopheles gambiae, Anopheles maculatus, and Anopheles stephensi resulted in prepatent periods ranging from 9 to 39 days (median of 15 days). Importantly, relapse was demonstrated in 5 of 5 sporozoite-induced infections with the Rossan strain following treatment with chloroquine.

Observations on the sporozoite transmission of Plasmodium vivax to monkeys.

Saimiri boliviensis monkeys were infected via sporozoites with the Salvador I strain of Plasmodium vivax that had been stored frozen for periods ranging from 12 to 5,312 days. Prepatent periods ranged from 16 to 53 days.

Adaptation of a multi-drug resistant strain of Plasmodium falciparum from Peru to Aotus lemurinus griseimembra, A. nancymaae, and A. vociferans monkeys.

A strain of Plasmodium falciparum from Peru was adapted to splenectomized Aotus nancymaae and Aotus vociferans monkeys. The Peru 134/CDC strain of P. falciparum was shown to be resistant to treatment with chloroquine in monkeys and partially resistant to mefloquine and malarone. Genetic mutations in crt, dhfr, dhps, and cytochrome b genes conferring drug resistance were also determined for this Peruvian strain of P. falciparum.

Isolates of Plasmodium inui adapted to Macaca mulatta monkeys and laboratory-reared anopheline mosquitoes for experimental study.

Plasmodium inui is a parasite of macaques and other nonhuman primates in Asia that is studied as a model for the human malaria parasite P. malariae. Presented here are descriptions of the isolation, passage histories into Macaca mulatta monkeys, and infectivity to different Anopheles spp. mosquitoes of 18 different isolates of this parasite.

Studies on sporozoite-induced and chronic infections with Plasmodium fragile in Macaca mulatta and New World monkeys.

Plasmodium fragile continues to be investigated because of its biologic similarities to the human malaria parasite, Plasmodium falciparum. Two strains of P. fragile are available for study; one strain is able to infect mosquitoes, whereas the other strain is transmissible only by blood inoculation. The Sri Lanka strain of P. fragile was transmitted to Macaca mulatta, Macaca fascicularis, Aotus lemurinus griseimembra, Aotus nancymaae, Aotus vociferans, and Saimiri boliviensis monkeys via sporozoites that developed to maturity only in Anopheles dirus mosquitoes. The prepatent periods ranged from 12 to 35 days for macaques and from 15 to 30 days for New World monkeys after intravenous injection of sporozoites. Eight rhesus monkeys were infected with the Nilgiri strain and followed for 482 days. Parasitemia in 6 animals persisted at relatively high density through the period of observation. Erythrocyte, hematocrit, and hemoglobin values reached their lowest levels 3 wk after infection and slowly recovered; however, the values did not approach preinfection levels as long as parasitemia persisted in the monkeys. The mean corpuscular volume and corpuscular hemoglobin concentration reached their peak and lowest values, respectively, at day 38 and then returned to the preinfection level. The mean corpuscular hemoglobin value decreased to its lowest level at day 87 and then returned to preinfection level.

Observations on the exoerythrocytic stages of different isolates of Plasmodium cynomolgi in hepatocytes of New World aotus and Saimiri monkeys.

Sporozoites of 3 isolates of Plasmodium cynomolgi dissected from the salivary glands of Anopheles dirus and Anopheles quadrimaculatus were injected intravenously into 9 New World monkeys. Liver stage parasites were demonstrated in all 9 animals; 7 of these animals also produced blood stages after prepatent periods of 9 to 23 days.

Aotus nancymaae as a potential model for the testing of anti-sporozoite and liver stage vaccines against Plasmodium falciparum.

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.

Assessment of transmission-blocking activity of candidate Pvs25 vaccine using gametocytes from chimpanzees.

Macaca mulatta monkeys were immunized with the candidate transmission-blocking vaccine against Plasmodium vivax, Pvs25, combined with alum or Montanide ISA 720. Efficacy was measured by combining post-immunization sera with gametocytes obtained from infections induced in chimpanzees using membrane-feeding techniques. The results indicate that immunization of M. mulatta monkeys with Pvs25 and Montanide ISA 720 was more effective than with alum in efficacy and resulted in the maintenance of a lasting transmission-blocking immunity to P. vivax. This was evident two weeks after the second immunization, and more strongly demonstrable 62 and 152 days after the second immunization. This transmission-blocking activity was strongly reinforced by a third immunization given 181 days after the primary immunization, as measured three weeks later by indirect membrane feeding. The use of gametocytes of P. vivax derived from infections induced in chimpanzees can contribute to the selection of appropriate constructs, formulations, and immunization regimens for the development of effective transmission-blocking vaccines.

