Safety, Immunogenicity, and Protective Efficacy against Controlled Human Malaria Infection of Plasmodium falciparum Sporozoite Vaccine in Tanzanian Adults

1In 2015 and in 2016, there were an estimated 429,000–730,500 deaths caused by malaria.– Plasmodium falciparum (Pf) is the cause of > 98% of malaria deaths and > 80% of malaria cases in sub-Saharan Africa. Our goal is to field a vaccine that will prevent infection with Pf and thereby prevent all manifestations of Pf malaria and parasite transmission from humans to mosquitoes.

2Plasmodium falciparum sporozoites (SPZ) are the only immunogens that have ever prevented Pf infection in > 90% of subjects.– Sanaria® PfSPZ Vaccine (Sanaria Inc., Rockville, MD) is composed of radiation-attenuated, aseptic, purified, cryopreserved PfSPZ., When administered by rapid intravenous injection, PfSPZ Vaccine protected 100% (6/6) of malaria-naïve subjects in the United States against mosquito bite–controlled human malaria infection (CHMI) with Pf parasites similar to those in the vaccine (homologous) 3 weeks after the last immunization, and 65% at 24 weeks. Protection was durable against homologous mosquito bite CHMI for at least 59 weeks and heterologous (parasites different than in vaccine) mosquito bite CHMI for at least 33 weeks. PfSPZ Vaccine also prevented naturally transmitted heterogeneous Pf in adults in Mali for at least 24 weeks (vaccine efficacy [VE] 52% by time to event and 29% by proportional analysis).

3We used the same dosage regimen as in the United States and Mali to evaluate the tolerability, safety, immunogenicity, and VE of PfSPZ Vaccine in young adult male Tanzanians. Previously, we had conducted the first modern CHMI in Africa and showed that injection of aseptic, purified, cryopreserved PfSPZ, Sanaria® PfSPZ Challenge, consistently infected Tanzanian volunteers and subsequently repeated in multiple other countries.– In this study, we took advantage of this capability to assess VE of PfSPZ Vaccine by CHMI with PfSPZ Challenge (NF54). The same PfSPZ Vaccine dosage regimen was less immunogenic and protective in Tanzanians than in Americans, and VE against homologous CHMI in Tanzania was lower (or similar) to VE against intense field exposure to heterogeneous Pf parasites in Mali.

4This double-blind, randomized, controlled trial was conducted in Bagamoyo, Tanzania, between April 2014 and August 2015. Sixty-seven healthy male volunteers of 18–35 years of age were recruited from higher learning institutions in Dar es Salaam. After initial screening, prospective volunteers were invited to the Bagamoyo Clinical Trial Unit of the Ifakara Health Institute (IHI) to complete informed consent and screening.

5All had to complete a 20-question assessment of trial understanding with a 100% correct response rate on the first or second attempt (Supplemental Table 1) to be eligible. Volunteers were screened using predetermined inclusion and exclusion criteria (Supplemental Tables 2 and 3). History of malaria in the previous 5 years or antibodies to Pf exported protein 1 (PfEXP1) by an enzyme-linked immunosorbent assay (ELISA) above a level associated with a single, recent Pf infection by CHMI (see the Antibody assays section) were the exclusion criteria. Hematology, biochemistry, and parasitology testing, including malaria thick blood smear (TBS), stool, and urine by microscopy was carried out. Tests for human immunodeficiency virus and hepatitis B and C were performed after counseling; volunteers were excluded if positive and referred for evaluation and management by appropriate local physicians. Volunteers were excluded if they had significant abnormalities on electrocardiograms.

6The trial was performed in accordance with Good Clinical Practices. The protocol was approved by institutional review boards (IRBs) of the IHI (Ref. No. IHI/IRB/No:02-2014), the National Institute for Medical Research Tanzania (NIMR/HQ/R.8a/Vol.IX/1691), the Ethikkommission Nordwest-und Zentralschweiz, Basel, Switzerland (reference number 261/13), and by the Tanzania Food and Drug Authority (Ref. No. TFDA 13/CTR/0003); registered at Clinical Trials.gov (NCT02132299); and conducted under U.S. FDA IND application.

