Bruce T. Wellde

A Preliminary Evaluation of a Recombinant Circumsporozoite Protein Vaccine against Plasmodium falciparum Malaria

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.

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 parasites 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.

Trypanosoma congolense. I. Clinical observations of experimentally infected cattle.

Abstract The course of disease was studied in 8 cattle infected with Trypanosoma congolense . Although the onset of patency was dependent on the numbers of infecting organisms, the duration of the infection was not. High fevers were present on the day of or the day after initial patency. Succeeding peaks of parasitemia, and a progressive weight loss of over 30% occurred. A decrease in packed cell volume (PCV) beginning the first week after infection was observed. Early in the course of the developing anemia, many polychromatophilic erythrocytes and occasional normoblasts were found in the blood. A leucopenia persisted for the duration of the disease. Total serum protein concentrations fell sharply during the first 5 weeks of infection, then gradually increased to low normal levels. Serum albumin levels followed a similar pattern for the first 5 weeks, and remained at a relatively low level. Although gamma globulin levels also declined during the first 5 weeks, their levels gradually surpassed those of preinfection samples. No marked changes in serum glucose were noted. A mild elevation of serum urea nitrogen values occurred early during infection, but subsided. The animals dying early after infection developed elevated total bilirubin levels.

Prophylaxis of Plasmodium falciparum Malaria with Azithromycin Administered to Volunteers