Preliminary observations on the efficacy of a recombinant multistage Plasmodium falciparum vaccine in Aotus nancymai monkeys.

A vaccine trial was conducted to determine the efficacy of a multicomponent candidate vaccine, FALVAC-1, against Plasmodium falciparum in Aotus nancymai monkeys. After two immunizations, animals were challenged intravenously with parasites of the Vietnam Oak Knoll (FVO) strain of P. falciparum. The primary outcome was to determine the protective response of the monkeys to immunization with the FALVAC-1 antigen produced in baculovirus when combined with different adjuvants (alum, QS-21, ASO2a, CRL1005/oil, and CRL1005/saline) as compared with FALVAC-1 with FCA/FIA and antigen alone. When compared with the monkeys immunized with FALVAC-1 alone, FALVAC-1 with FCA/FIA reduced the mean parasite count (to Day 11), reduced the mean accumulated parasitemia (through Day 11), and extended the number of days to treatment. None of the other 5 antigen-adjuvant combinations were able to provide discernable levels of protection based on log(parasitemia) and log(cumulative parasitemia) to Day 11.

Plasmodium simium and Saimiri boliviensis as a model system for testing candidate vaccines against Plasmodium vivax.

Observations on Plasmodium simium infections in Saimiri boliviensis boliviensis monkeys suggest that this host-parasite combination would be a suitable model for the testing of candidate vaccines against Plasmodium vivax. To evaluate the normal course of infections, parasitemia in 52 splenectomized S. boliviensis boliviensis monkeys infected with P. simium were analyzed. The mean maximum parasite count for 31 monkeys after injection with trophozoite-infected erythrocytes was 77,580/microL. Twenty-one monkeys were infected via sporozoites, and prepatent periods ranged from 14 to 24 days with a median of 15 days. The mean maximum parasite count was 29,234/microL. The mean maximum parasite count for monkeys previously infected with Old World P. vivax was 26,337/microL versus 56,362/microL for those previously infected with New World P. vivax, possibly suggesting a closer antigenic relationship between P. simium and the Old World parasites.

Studies on two strains of Plasmodium cynomolgi in New World and Old World monkeys and mosquitoes.

Infections that cause the Gombak and Smithsonian strains of Plasmodium cynomolgi were induced in Macaca mulatta, Aotus lemurinus griseimembra, Aotus nancymai, and Saimiri boliviensis monkeys. Transmission of the Gombak strain to Aotus spp. monkeys was obtained by the injection of sporozoites dissected from the salivary glands of experimentally infected Anopheles dirus and by the bites of infected An. dirus and Anopheles farauti mosquitoes. Two S. boliviensis monkeys were infected via the injection of sporozoites dissected from An. dirus. Prepatent periods in New World monkeys ranged from 14 to 44 days, with a median of 18 days. The Smithsonian strain was transmitted via sporozoites to 1 A. lemurinus griseimembra and 9 A. nancymai monkeys. Prepatent periods ranged from 12 to 31 days.

Observations on the Vietnam Palo Alto strain of Plasmodium vivax in two species of Aotus monkeys.

Thirty-three splenectomized Aotus lemurinus griseimembra monkeys with no previous experience with malaria were infected with the Vietnam Palo Alto strain of Plasmodium vivax. The median maximum parasite count was 280,000/microl. Nine splenectomized monkeys with previous infection with Plasmodium falciparum had median maximum parasite counts of 120,000/microl. Splenectomized Aotus nancymai monkeys supported infections at a lower level. Transmission via the bites of Anopheles dirus mosquitoes was obtained in a splenectomized A. lemurinus griseimembra, with a prepatent period of 31 days. It is estimated that between 1.5 x 10(8) and 1.6 x 10(9) parasites can be removed from an infected animal for molecular or diagnostic antigenic studies.

Susceptibility of Anopheles farauti to infection with different species of Plasmodium.

A colony of Anopheles farauti, originally from the island of New Britain in Papua New Guinea, was tested for its receptivity to infection with different species of Plasmodium in comparison with An. freeborni and An. stephensi. This colony adapted well to feeding on monkeys and was infected with New World and Old World strains of P. vivax and P. falciparum, P. ovale, P. cynomolgi, and P. brasilianum.