Megan Dowler

Protection of Humans against Malaria by Immunization with Radiation-Attenuated Plasmodium falciparum Sporozoites

During 1989-1999, 11 volunteers were immunized by the bites of 1001-2927 irradiated mosquitoes harboring infectious sporozoites of Plasmodium falciparum (Pf) strain NF54 or clone 3D7/NF54. Ten volunteers were first challenged by the bites of Pf-infected mosquitoes 2-9 weeks after the last immunization, and all were protected. A volunteer challenged 10 weeks after the last immunization was not protected. Five previously protected volunteers were rechallenged 23-42 weeks after a secondary immunization, and 4 were protected. Two volunteers were protected when rechallenged with a heterologous Pf strain (7G8). In total, there was protection in 24 of 26 challenges. These results expand published findings demonstrating that immunization by exposure to thousands of mosquitoes carrying radiation-attenuated Pf sporozoites is safe and well tolerated and elicits strain-transcendent protective immunity that persists for at least 42 weeks.

Randomized, Double-Blind, Phase 2a Trial of Falciparum Malaria Vaccines RTS,S/AS01B and RTS,S/AS02A in Malaria-Naive Adults: Safety, Efficacy, and Immunologic Associates of Protection

BACKGROUND To further increase the efficacy of malaria vaccine RTS,S/AS02A, we tested the RTS,S antigen formulated using the AS01B Adjuvant System (GlaxoSmithKline Biologicals). METHODS In a double-blind, randomized trial, 102 healthy volunteers were evenly allocated to receive RTS,S/AS01B or RTS,S/AS02A vaccine at months 0, 1, and 2 of the study, followed by malaria challenge. Protected vaccine recipients were rechallenged 5 months later. RESULTS RTS,S/AS01B and RTS,S/AS02A were well tolerated and were safe. The efficacy of RTS,S/AS01B and RTS,S/AS02A was 50% (95% confidence interval [CI], 32.9%-67.1%) and 32% (95% CI, 17.6%-47.6%), respectively. At the time of initial challenge, the RTS,S/AS01B group had greater circumsporozoite protein (CSP)-specific immune responses, including higher immunoglobulin (Ig) G titers, higher numbers of CSP-specific CD4(+) T cells expressing 2 activation markers (interleukin-2, interferon [IFN]-gamma, tumor necrosis factor-alpha, or CD40L), and more ex vivo IFN-gamma enzyme-linked immunospots (ELISPOTs) than did the RTS,S/AS02A group. Protected vaccine recipients had a higher CSP-specific IgG titer (geometric mean titer, 188 vs 73 mug/mL; P < .001), higher numbers of CSP-specific CD4(+) T cells per 10(6) CD4(+) T cells (median, 963 vs 308 CSP-specific CD4(+) T cells/10(6) CD4(+) T cells; P < .001), and higher numbers of ex vivo IFN-gamma ELISPOTs (mean, 212 vs 96 spots/million cells; P < .001). At rechallenge, 4 of 9 vaccine recipients in each group were still completely protected. CONCLUSIONS The RTS,S/AS01B malaria vaccine warrants comparative field trials with RTS,S/AS02A to determine the best formulation for the protection of children and infants. The association between complete protection and immune responses is a potential tool for further optimization of protection. Trial registration. ClinicalTrials.gov identifier NCT00075049.

Efficacy of Recombinant Circumsporozoite Protein Vaccine Regimens against Experimental Plasmodium falciparum Malaria

After initial successful evaluation of the circumsporozoite-based vaccine RTS,S/SBAS2, developed by SmithKline Beecham Biologicals with the Walter Reed Army Institute of Research, protective efficacy of several regimens against Plasmodium falciparum challenge was determined. A controlled phase 1/2a study evaluated 1 or 2 standard doses of RTS,S/SBAS2 in 2 groups whose members received open-label therapy and 3 immunizations in blinded groups who received standard, one-half, or one-fifth doses. RTS,S/SBAS2 was safe and immunogenic in all groups. Of the 41 vaccinees and 23 control subjects who underwent sporozoite challenge, malaria developed in 7 of 10 who received 1 dose, in 7 of 14 who received 2 doses, in 3 of 6 who received 3 standard doses, in 3 of 7 who received 3 one-half doses, in 3 of 4 who received 3 one-fifth doses, and in 22 of 23 control subjects. Overall protective efficacy of RTS,S/SBAS2 was 41% (95% confidence interval, 22%-56%; P=.0006). This and previous studies have shown that 2 or 3 doses of RTS,S/SBAS2 protect against challenge with P. falciparum sporozoites.