New effective and well-tolerated prophylactic agents against P. falciparum malaria need to be developed. Chloroquine and proguanil are now relatively ineffective [1, 2]. Although the efficacy of mefloquine is approximately 90% [1], this rate may decline as resistance spreads [3], and mefloquine is currently not recommended for pregnant women, infants weighing less than 15 kg, and persons with neuropsychiatric disorders [2]. Sulfadoxine-pyrimethamine (Fansidar, Roche, Nutley, New Jersey) is no longer recommended for prophylaxis [2] because of the possibility of fatal toxic epidermal necrolysis. Doxycycline, 100 mg/d, is approximately 90% effective [4], but this agent is contraindicated in pregnant women and children younger than 8 years old and has gastrointestinal side effects. Plasmodium falciparum initially infects the liver, and, after approximately 7 days, parasites begin to emerge from the liver to parasitize red cells. A prophylactic agent against the liver stage of infection (causal prophylactic agent) needs to be administered for only approximately 7 days or less after infection, whereas an agent effective solely against the blood stage of the infection (suppressive prophylactic agent) needs to be administered for several weeks after infection. Mefloquine and doxycycline are insufficiently effective against liver-stage parasites, and patients require dosing for 4 weeks after leaving the endemic area. Azithromycin is an azalide analog of erythromycin that was recently licensed for the treatment of bacterial and chlamydial diseases. In a rodent model, we found that azithromycin has a causal prophylactic efficacy superior to that of doxycycline [5]. In addition, we did a study in which volunteers were exposed to the bites of P. falciparum-infected mosquitoes and given azithromycin for as long as 5 days after exposure to the bites. Azithromycin, 500 mg administered 2 days before exposure to the bites followed by 250 mg/d administered for 5 days after exposure to the bites, was successful prophylaxis for three of four persons [6]. Because the previous clinical study involved only 4 drug recipients, we did a new study of causal prophylaxis using 10 drug recipients. Because we obtained results showing unexpectedly poor efficacy, we subsequently entered another cohort of 10 drug recipients to determine the combined causal efficacy as well as the suppressive efficacy of azithromycin against P. falciparum malaria in the challenge model. Methods Study Design We did an open-label, controlled phase II study in which two cohorts were entered sequentially. In cohort 1, we evaluated the causal prophylactic efficacy of azithromycin. Ten persons received 500 mg of the drug on day 14 before the challenge, followed by 250 mg/d from day 13 before the challenge through day 7 after the challenge. Dosing from day 14 before the challenge to day 0 was intended to allow serum concentrations to reach a steady state; dosing from days 1 to 7 after the challenge was intended to provide causal prophylaxis. In the 12 preceding experiments at our clinical research center, all of approximately 40 control volunteers inoculated with this strain of P. falciparum became infected. In our present study, two controls did not receive azithromycin and were concomitantly infected to verify that the parasitologic challenge was infectious. In cohort 2, we evaluated the combined causal and suppressive prophylactic efficacy of azithromycin. Ten persons received 500 mg of the agent on day 14 before the challenge, followed by 250 mg/d from day 13 before the challenge through day 28 after the challenge. Dosing from days 8 through 28 was intended to provide suppressive prophylaxis. Two controls were concomitantly challenged. Inclusion and Exclusion Criteria Men and women aged 18 through 46 years were eligible if they were healthy on the basis of history, physical examination, and results of laboratory tests (complete blood count; serum levels of sodium, potassium, chloride, urea nitrogen, creatinine, calcium phosphate, total protein, albumin, aspartate aminotransferase, alkaline phosphatase, total bilirubin, lactate dehydrogenase, and uric acid) and had no history of exposure to malaria in the previous 2 years. Persons who did not object to the high likelihood of becoming parasitemic were permitted to be controls. Study Procedures For each cohort of 12 volunteers, all persons were infected [7] by exposure to the bites of five female Anopheles stephensi mosquitoes infected with the chloroquine-sensitive, mefloquine-resistant NF54 strain of P. falciparum. During drug administration, adverse reactions and symptoms of malaria were assessed by means of daily interviews for subjective symptoms (chills, headache, photophobia, back pain, muscle ache, stomach ache, anorexia, nausea, vomiting, diarrhea, vaginal itching), by daily recording of body temperature, and by repetition of the entrance laboratory tests on day 4 before the challenge and day 5 after the challenge. To make the definitive diagnosis of malaria, on days 5 through 28 (for cohort 1) or on days 5 through 35 (for cohort 2), thick blood smears were obtained, stained with Giemsa, and examined for P. falciparum by an investigator blinded to whether or not the study participant had received azithromycin. Positive results were confirmed by two other investigators. In addition, all persons not already diagnosed as having malaria were instructed to contact study personnel until day 70 if they had symptoms that suggested malaria. In each thick smear, 200 fields were examined. Patent malaria was defined as the presence of at least two parasites per smear. If the volunteer had patent parasitemia, he or she was treated with chloroquine (1500-mg base given over 3 days). Treatment Agents Azithromycin in the form of 250-mg tablets was provided by Pfizer Central Research. Groton, Connecticut. Chloroquine in the form of Aralen Phosphate tablets was purchased from Winthrop Pharmaceuticals. New York, New York. Protocol Approval Our protocol was approved by the Office of the Surgeon General of the United States Army and filed under IND with the U.S. Food and Drug Administration. All volunteers gave written informed consent. Statistical Methods Computation of exact CIs of a proportion was done using the binomial distribution. Results Prophylactic Efficacy of Azithromycin Cohort 1 consisted of 10 volunteers who received azithromycin until 7 days after challenge with P. falciparum sporozoites and 2 volunteers who were challenged with sporozoites but received no treatment. Both of the controls who received no treatment and 6 of the 10 volunteers who received treatment with the drug developed parasitemia. One participant was removed from the study on day 25 because of noncompliance with follow-up. He was not patent (parasitemic) on that day, and no other participant became patent between day 23 and the end of the study on day 70. We consider this participant to be a prophylactic success. All but one of the persons who received treatment with the drug and in whom prophylaxis failed had delayed patency (mean day of patency, 19 days; range, 11 to 23 days) compared with the controls (mean day of patency, 10 days; range, 9 to 11 days). In cohort 1, the efficacy of treatment was 40% (95% CI, 12% to 74%). Cohort 2 consisted of 10 persons who received azithromycin until 28 days after exposure to sporozoites and 2 controls who received no treatment. Both controls became patent (on days 11 and 13); none of the participants who received drug treatment became patent. In cohort 2, the efficacy of azithromycin was 100% (lower 95% CI, 70%). Of the 10 participants in both cohorts who became parasitemic, only 3 had temperatures greater than 37.7 C, and 7 had symptoms characterized by chills, headache, myalgia, and anorexia. No volunteer required hospitalization. Adverse Effects of Azithromycin Although each day during the period of drug administration, study participants were interviewed for subjective side effects, we found only four instances of headache, four instances of stomach ache, and one instance of diarrhea not temporally linked to parasitemia. Each of these events was mild and lasted at most 1 day. In addition, one patient developed flulike symptoms 2 days after receiving the last dose of the drug. Neither the one woman in cohort 1 nor the three women in cohort 2 reported symptoms of vaginitis. The only abnormality in laboratory variables was total bilirubin values somewhat higher than the upper limit of normal (24 mol/L) in 3 participants. In 1 volunteer, the value on day 4 before the challenge (37.6 mol/L) had returned to normal by day 5 after the challenge (15.4 mol/L) while the participant was still receiving the drug. In 1 participant, the values were relatively constant during therapy (27.4 mol/L on day 4 before the challenge and 34.2 mol/L on day 5 after the challenge) and then returned to normal after therapy. In the third participant, a value of 30.8 mol/L was first recorded on day 5 and levels returned to normal after therapy. Discussion A causal and suppressive prophylactic regimen of azithromycin, 250 mg/d for 28 days after a single challenge with P. falciparum sporozoites, was successful in 10 of 10 persons studied. In contrast, a causal prophylactic regimen of azithromycin, 250 mg/d for 7 days after challenge, was successful in only 4 of 10 persons. We had hoped that azithromycin would be sufficiently concentrated in the liver to be completely causally prophylactic, because it has been reported that azithromycin liver concentrations are approximately 10 times that of serum concentrations [8]. The results of treatment for 7 days after the challenge show that, as with doxycycline [9], azithromycin has only partial causal prophylactic activity in the human challenge model. The mean percent efficacy determined here (40% [CI, 12% to 74%]) is much less than that recently achieved by Kuschner and colleagues [6] in a tria

Resistance produced in rats and mice by exposure to irradiated Plasmodium berghei.

