Rhesus monkey model for Leishmania major transmitted by Phlebotomus papatasi sandfly bites.

Leishmaniasis research needs a near-human model for investigations of natural infection processes, immunological responses and evaluation of treatments. Therefore, we developed a reproducible system using Leishmania major Yakimoff & Schokhor (Trypanosomatidae: Kinetoplastida), the cause of Old World zoonotic cutaneous leishmaniasis (ZCL), transmitted to rhesus monkeys Macaca mulatta (Zimmerman) (Primates: Cercopithecidae) by sandfly bites of experimentally infected Phlebotomus papatasi (Scopoli) (Diptera: Psychodidae). Eight monkeys of presumed Indian origin (Leishmania naive) were exposed to bites of female sandflies that had been infected with L. major by membrane-feeding on human blood seeded with amastigotes isolated from hamster footpad lesions. Infection rates of membrane-fed sandflies averaged > 85% seven days after the infective feed, with uniformly high numbers of promastigotes in the stomodaeal valve region of the sandfly gut. Nodules and ulcerating dermal lesions developed on 7/8 monkeys 2-4 weeks post-bite and persisted for 3-7 months. Monkeys also developed satellite lesions beyond the area of sandfly bites on the head, but not on the chest. Three re-challenged monkeys developed lesions that healed faster than lesions from their primary challenges. After infection, monkeys developed delayed type hypersensitivity (DTH) responses to a panel of Leishmania skin test antigens (LSTA) and, when tested by ELISA and IFA, showed significant post-infection antibody titres which typically rose for approximately 170 days and then gradually receded during the next 100 days following the first challenge. After the second challenge, antibody titres spiked higher within approximately 50 days and receded more rapidly. In contrast, four rhesus macaques of Chinese origin developed no lesions following infected sandfly bites, although they raised antibodies and LSTA reactions, indicating subclinical infection.

Long-term efficacy and immune responses following immunization with the RTS,S malaria vaccine.

The malaria sporozoite vaccine candidate RTS,S, formulated with an oil-in-water emulsion plus the immunostimulants monophosphoryl lipid A and the saponin derivative QS21 (vaccine 3), recently showed superior efficacy over two other experimental formulations. Immunized volunteers were followed to determine the duration of protective immune responses. Antibody levels decreased to between one-third and one-half of peak values 6 months after the last dose of vaccine. T cell proliferation and interferon-gamma production in vitro were observed in response to RTS,S or hepatitis B surface antigen. Seven previously protected volunteers received sporozoite challenge, and 2 remained protected (1/1 for vaccine 1, 0/1 for vaccine 2, and 1/5 for vaccine 3). The prepatent period was 10.8 days for the control group and 13.2 days for the vaccinees (P < .01). Immune responses did not correlate with protection. Further optimization in vaccine composition and/or immunization schedule will be required to induce longer-lasting protective immunity.

Phase I/IIa safety, immunogenicity, and efficacy trial of NYVAC-Pf7, a pox-vectored, multiantigen, multistage vaccine candidate for Plasmodium falciparum malaria.

Candidate malaria vaccines have failed to elicit consistently protective immune responses against challenge with Plasmodium falciparum. NYVAC-Pf7, a highly attenuated vaccinia virus with 7 P. falciparum genes inserted into its genome, was tested in a phase I/IIa safety, immunogenicity, and efficacy vaccine trial in human volunteers. Malaria genes inserted into the NYVAC genome encoded proteins from all stages of the parasite's life cycle. Volunteers received three immunizations of two different dosages of NYVAC-Pf7. The vaccine was safe and well tolerated but variably immunogenic. While antibody responses were generally poor, cellular immune responses were detected in >90% of the volunteers. Of the 35 volunteers challenged with the bite of 5 P. falciparum-infected Anopheles mosquitoes, 1 was completely protected, and there was a significant delay in time to parasite patency in the groups of volunteers who received either the low or high dose of vaccine compared with control volunteers.

Passive transfer of growth-inhibitory antibodies raised against yeast-expressed recombinant Plasmodium falciparum merozoite surface protein-1(19).