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 1/2a Study of the Malaria Vaccine Candidate Apical Membrane Antigen-1 (AMA-1) Administered in Adjuvant System AS01B or AS02A

Background This Phase 1/2a study evaluated the safety, immunogenicity, and efficacy of an experimental malaria vaccine comprised of the recombinant Plasmodium falciparum protein apical membrane antigen-1 (AMA-1) representing the 3D7 allele formulated with either the AS01B or AS02A Adjuvant Systems. Methodology/Principal Findings After a preliminary safety evaluation of low dose AMA-1/AS01B (10 µg/0.5 mL) in 5 adults, 30 malaria-naïve adults were randomly allocated to receive full dose (50 µg/0.5 mL) of AMA-1/AS01B (n = 15) or AMA-1/AS02A (n = 15), followed by a malaria challenge. All vaccinations were administered intramuscularly on a 0-, 1-, 2-month schedule. All volunteers experienced transient injection site erythema, swelling and pain. Two weeks post-third vaccination, anti-AMA-1 Geometric Mean Antibody Concentrations (GMCs) with 95% Confidence Intervals (CIs) were high: low dose AMA-1/AS01B 196 µg/mL (103–371 µg/mL), full dose AMA-1/AS01B 279 µg/mL (210–369 µg/mL) and full dose AMA-1/AS02A 216 µg/mL (169–276 µg/mL) with no significant difference among the 3 groups. The three vaccine formulations elicited equivalent functional antibody responses, as measured by growth inhibition assay (GIA), against homologous but not against heterologous (FVO) parasites as well as demonstrable interferon-gamma (IFN-γ) responses. To assess efficacy, volunteers were challenged with P. falciparum-infected mosquitoes, and all became parasitemic, with no significant difference in the prepatent period by either light microscopy or quantitative polymerase chain reaction (qPCR). However, a small but significant reduction of parasitemia in the AMA-1/AS02A group was seen with a statistical model employing qPCR measurements. Significance All three vaccine formulations were found to be safe and highly immunogenic. These immune responses did not translate into significant vaccine efficacy in malaria-naïve adults employing a primary sporozoite challenge model, but encouragingly, estimation of parasite growth rates from qPCR data may suggest a partial biological effect of the vaccine. Further evaluation of the immunogenicity and efficacy of the AMA-1/AS02A formulation is ongoing in a malaria-experienced pediatric population in Mali. Trial Registration www.clinicaltrials.gov NCT00385047

Preerythrocytic, live-attenuated Plasmodium falciparum vaccine candidates by design

Falciparum malaria is initiated when Anopheles mosquitoes transmit the Plasmodium sporozoite stage during a blood meal. Irradiated sporozoites confer sterile protection against subsequent malaria infection in animal models and humans. This level of protection is unmatched by current recombinant malaria vaccines. However, the live-attenuated vaccine approach faces formidable obstacles, including development of accurate, reproducible attenuation techniques. We tested whether Plasmodium falciparum could be attenuated at the early liver stage by genetic engineering. The P. falciparum genetically attenuated parasites (GAPs) harbor individual deletions or simultaneous deletions of the sporozoite-expressed genes P52 and P36. Gene deletions were done by double-cross-over recombination to avoid genetic reversion of the knockout parasites. The gene deletions did not affect parasite replication throughout the erythrocytic cycle, gametocyte production, mosquito infections, and sporozoite production rates. However, the deletions caused parasite developmental arrest during hepatocyte infection. The double-gene deletion line exhibited a more severe intrahepatocytic growth defect compared with the single-gene deletion lines, and it did not persist. This defect was assessed in an in vitro liver-stage growth assay and in a chimeric mouse model harboring human hepatocytes. The strong phenotype of the double knockout GAP justifies its human testing as a whole-organism vaccine candidate using the established sporozoite challenge model. GAPs might provide a safe and reproducible platform to develop an efficacious whole-cell malaria vaccine that prevents infection at the preerythrocytic stage.