7The IPs were Sanaria® PfSPZ Vaccine– and Sanaria® PfSPZ Challenge.– PfSPZ Vaccine consists of aseptic, purified, vialed, metabolically active, nonreplicating (radiation attenuated), cryopreserved PfSPZ (NF54 strain). It was stored, thawed, diluted, and administered by direct venous inoculation (DVI) in 0.5 mL through a 25-gauge needle.,,, PfSPZ Challenge is identical to PfSPZ Vaccine except it is not radiation attenuated. It was handled and administered like PfSPZ Vaccine. Preparation of IPs was supervised by the study pharmacist. After labeling the syringe, the pharmacist handed it to the clinical team through a window.

8Volunteers were allocated to five groups (Table 1; Figure 1). Forty-nine received PfSPZ Vaccine and eight normal saline (NS). Ten were additional infectivity controls. The clinical team and volunteers were blinded to assignment to vaccine or NS until study end.

9Three volunteers received consecutive doses of 3 × 104, 1.35 × 105, and 2.7 × 105 PfSPZ of PfSPZ Vaccine at 4-week intervals to assess safety (Group 1).

10Volunteers were randomized to receive 1.35 × 105 PfSPZ of PfSPZ Vaccine (N = 20) or NS (N = 4) (Group 2), or 2.7 × 105 PfSPZ of PfSPZ Vaccine (N = 20) or NS (N = 4) (Group 3) at 0, 4, 8, 12, and 20 weeks.

11Six volunteers were immunized with 2.7 × 105 PfSPZ of PfSPZ Vaccine on the same schedule as Group 3.

12Ten volunteers served as unblinded infectivity controls during CHMIs (see in the following paragraph): two with CHMI #1, two with CHMI #2, and six with CHMI #3.

13Vaccine efficacy was assessed by CHMI by DVI of 3.2 × 103 PfSPZ of PfSPZ Challenge. Controlled human malaria infection #1 was 3 weeks after the last immunization in Group 2. Controlled human malaria infection #2 was 3 weeks after the last immunization in Group 3. Controlled human malaria infection #3 was 24 weeks after the last immunization in Groups 3 and 4 and included the four volunteers in Group 3 who did not develop parasitemia after CHMI #2 and the six Group 4 volunteers. Volunteers were inpatients from day 9 after PfSPZ Challenge injection for observation until diagnosed and treated for malaria or until day 21; daily outpatient monitoring for TBS-negative volunteers continued until day 28. Thick blood smears were obtained every 12 hours on days 9–14 after CHMI and daily on days 15–21 until positive or until day 21. Thick blood smears could be performed more frequently, if volunteers had symptoms/signs consistent with malaria. After initiation of treatment, TBSs were assessed until two consecutive daily TBSs were negative and on day 28.

14Slide preparation and reading for TBSs were performed as described. Sensitivity was 2 parasites/μL blood unless the volunteer was symptomatic, in which case four times as many fields were read. Parasitemia was also determined by quantitative polymerase chain reaction (qPCR) with sensitivity of 0.1 parasites/μL blood based on a multiplex assay detecting Plasmodium spp. 18S genes and the human RNaseP gene as endogenous control. A second, more sensitive qPCR assay with a sensitivity of 0.05 parasites/μL blood and targeting the Pf-specific telomere-associated repetitive element 2 was used to reanalyze all samples that were negative by 18S-based qPCR. After the start of CHMI, the time of first blood sample positivity by qPCR was used to determine infection status and for the calculation of prepatent period. Volunteers were continuously monitored by qPCR until malaria treatment based on TBS positivity. The World Health Organization International Standard for Pf DNA Nucleic Acid Amplification Techniques (NIBSC, Hertfordshire, United Kingdom) was used as standard for calculation of parasite densities. DNA was extracted from 100 μL whole blood and eluted with 50 μL Elution Buffer using Quick-gDNA Blood MicroPrep Kit (Zymo Research, Irvine, CA). Blood samples were analyzed retrospectively by qPCR after storing at −80°C after the conclusion of CHMIs. To exclude field strain infections, parasite genotyping was performed on samples randomly chosen as described. In all cases in which TBS was negative and qPCR was considered positive, two consecutive samples were positive by qPCR.