Abstract Rats infected with Plasmodium berghei parasitized erythrocytes exposed to doses up to and including 16,000 r developed progressive parasitemias with obvious parasite multiplication and reinvasion of RBCs. Some rats receiving parasitized RBCs irradiated at levels of 17,000 and 18,000 r did not develop progressive infections. Infections were aborted by exposure to irradation at 19,000 r or higher. Inoculation with parasitized blood exposed to 20,000 r stimulated a resistance to a challenge infection with nonirradiated parasites. The peak parasitemias reached in the immunized animals were significantly lower than those of the nonimmunized controls and reduction in parasitemias began much earlier. The degree of acquired resistance induced by inoculation with irradiated parasites was influenced by the number of immunizing exposures before the challenging infection. The acquired resistance induced by irradiated infected RBCs could be detected also in a more susceptible animal such as the mouse. Twice-weekly inoculations of irradiated parasitized cells given three, five, and ten times extended the mean survival time after challenge considerably; a significant number of mice survived otherwise lethal infections. In both rats and mice the degree of acquired immunity was directly proportional to the number of immunizing doses. Similar results were obtained when immunizations occurred at weekly intervals or when given twice weekly. Likewise, no significant differences were detected when the challenge took place 1 or 2 weeks after the last immunizing dose.

Trypanosoma congolense: Natural and acquired resistance in the bovine

Abstract A total of 42 animals of various ages were infected with Trypanosoma congolense to investigate age resistance. Ten of eleven animals between 4 months and 1 year of age survived the infection without treatment. Two of eleven animals in the age range of 1 to 2 years also survived the infection whereas all 20 animals between 2 and 5 years of age died or needed treatment to survive. Young animals which needed no treatment to survive were refractive to challenge for at least 1 year after their last patent parasitemia. Older animals which required treatment to survive were also challenged at intervals after therapy. Three animals infected for 49 to 75 days before treatment were rechallenged 198 to 296 days later. Extensions in prepatent periods ranged from 5 to 13 days when compared to controls and the resulting infections were of a relapsing nature followed by self-cure. Effects of this disease on clinical parameters of previously infected animals were minimal. One animal infected for 196 days and rechallenged 501 days later had a prepatent period of 14 days as compared to 5 days for controls. This animal developed a brief relapsing infection followed by self-cure. Animals which were infected for periods of 41 to 77 days, received treatment, and were then rechallenged from 600 to 900 days later, showed some resistance to infection. Prepatent periods were extended from 1 to 3 days over those of control animals and although the resulting disease was severe, one of four animals self-cured without treatment. When animals which had self-cured primary challenges were rechallenged at periods up to 2 years later, they were completely refractory. When 12 animals which were presumed to be immune to syringe-passaged T. congolense were challenged by tsetse fly bite with the same strain of trypanosome, an appreciable immunity was evident. Five of twelve immune animals did not become patent while the other seven developed mild infections without severe clinical signs. All nine controls developed severe infections with eight requiring treatment to survive. When animals immune to the Trans-Mara I strain of T. congolense were challenged either by syringe or tsetse fly bite with a heterologous strain of T. congolense obtained from a different geographical area, no evidence of immunity was detected.

Trypanosoma congolense: immunization of mice, dogs, and cattle with gamma- irradiated parasites.

Abstract In a series of experiments involving 21 groups of mice, between 50 and 100 per cent of those inoculated with irradiated Trypanosoma congolense were completely protected from challenge inoculations of unirradiated T. congolense . Of 37 mice given a second or third challenge after 9 and 26 weeks, 23 remained parasite-free. Unimmunized control mice invariably developed parasitemias and most of them died as a result of the infection. Dogs and cattle failed to withstand challenge inoculations after they were treated with irradiated trypanosomes, though prepatency in these animals was longer than that of the controls.

Variants of Plasmodium berghei resistant to passive transfer of immune serum.

Abstract Antiserum from infected rats suppressed stock strain P. berghei infections in mice, the degree of suppression depending in part on the nature and amount of antiserum used. Relatively large amounts of antiserum did not, however, produce more than a transient suppression of parasitemia, which eventually increased rapidly and killed the host. Parasites multiplying in antiserum-treated hosts were subinoculated into normal mice; although these parasites showed no apparent alteration in virulence, they did show a decreased susceptibility to the same antiserum. The present work suggests that there is some biological variability in populations of mouse P. berghei and that rat antiserum can change the immunological nature of these populations, i.e., render them relatively antiserum resistant.

Trypanosoma rhodesiense: variant specificity of immunity induced by irradiated parasites.

Abstract Rats immunized with irradiated Trypanosoma rhodesiense resisted infection with the homologous strain. When similarly immunized rats were challenged with parasites obtained from rhesus monkeys infected with the same strain, resistance depended on when parasites were obtained from the donor monkeys. Immunized rats challenged with trypanosomes obtained from a monkey during the first peak of parasitemia were solidly immune; immunized rats challenged with trypanosomes obtained from monkeys after their initial peak of parasitemia all succumbed to the challenging infection. These observations indicate that parasites of a variant antigenic specificity arose during the course of the monkey infections. Neutralization tests performed on the various isolates from rats and monkeys using antiserum obtained from immunized rats confirmed that the immunity produced by irradiated trypanosomes was variant specific.