Purified rabbit immunoglobulin raised against yeast-expressed recombinant FVO or 3D7 Plasmodium falciparum merozoite surface protein-1 (MSP-1) 19k-D C terminal fragment (MSP-1(19)) was transfused into malaria-naive Aotus nancymai monkeys that were immediately challenged with FVO asexual stage malaria parasites. Control monkeys received rabbit immunoglobulin raised against the sexual stage antigen Pfs25 or Aotus hyperimmune serum obtained from monkeys immunized by P. falciparum infection and drug cure. Passive transfer of rabbit anti-MSP-1(19) failed to protect against homologous or heterologous challenge and, when compared with negative controls, there were no differences in prepatent periods or time to treatment. Interestingly, rabbit anti-MSP-1(19), but not anti-Pfs25, immunoglobulin, and immune monkey serum prevented the development of antibodies directed against MSP-1(19) fragment by infected monkeys, indicating that the antibodies were reactive with native MSP-1(19) antigen in vivo. The prepatent period and time to treatment was greatly delayed in the two monkeys that received Aotus immune serum, both of which developed a chronic intermittent low level infection. In vitro parasite growth inhibition assays (GIAs) confirmed the presence of inhibitory activity (40% maximum inhibition) in concentrated anti-MSP-1(19) immunoglobulin (4.8 mg/ml), but the peak concentrations we achieved in vivo (1 mg/ml) were not inhibitory in vitro. Subinhibitory levels of anti-MSP-1(19) antibodies achieved by passive transfer were not protective against P. falciparum challenge.

Plasmodium vivax infections in U.S. Army troops: failure of primaquine to prevent relapse in studies from Somalia.

Different strains of Plasmodium vivax vary in their sensitivity to primaquine, the only drug that prevents relapses. Described are the clinical data and relapse pattern for 75 soldiers treated for vivax malaria since returning from Somalia. Following their initial attack of malaria, 60 of the 75 cases received a standard course of primaquine (15 mg base daily for 14 days). Twenty-six of the 60 soldiers subsequently relapsed for a failure rate of 43%. Eight soldiers had a second relapse following primaquine therapy after both the primary attack and first relapse. Three of these soldiers had received a higher dosage of primaquine (30 mg base daily for 14 days) after their second attack. The apparent ineffectiveness of primaquine therapy in preventing relapses suggests the presence of primaquine-resistant P. vivax strains in Somalia.

A preliminary evaluation of a recombinant circumsporozoite protein vaccine against Plasmodium falciparum malaria. RTS,S Malaria Vaccine Evaluation Group.

BACKGROUND: The candidate vaccines against malaria are poorly immunogenic and thus have been ineffective in preventing infection. We developed a vaccine based on the circumsporozoite protein of Plasmodium falciparum that incorporates adjuvants selected to enhance the immune response. METHODS: The antigen consists of a hybrid in which the circumsporozoite protein fused to hepatitis B surface antigen (HBsAg) is expressed together with unfused HBsAg. We evaluated three formulations of this antigen in an unblinded trial in 46 subjects who had never been exposed to malaria. RESULTS: Two of the vaccine formulations were highly immunogenic. Four subjects had adverse systemic reactions that may have resulted from the intensity of the immune response after the second dose, which led us to reduce the third dose. Twenty-two vaccinated subjects and six unimmunized controls underwent a challenge consisting of bites from mosquitoes infected with P. falciparum. Malaria developed in all six control subjects, seven of eight subjects who received vaccine 1, and five of seven subjects who received vaccine 2. In contrast, only one of seven subjects who received vaccine 3 became infected (relative risk of infection, 0.14; 95 percent confidence interval, 0.02 to 0.88; P<0.005). CONCLUSIONS: A recombinant vaccine based on fusion of the circumsporozoite protein and HBsAg plus a potent adjuvant can protect against experimental challenge with P. falciparum sporozoites. After additional studies of protective immunity and the vaccination schedule, field trials are indicated for this new vaccine against P. falciparum malaria.