15Volunteers were observed as inpatients for 48 hours after administration of IP and discharged with diaries and thermometers for recording AEs and temperatures and followed with daily telephone calls. Symptoms and signs (solicited and unsolicited) were recorded and graded by physicians: mild (easily tolerated), moderate (interfere with normal activity), severe (prevents normal activity), or life threatening. Axillary temperature was grade 1 (> 37.5–38.0°C), grade 2 (> 38.0–39.0°C), grade 3 (> 39.0–40.0°C), or grade 4 (> 40.0°C). Hematological and biochemical abnormalities were also assessed using standard clinical assays.

16During the first 7 days after injection of IPs, prespecified local (site of injection) and systemic AEs were solicited. Open-ended questioning was used to identify unsolicited AEs through day 28 (Supplemental Table 4). All AEs were assessed for severity and relatedness to IP administration. Adverse events were classified as definitely related, probably related, possibly related, unlikely to be related, and not related. Definitely, probably, and possibly were considered to be related. Unlikely to be related and not related were considered to be unrelated. For CHMIs, volunteers returned on day 9 for admission to the ward for diagnosis and treatment of malaria. Events during the 8–28 day period were assessed for relationship to Pf infection and considered related if the event was within 3 days before and 7 days after TBS was first positive.

17Sera were assessed for antibodies by ELISA, immunofluorescence assay (aIFA), and inhibition of sporozoite invasion (ISI) assay as described (see Supplemental Table 5). For ELISAs, the results are reported as the serum dilution at which the optical density (OD) was 1.0. Enzyme-linked immunosorbent assay for PfEXP1 was used to screen volunteers for possible malaria exposure (Supplemental Table 6). Any subject with an OD 1.0 of ≥ 600 was excluded. This was because we had previously determined in Tanzanians who underwent CHMI that antibodies to PfEXP1 at this level were a sensitive indicator of recent Pf infection (unpublished).

18T-cell responses in cryopreserved peripheral blood mononuclear cells (PBMC) were measured by flow cytometry in a single batch after the study as described. After stimulation, cells were stained as described. The staining panels are in Supplemental Table 7 and antibody clones and manufacturers are in Supplemental Table 8. All antigen-specific frequencies are reported after background subtraction of identical gates from the same sample incubated with control antigen. Data were analyzed with FlowJo v9.9.3 (TreeStar, Ashland, OR) and graphed in Prism v7.0a (GraphPad, San Diego, CA).

19Comparisons of categorical variables between groups were analyzed using 2-tailed Fisher’s exact test. Comparisons of continuous variables between groups were analyzed by 2-tailed nonparametric tests. For multiple group comparisons, the Kruskal–Wallis test was used. Time to event was assessed by the Kaplan–Meier curves and log-rank test. Vaccine efficacy by time to event was quantified using Cox proportional hazards ratios. Time to event data were analyzed from CHMI injection until positive TBS result or positive qPCR result. Controlled human malaria infection follow-up period lasted until day 28 after CHMI injection. Analyses of immunological data are described with the data.

20The funders were involved in study design, study management, data collection, data analysis, data interpretation, and writing the report. Salim Abdulla and Stephen L. Hoffman had full access to all data in the study and final responsibility for decision to submit for publication.

21Fifty-seven Tanzanian men (Table 1; Figure 1) met the criteria (Supplemental Tables 2 and 3) and received PfSPZ Vaccine (N = 49) or NS (N = 8). All volunteers had AA hemoglobin and normal G6PD activity. Thirty-one volunteers (46%) were heterozygous for α-thalassemia; 21 had evidence of latent tuberculosis infection by Quantiferon testing, but showed no evidence of active tuberculosis. One volunteer (group 2, NS) had Strongyloides stercoralis on screening and was successfully treated before vaccination (Table 1).

22Of 237 immunizations with PfSPZ Vaccine, 234 were completed with a single injection (98.7%). Two hundred and thirty injections (97.0%) were considered painless by the volunteer. For NS subjects, 39 of 40 immunizations (97.5%) were completed in a single injection and 39 of 40 (97.5%) considered painless by the volunteer. The nurse performing immunizations considered the procedure to be simple in 265 of 273 single injections (97.1%).