Phase 2a Trial of 0, 1, and 3 Month and 0, 7, and 28 Day Immunization Schedules of Malaria Vaccine RTS,S/AS02 in Malaria-Naive Adults at the Walter Reed Army Institute of Research

BACKGROUND Immunization with RTS,S/AS02 consistently protects some vaccinees against malaria infection in experimental challenges and in field trials. A brief immunization schedule against falciparum malaria would be compatible with the Expanded Programme on Immunization, or in combination with other prevention measures, interrupt epidemic malaria or protect individuals upon sudden travel to an endemic area. METHODS We conducted an open label, Phase 2a trial of two different full dose schedules of RTS,S/AS02 in 40 healthy malaria-naïve adults. Cohort 1 (n=20) was immunized on a 0, 1, and 3 month schedule and Cohort 2 (n=20) on a 0, 7, and 28 day schedule. Three weeks later, 38 vaccinees and 12 unimmunized infectivity controls underwent malaria challenge. RESULTS Both regimens had a good safety and tolerability profile. Peak GMCs of antibody to the circumsporozoite protein (CSP) were similar in Cohort 1 (78 microg/mL; 95% CI: 45-134) and Cohort 2 (65 microg/mL; 95% CI: 40-104). Vaccine efficacy for Cohort 1 was 45% (95% CI: 18-62%) and for Cohort 2, 39% (95% CI: 11-56%). Protected volunteers had a higher GMC of anti-CSP antibody (114 microg/mL) than did volunteers with a 2-day delay (70 microg/mL) or no delay (30 microg/mL) in the time to onset of parasitemia (Kruskal-Wallis, p=0.019). A trend was seen for higher CSP-specific IFN-gamma responses in PBMC from protected volunteers only in Cohort 1, but not in Cohort 2, for ex vivo and for cultured ELISPOT assays. CONCLUSION In malaria-naïve adults, the efficacy of three-dose RTS,S/AS02 regimens on either a 0, 1, and 3 month schedule or an abbreviated 0, 7, and 28 day schedule was not discernibly different from two previously reported trials of two-dose regimens given at 0, 1 month that conferred 47% (95% CI: -19 to 76%) protection and in another trial 42% (95% CI: 5-63%). A strong association of CSP-specific antibody with protection against malaria challenge is observed and confirms similar observations made in other studies. Subsequent trials of adjuvanted RTS,S in African children and infants on a 0, 1, and 2 month schedule have demonstrated a favorable safety and efficacy profile.

First-in-human evaluation of genetically attenuated Plasmodium falciparum sporozoites administered by bite of Anopheles mosquitoes to adult volunteers

BACKGROUND Immunization with genetically engineered, attenuated malaria parasites (GAP) that arrest during liver infection confers sterile protection in mouse malaria models. A first generation Plasmodium falciparum GAP (Pf p52(-)/p36(-) GAP) was previously generated by deletion of two pre-erythrocytic stage-expressed genes (P52 and P36) in the NF54 strain. METHODS A first-in-human, proof-of-concept, safety and immunogenicity clinical trial in six human volunteers was conducted. Exposure consisted of delivery of Pf p52(-)/p36(-) GAP sporozoites via infected Anopheles mosquito bite with a five-bite/volunteer exposure followed by an approximately 200-bite exposure/volunteer one month later. RESULTS The exposures were well tolerated with mild to moderate local and systemic reactions. All volunteers remained blood stage negative after low dose exposure. Five volunteers remained blood stage negative after high dose exposure. One volunteer developed peripheral parasitemia twelve days after high dose exposure. Together the findings indicate that Pf p52(-)/p36(-) GAP was severely but not completely attenuated. All six volunteers developed antibodies to CSP. Furthermore, IFN-γ responses to whole sporozoites and multiple antigens were elicited in 5 of 6 volunteers, with both CD4 and CD8 cell cytokine production detected. CONCLUSION Severe attenuation and favorable immune responses following administration of a first generation Pf p52(-)/p36(-) GAP suggests that further development of live-attenuated strains using genetic engineering should be pursued.