Immunization of Aotus nancymai with recombinant C terminus of Plasmodium falciparum merozoite surface protein 1 in liposomes and alum adjuvant does not induce protection against a challenge infection.

Merozoite surface protein 1 (MSP-1) of Plasmodium falciparum is an antimalarial vaccine candidate. The highly conserved 19-kDa C-terminal processing fragment of MSP-1 (MSP-1(19)) is of particular interest since it contains epitopes recognized by monoclonal antibodies which inhibit the invasion of erythrocytes in vitro. The presence of naturally acquired anti-MSP-1(19) antibodies in individuals exposed to malaria has been correlated with reduced morbidity, and immunization with an equivalent recombinant P. yoelii antigen induces substantial protection against this parasite in mice. We have expressed P. falciparum MSP-1(19) in Escherichia coli as a correctly folded protein and immunized Aotus nancymai monkeys by using the protein incorporated into liposomes and adsorbed to alum. After vaccination, the sera from these animals contained anti-MSP-1(19) antibodies, some of which competed for binding to MSP-1(19) with monoclonal antibodies that inhibit parasite invasion of erythrocytes in vitro. However, after challenge with either a homologous or a heterologous strain of parasite, all animals became parasitemic and required treatment. The immunization did not induce protection in this animal model.

NYVAC-Pf7: a poxvirus-vectored, multiantigen, multistage vaccine candidate for Plasmodium falciparum malaria.

The highly attenuated NYVAC vaccinia virus strain has been utilized to develop a multiantigen, multistage vaccine candidate for malaria, a disease that remains a serious global health problem and for which no highly effective vaccine exists. Genes encoding seven Plasmodium falciparum antigens derived from the sporozoite (circumsporozoite protein and sporozoite surface protein 2), liver (liver stage antigen 1), blood (merozoite surface protein 1, serine repeat antigen, and apical membrane antigen 1), and sexual (25-kDa sexual-stage antigen) stages of the parasite life cycle were inserted into a single NYVAC genome to generate NYVAC-Pf7. Each of the seven antigens was expressed in NYVAC-Pf7-infected culture cells, and the genotypic and phenotypic stability of the recombinant virus was demonstrated. When inoculated into rhesus monkeys, NYVAC-Pf7 was safe and well tolerated. Antibodies that recognize sporozoites, liver, blood, and sexual stages of P. falciparum were elicited. Specific antibody responses against four of the P.falciparum antigens (circumsporozoite protein, sporozoite surface protein 2, merozoite surface protein 1, and 25-kDa sexual-stage antigen) were characterized. The results demonstrate that NYVAC-Pf7 is an appropriate candidate vaccine for further evaluation in human clinical trials.

Plasmodium falciparum: passive immunization of Aotus lemurinus griseimembra with immune serum.

Owl monkeys (Aotus lemurinus griseimembra) were immunized against Plasmodium falciparum by infection and drug cure. After challenge, 3 of 4 monkeys developed extended prepatent periods and low grade parasitemias followed by self cure. The fourth monkey did not develop a patent infection. Immune monkey serum passively transferred at the time of challenge conferred immunity to 20 naive monkeys. Immunity was characterized by extended prepatent periods in 19 monkeys, low levels of parasitemia (< or = 1%) followed by self cure in 12 animals, and lack of detectable infection in 3 recipient monkeys. Immune serum collected from monkeys undergoing repeated challenges afforded more protection than serum from singly infected monkeys. However, single doses of hyperimmune serum appeared to be as effective as multiple doses. Normal serum had no effect on the course of infection in 12 monkeys. These studies confirm that owl monkeys can be immunized by infection and cure and demonstrate that this immunity can, in large part, be transferred to nonimmune recipients with serum from immune donors.

Azithromycin prophylaxis against a chloroquine-resistant strain of Plasmodium falciparum.