23One subject in Group 2 received four immunizations. The third immunization was withheld while the subject was evaluated for what was diagnosed as benign ethnic neutropenia., One subject in Group 4 missed his second immunization when he left town. All other subjects (other than Group 1 and added infectivity controls) received five immunizations.

24Among 49 volunteers who received 237 doses of PfSPZ Vaccine, there were 17 solicited AEs possibly related to IP (17/237 = 7.2%) in 10 of the 49 vaccinees (20.4%) (Table 2). Among eight volunteers who received 40 doses of NS, there were two solicited AEs possibly related to IP (2/40 = 5.0%) in one of the eight controls (12.5%) (Table 2). There were no AEs considered by the clinicians to be probably or definitely related to IP. There were no local or serious AEs. One episode each of headache and fever were grade 2; all other solicited AEs were grade 1. None of the comparisons of AEs between vaccinees and controls or between Group 2 (1.35 × 105 PfSPZ) and Groups 3 and 4 (2.7 × 105 PfSPZ) showed statistically significant differences (Table 2). Twenty-six of 49 vaccinees (53.1%) experienced 43 unsolicited AEs (0.88/individual) in the 28 days following injections #1–#4 and the 21 days before CHMI after injection #5. Seven of eight controls (87.5%) experienced 14 unsolicited AEs (2/individual) during this period. None of these unsolicited AEs recorded within 28 days of an immunization was considered related to IP.

25Laboratory abnormalities occurred at roughly equal rates comparing PfSPZ Vaccine recipients and controls, except for leukocytosis and eosinophilia, which were more frequent in controls (Table 3). There was no apparent explanation for these differences. A cyclic variation in total bilirubin following each immunization was observed equally in volunteers receiving vaccine or NS that was attributed to enriched diet, as the volunteers were transported to Bagamoyo from Dar es Salaam during the periods of immunization and CHMI and were amply fed (see Supplemental Figure 1). In Dar es Salaam, malaria transmission is low. No volunteer had malaria during screening or during the trial other than from CHMI.

26Forty-six vaccinees, eight NS controls, and 10 added infectivity controls underwent homologous CHMI. All subjects were negative by TBS and qPCR for Pf infection on the day of CHMI. Two volunteers were excluded from primary analysis—a Group 2 volunteer who left the area 2 days after administration of PfSPZ Challenge and a Group 4 volunteer who left 9 days after. Both volunteers were located and treated preemptively.

27Controlled human malaria infection was well tolerated with no local solicited AEs and three systemic solicited AEs (grade 1 headache in Group 3, grade 2 headache in Group 4, and grade 1 arthralgia in an infectivity control) in the 7 days post-administration of PfSPZ Challenge.

28The 18 NS and infectivity controls developed Pf infection after CHMI (16 TBS and qPCR positive and two TBS negative and qPCR positive) (Figure 2A–D and Supplemental Table 9). These included four NS and two infectivity controls in CHMI #1, the same in CHMI #2, and six infectivity controls in CHMI #3. All received the same lot of PfSPZ Challenge. One isolate of those positive from CHMI #1, one from CHMI #2, and four from CHMI #3 were genotyped, and all parasites tested were PfNF54. Vaccine efficacy was calculated based on the results of qPCR assays from the six controls in CHMI #1, CHMI #2, and CHMI #3 individually (Figure 2D).

29Seventeen of 18 volunteers who received five doses and 1/1 volunteer who received four doses developed parasitemia (Figure 2A), 15 positive by TBS and qPCR, and 3 by qPCR only (CHMI #1) (Supplemental Table 10). One volunteer was negative through day 28 by TBS and qPCR. Vaccine efficacy by proportional analysis was 5.56% (95% confidence interval [CI]: 3.61%, 14.73%; P > 0.99, Fisher’s exact test, 2-tailed). There was no significant delay in parasitemia by qPCR in the vaccinees as compared with controls (P = 0.4481 by log rank).

30First CHMI at 3 weeks (CHMI #2): 16/20 volunteers who received five doses developed parasitemia (Figure 2B), all positive by TBS and qPCR; four volunteers were negative through day 28 by TBS and qPCR. Vaccine efficacy by proportional analysis was 20% (95% CI: 4.62%, 35.38%; P = 0.543). There was a delay in the onset of parasitemia in vaccinees as compared with controls (P = 0.015 by log rank).