Mosquito bisection as a variable in estimates of PCR-derived malaria sporozoite rates

BackgroundHighly sensitive polymerase chain reaction (PCR) methods offer an alternative to the light microscopy examination of mosquito salivary glands for the determination of malaria sporozoite rates in wild caught female Anopheles. Removal of mosquito abdomens is assumed to eliminate false positives caused by malaria oocyst DNA in the midgut. This assumption has not been tested with current gold standard PCR assays, and for the variety of conditions that specimens could encounter in the laboratory and field.MethodsLaboratory Anopheles stephensi were used that had been infected with Plasmodium falciparum 6–7 days and 14 days post infection (p.i.), when oocysts only and oocysts + sporozoites, respectively, are developed. Mosquitoes were killed and immediately frozen, air dried before being frozen, or stored under humid conditions overnight before being frozen, to simulate a range of conditions in the field. Additionally, abdomens were removed anterior to, at, or posterior to the junction of the abdomen and thorax, and both portions were processed using a standard nested PCR of the small sub-unit nuclear ribosomal genes (ssrDNA) with products visualized on agarose gels.ResultsOverall, 4.1 % (4/97) of head + thorax samples that were 6–7 days p.i. gave apparent false positives for sporozoites, compared to 9.3 % (9/97) that were positive for abdomens. No positives (0/52) were obtained when similar specimens were bisected anterior to the junction of the thorax and abdomen, compared to 21.2 % (11/52) that were positive for posterior portions. Multiple bands were noted for positives from the ‘Frozen’ treatment and the rate of false negatives due to DNA degradation appears higher under the ‘Humid’ treatment. Reproducibility of results for the ‘Frozen’ treatment was 90 %.ConclusionsDespite the importance of specimen condition and the bisection step in determining sporozoite rates, little attention has been paid to them in the literature. Recommendations from this study are that: 1) care needs to be taken to reduce DNA degradation in the field; 2) mosquito abdomens be separated anterior to the junction of the thorax and abdomen; and 3) DNA sequencing of a subsample of positive results should be undertaken if possible.

Using infective mosquitoes to challenge monkeys with Plasmodium knowlesi in malaria vaccine studies

BackgroundWhen rhesus monkeys (Macaca mulatta) are used to test malaria vaccines, animals are often challenged by the intravenous injection of sporozoites. However, natural exposure to malaria comes via mosquito bite, and antibodies can neutralize sporozoites as they traverse the skin. Thus, intravenous injection may not fairly assess humoral immunity from anti-sporozoite malaria vaccines. To better assess malaria vaccines in rhesus, a method to challenge large numbers of monkeys by mosquito bite was developed.MethodsSeveral species and strains of mosquitoes were tested for their ability to produce Plasmodium knowlesi sporozoites. Donor monkey parasitaemia effects on oocyst and sporozoite numbers and mosquito mortality were documented. Methylparaben added to mosquito feed was tested to improve mosquito survival. To determine the number of bites needed to infect a monkey, animals were exposed to various numbers of P. knowlesi-infected mosquitoes. Finally, P. knowlesi-infected mosquitoes were used to challenge 17 monkeys in a malaria vaccine trial, and the effect of number of infectious bites on monkey parasitaemia was documented.ResultsAnopheles dirus, Anopheles crascens, and Anopheles dirus X (a cross between the two species) produced large numbers of P. knowlesi sporozoites. Mosquito survival to day 14, when sporozoites fill the salivary glands, averaged only 32% when donor monkeys had a parasitaemia above 2%. However, when donor monkey parasitaemia was below 2%, mosquitoes survived twice as well and contained ample sporozoites in their salivary glands. Adding methylparaben to sugar solutions did not improve survival of infected mosquitoes. Plasmodium knowlesi was very infectious, with all monkeys developing blood stage infections if one or more infected mosquitoes successfully fed. There was also a dose-response, with monkeys that received higher numbers of infected mosquito bites developing malaria sooner.ConclusionsAnopheles dirus, An. crascens and a cross between these two species all were excellent vectors for P. knowlesi. High donor monkey parasitaemia was associated with poor mosquito survival. A single infected mosquito bite is likely sufficient to infect a monkey with P. knowlesi. It is possible to efficiently challenge large groups of monkeys by mosquito bite, which will be useful for P. knowlesi vaccine studies.