Azithromycin has antimalarial activity and favourable pharmacokinetic properties for a prophylactic antimalarial agent. We investigated the ability of azithromycin to prevent malaria in volunteers infected with a chloroquine-resistant strain of Plasmodium falciparum. 4 volunteers received oral azithromycin 500 mg followed by 250 mg daily for 7 further days. Subjects were infected on the third day of azithromycin. 3 subjects were protected compared with none of 15 controls. The volunteer not protected by azithromycin had unquantifiable plasma levels of azithromycin, probably because of poor absorption. Azithromycin could be a promising prophylactic agent for P falciparum malaria.

A history of sleeping sickness in Kenya.

Gambian sleeping sickness entered what is now Kenya from Uganda in about 1901 and quickly spread along the Kenyan shores and islands of Lake Victoria, reaching Tanzania in 1902. By 1910 the disease had spread 25 miles inland along the Kuja and Migori rivers and their tributaries. Sleeping sickness waxed and waned in these areas despite attempts to control tsetse fly populations by various methods. It was not until 1950, when the use of insecticides (DDT) applied by backpack sprayer proved successful against Glossina fuscipes at Kibigori, that eradication of G. fuscipes and Gambian sleeping sickness seemed possible. Subsequently the Kuja-Migori endemic area was cleared of flies and disease, as well as the South and Central Nyanza lake shores and islands. By 1965 Gambian sleeping sickness had virtually disappeared from Kenya. A more virulent form of the disease, Rhodesian sleeping sickness, may have also spread to Kenya from Uganda, although its appearance in diverse areas of the Gambian disease suggest that local ecological factors may have played a role in enhanced virulence of trypanosomes stocks. The Rhodesian form of sleeping sickness appeared in the Lambwe Valley, South Nyanza, in about 1959, and despite attempts to eradicate this disease it still persists as the only remaining endemic area in Kenya at this time. The usual transmission of Rhodesian sleeping sickness by G. pallidipes in Kenya was altered when an outbreak occurred at Alego, in Central Nyanza, in 1964. It was discovered that G. fuscipes was the vector and that domestic cattle were an important reservoir of infection. Glossina fuscipes was also the vector of Rhodesian sleeping sickness in an outbreak in Samia in 1976 and another along the lakeshore in South Nyanza in 1981. Sleeping sickness has been restricted primarily to the Western and Nyanza Provinces of Kenya (Fig. 1).

Sleeping sickness in the Lambwe Valley in 1978.

Even though tsetse control measures were discontinued in the Lambwe Valley in 1974 the prevalence of Rhodesian sleeping sickness remained at low levels. A survey conducted in 1978 verified a low prevalence of disease (0.1%). Thirty-four per cent of the individuals tested were positive for malaria with the highest prevalence (44%) in children aged 0-9 years. Thirteen of 1340 individuals (0.97%) tested and found negative for sleeping sickness in 1978 developed the disease by 1985. Fourteen individuals with moderate titres (2+) in the IFAT but who showed no evidence of disease were traced and found to be alive and well seven years later. Three of these patients still had positive titres but the others had converted to negative. Sera from four patients infected and treated in 1978 were also positive, but only one of five patients treated in 1977 reacted in the test. The CFT as described did not appear useful as a diagnostic test.

Diagnosis of Rhodesian sleeping sickness in the Lambwe Valley (1980-1984).

In primary Rhodesian sleeping sickness patients, parasitological diagnosis was best performed by rodent inoculation of blood (98.5%+) followed by Giemsa-stained thick blood smears (93.3%+). Parasitological diagnosis in relapse patients was sometimes impossible and clinical diagnosis based on CSF examination was necessary. Early during a disease outbreak in 1980, 89% of the infections were detected by mobile field teams, but once established in the endemic area a stationary diagnostic facility detected most of the cases. A total number of 23,751 examinations for Rhodesian sleeping sickness and malaria were made by mobile field teams during 1980-1984; 102 primary cases (0.43%) and 25 (0.10%) relapse cases were diagnosed. A total of 9339 individuals (39%) had patent malaria infections. The IFAT was positive in 89% of the primary sleeping sickness patients and 77% of the relapse patients. Seventy-nine per cent of the primary patients were positive in a CFT test, and 77% of the relapse patients were considered positive.