31Second CHMI at 24 weeks (CHMI #3): The four uninfected volunteers from the first CHMI underwent a second CHMI 24 weeks after the last vaccine dose (Figure 2C). Three were negative by TBS and qPCR through day 28 day. The fourth volunteer, who was asymptomatic, was reported to have a positive TBS on day 12 and treated. The sample with positive TBS was negative by retrospective qPCR. Reevaluation of the TBS indicated an error in slide reading (false-positive). Vaccine efficacy by proportional analysis at this time point was 100% (for 3/3 and 4/4 protected: 95% CI: 43.8%, 100%, and 51.01%, 100%; P = 0.012 and 0.005, respectively). However, given the 20% VE at 3 weeks by proportional analysis, overall VE by proportional analysis was considered to be 20%.

32First CHMI at 24 weeks after the last vaccine dose (CHMI #3): 4/5 vaccinees developed parasitemia by TBS and qPCR. The fifth was negative by TBS, but positive by qPCR (see Supplemental Table 10). There was one excluded volunteer (see the previous paragraph). Vaccine efficacy by proportional analysis was 0% (P > 0.99%). There was a significant delay in the onset of parasitemia by qPCR in vaccinees as compared with controls (P = 0.001 by log rank).

33Volunteers heterozygous for α-thalassemia were no more likely to be TBS negative and qPCR positive than volunteers without α-thalassemia (three of 27 versus three of 34, P = 1.0). Protection from CHMI did not correlate with α-thalassemia status; 3/37 with normal hemoglobin and 2/29 heterozygous for α-thalassemia were protected.

34Although the median prepatent periods by TBS in controls in each CHMI group (12.5, 13.0, and 12.0, respectively) were shorter than in the vaccinees in Groups 2–4 (14.0, 14.0, and 15.3 days, respectively), these differences did not reach the level of statistical significance (P = 0.486, P = 0.491, and P = 0.333, respectively) (Supplemental Table 9). The prepatent periods by qPCR in vaccineees in Group 3 (3 and 24 week CHMIs) and Group 4 (24 week CHMI) were significantly longer than in the respective controls (Figure 2D). The parasite densities by qPCR and TBS at the time of diagnosis for each individual are in Supplemental Table 10. The median parasite density in controls versus vaccinees at the time of first positivity were 0.5 versus 0.4 parasites/μL for qPCR (P = 0.5714) and 11.2 versus 15.0 parasites/μL for TBS (P = 0.1492).

35Sixteen controls developed parasitemia by TBS; 9 (56%) never had symptoms (Supplemental Table 11). Headache occurred in 7/7 symptomatic individuals. One of two control volunteers only positive by qPCR did not have any symptoms; the second had headache 8 days after qPCR spontaneously reverted to negative. No volunteer had symptoms at the time of first positive qPCR.

36Thirty-five immunized volunteers developed parasitemia by TBS; 20 (57%) never had symptoms. Three volunteers had temperature > 39.0°C; all other clinical manifestations were grade 1 or 2. Fever (28.6%) and headache (31.4%) were most common. Compared with controls, elevated temperature was more common in vaccinees with positive TBSs (9/35 versus 0/16, P = 0.043). There was no significant difference in the frequency of headache between controls and vaccinees. In the three volunteers in Group 2 who were qPCR positive and TBS negative, one developed headache 3 days after qPCR positivity. No volunteer had symptoms at the time of first positive qPCR.

37No unexpected changes were observed following CHMI. Declines in lymphocyte counts were observed in TBS positive controls and vaccinees (mean decline 1,110 ± 720 cells/μL and 1,180 ± 680 cells/μL, respectively) on day of first positive TBS. Absolute lymphocyte counts less than 1,000 cells/μL were observed in 8/16 and 16/35 TBS positive controls and vaccinees. All lymphocyte counts returned to the baseline by day 28. There were mild decreases in platelet counts in TBS positive subjects, but all platelet counts were > 100 × 103 cells/μL.

38Volunteers with positive TBSs were treated with either atovaquone/proguanil (N = 43) or artemether/lumefantrine (N = 8) within 24 hours of first positive TBS. Normal saline and infectivity controls who were TBS negative (N = 2) were treated at day 28.