Immunodiagnostic tests on cerebrospinal fluid in the diagnosis of meningoencephalitic Trypanosoma brucei rhodesiense infection.

Fourteen cerebrospinal fluid (CSF) samples obtained from Rhodesian sleeping sickness patients from the Lambwe Valley at relapse were positive for the presence of anti-trypanosomal antibody by both IFAT and ELISA. The mean optical density (o.d.) in the ELISA test was 0.804 +/- 0.362 and ranged from 0.258 to 1.363. CSF from five patients from the same area without evidence of meningoencephalitis were all negative by ELISA (mean o.d. 0.023 +/- 0.016, range 0.011-0.051). Control CSF samples from U.K. patients without Rhodesian sleeping sickness but with elevated levels of CSF total protein were also negative. Antibody detected by ELISA declined after Mel-B treatment of relapse and most samples had returned to negative within two years of treatment. We present evidence that serological evaluation of the CSF by ELISA and/or IFAT can provide supportive evidence of the trypanosomal origin of the infection. This is especially important at the time of relapse, when parasitological diagnosis may be impossible and records of treatment for the primary infection may not be available.

Parasite survey of eight wild animals in the Ruma National Park.

Eight game animals representing seven species in the Ruma National Park in South Nyanza, Kenya, were examined for the presence of blood protozoa, ectoparasites, and helminthic and coccidian endoparasites using standard parasite-identification methods. Haematological parameters were also determined. The oribi was positive for Trypanosoma brucei ssp. and the reedbuck for T. congolense. No other blood protozoans were found. Strongyle eggs were found in the faeces of all species except the water buck. Five of eight animals harboured liver flukes and five were parasitized by ticks of the genus Amblyomma. One roan antelope was anaemic, but the other animals had haemoglobin levels within the normal range and appeared to be in a good state of health.

Review of tsetse control measures taken in the Lambwe Valley in 1980-1984.

During an outbreak of Rhodesian sleeping sickness in the Lambwe Valley in 1980 initial tsetse control measures consisted of applications of dieldrin to the periphery of the Ruma National Park. This activity had a marked effect on the prevalence of sleeping sickness. Concern about the use of dieldrin caused the cessation of this programme and justified an aerial spray programme using endosulfan. Although the Lambwe Valley did not appear to be a good candidate for aerial spray, the endosulfan had a marked effect on tsetse fly levels and on the prevalence of sleeping sickness. Sleeping sickness cases were detected in decreasing numbers for eight months following the endosulfan programme, but the subsequent five months yielded no cases of sleeping sickness in the area. Some flies persisted, however, and they had regained high levels in about a year. As the prevalence of sleeping sickness increased another aerial spray programme was initiated in 1983, using pyrethrum as insecticide. The pyrethrum aerial spray programme did not make significant reductions in the Glossina pallidipes population or in the prevalence of sleeping sickness. A subsequent ground control programme using insecticides (dieldrin and cypermethrin) and bush clearing, conducted primarily within the National Park, has subsequently limited the prevalence of sleeping sickness to low levels.

Mechanical transmission of Trypanosoma brucei rhodesiense by Glossina morsitans morsitans (Diptera:Glossinidae).

Interrupted feedings of teneral, laboratory-reared Glossina morsitans morsitans were used to study mechanical transmission of Trypanosoma brucei rhodesiense. Intervals between exposure of individual flies on parasitaemic rats and refeeding on clean rats were varied from five minutes to 24 hours. Direct transmissions were demonstrated at each interval up to 160 minutes after exposure. Proboscis dissections showed that active trypanosomes were present up to 320 minutes after exposure. No mechanical transmissions from bovine to bovine occurred in 39 attempts, when groups of 20-120 flies exposed on parasitaemic bovines were transferred immediately to uninfected cattle, but two of 40 individual flies exposed on parasitaemic bovines mechanically transmitted trypanosomes to clean rats. Proboscis dissections made immediately after flies were exposed to a bovine with a parasitaemia of 4.8 x 10(-4) trypanosomes/microliters of blood showed that 11 of 20 (55%) had active trypanosomes in the food canal. The mean number of trypanosomes per proboscis was 29.4 (+/- 20.5). Of 20 flies exposed on a bovine with a low parasitaemia, however, only one trypanosome was seen in proboscis dissections. The parasitaemia of the infected donor was an important factor in mechanical transmission. The mechanical transmission of trypanosomes from one host to another may largely depend on the infectivity threshold of the recipient host, and individual mechanically-infected tsetse flies may not transmit an infective dose.

The Lambwe Valley and its people.

By 1936 the Lambwe Valley, which had been heavily populated in the early years of this century, was nearly devoid of people. Population since that time has increased markedly as a result of a settlement scheme and efforts made to control and eradicate Glossina pallidipes and trypanosomiasis. The formation of a game reserve (now a National Park) prevented the completion of a tsetse eradication programme and has provided an unmolested habitat for both G. pallidipes and large numbers of game animals which act as a reservoir for trypanosomiasis. Rhodesian sleeping sickness, as well as animal trypanosomiasis, have been severe problems for the local farmers who live around the boundaries of the National Park.

Experimental infection of cattle with Trypanosoma brucei rhodesiense.

Infection of cattle with various stocks of Trypanosoma brucei rhodesiense indicated that 49% developed a fatal CNS disease comparable to that found in man. Duration of disease ranged from 85 to 1613 days post infection. All eight stocks of T. b. rhodesiense tested, including those from Ethiopia and Tanzania, induced CNS disease. Blood became positive three to five days after inoculation, and after an initial peak of parasitaemia remained positive for three to five months. Subinoculation of blood into rodents subsequently became negative, although trypanosomes persisted in the lymph nodes for at least 56 to 1613 days. Only animals with CNS disease had detectable parasites in the CSF, usually after the animals had undergone severe deterioration. At post mortem examination trypanosomes could usually be found in the lymph nodes and CSF, and occasionally in the blood. Clinical signs included fever, hyperkinesia, weight loss, cerebellar ataxia, tremor, salivation and hyperaesthesia. A mild to moderate anaemia accompanied a transient thrombocytopenia and leucopenia. Animals subsequently developed leucocytosis. A pleocytosis and elevated total protein in the CSF was found, which persisted in some animals for long periods. Histopathological examination of the brain showed prominent generalized perivascular infiltrates consisting mainly of lymphocytes and plasma cells. Mott's cells were regularly observed. Vascular changes were characterized by swollen endothelium, infiltration of the vascular wall by inflammatory cells, and in some instances perivascular oedema. In the most severe cases evidence of ischaemia consisted of large numbers of astrocytes, rarefaction of the parenchyma, and areas of necrosis with loss of normal architecture. Demyelination was limited to perivascular areas. Occasionally a moderate to severe pancarditis was found.

Cerebral trypanosomiasis in naturally-infected cattle in the Lambwe Valley, south Nyanza, Kenya.

Surveys in Zebu cattle in the Lambwe Valley in 1980 indicated that many (up to 70%) were infected with trypanosomes. The predominant parasite was Trypanosoma brucei sspl followed by T. congolense. Cerebrospinal fluid (CSF) analysis showed a high proportion of animals with pleocytosis and elevated total CSF protein. Trypanosomes were detected in CSF and signs of a central nervous system (CNS) disease were observed. Histopathological lesions in the CNS were identical to those found in experimentally-infected cattle and consisted of perivascular infiltrations, swollen endothelium of vessels, infiltration of the vascular wall, and perivascular oedema. The severest cases showed rarefaction, astrocytosis and areas of necrosis. Infected cattle transported to the Veterinary Research Laboratory were studied for up to four years. Absence of trypanosomes from the peripheral blood was common, and even subinoculation of lymph node aspirates and CSF were usually negative. Death was preceded by a period of weight loss and the development of severe CNS signs. An attempt to cure animals with Mel-B treatment failed. Serum from naturally-infected cattle neutralized T. b. rhodesiense stocks collected in the same area.