39Antibodies against PfCSP by ELISA 1), PfSPZ by aIFA 2), and PfSPZ by ISI 3) in sera taken 2 weeks after the last vaccine dose and just before CHMI (20–23 days after the last dose) for Groups 2 (CHMI #1) and 3 (CHMI #2) are in Figure 3A–C. The median responses and those uninfected and infected by qPCR are shown.

40For all three assays, median antibody responses before first CHMI were higher in uninfected than in infected vaccinees. There was a significant difference in median net aIFA responses between infected and uninfected volunteers in Group 3 before CHMI #1 (P = 0.0499, Wilcoxon Rank-Sum Test), but not PfCSP (P = 0.290) or for ISI (P = 0.249).

41In sera collected before CHMI #3 (170–171 days after the last vaccine dose), antibodies by the three assays for Group 4 and for the four volunteers in Group 3 uninfected in CHMI #1 who underwent CHMI #2 are in Figure 3D–F. All data appear in Supplemental Table 12.

42After the fifth dose, in the PfCSP ELISA, volunteers were considered to have seroconverted if their net OD 1.0 and OD 1.0 ratio calculated, respectively, by subtracting or dividing by the prevaccination antibody OD 1.0, were ≥ 50 and ≥ 3.0. By these criteria, 15/18 volunteers (83%) in Group 2, 20/20 (100%) in Group 3, and 5/5 (100%) in Group 4 seroconverted, median net OD 1.0 of positives of 1,189, 2,685, and 961, and median OD 1.0 ratio of positives of 11.50, 21.15, and 37.83, respectively (Supplemental Table 13). In the aIFA, volunteers with a net arbitrary fluorescence unit (AFU) 2 × 105 of ≥ 150 and a ratio of post- to pre-AFU 2 × 105 of ≥ 3.0 were considered positive (Supplemental Table 13). By these criteria, 17/18 volunteers (94%) in Group 2, 18/20 (90%) in Group 3, and 5/5 (100%) in Group 4 seroconverted, median net OD 1.0 of positives of 2,844, 1,165, and 1,820, and median OD 1.0 ratio of positives of 1,193.00, 552.88, and 224.86, respectively (Supplemental Table 13). For the ISI, volunteers with a net ISI activity of ≥ 10% and a ratio of post- to pre-ISI activity of ≥ 3.0 were considered positive. By these criteria, 3/18 volunteers (17%) in Group 2, 8/20 (40%) in Group 3, and 3/5 (60%) in Group 4 were positive, median net OD 1.0 of positives of 22.05, 38.92, and 12.44, and median OD 1.0 ratio of positives of 19.79, 12.53, and 13.44, respectively (Supplemental Table 13).

43Two weeks after the fifth dose in Group 2 (1.35 × 105 PfSPZ) and groups 3 and 4 (2.7 × 105 PfSPZ), there were antibodies to PfCSP in 15/18 and 25/25 subjects, respectively. Ten of 25 volunteers immunized with 2.7 × 105 PfSPZ made antibodies to Pf apical membrane antigen 1 and 4–16% responded to PfCelTOS, PfMSP5, PfMSP1, or Pf erythrocyte binding antigen 175 (PfEBA175) (Supplemental Table 14). The presence of antibodies, albeit at low incidence, against proteins first expressed in late liver stages (PfMSP1 and PfEBA175) was unexpected; results were confirmed by repeating the assays. No antibody responses were associated with protection.

44T cells against liver-stage malaria parasites in mice and nonhuman primates immunized with radiation-attenuated SPZ mediate protection,– and it is likely this is the case in humans. CD8 and CD4 T-cell responses generally peak after the first vaccination with PfSPZ Vaccine. In this trial, T-cell responses were measured before immunization, 2 weeks after the first and 2 weeks after the final immunization in Group 2 (1.35 × 105 PfSPZ). For technical reasons (loss of viability), the other groups could not be studied.

45After the first vaccination, the percent of Pf red blood cell (PfRBC)-specific and PfSPZ-specific cytokine-producing memory CD4 T-cell responses increased by 0.25 ± 0.06 (mean ± SEM) and 0.24 ± 0.04, respectively (Figure 4A, B). Throughout, “naïve T cell” refers to cells that co-express CCR7 and CD45RA, and “memory T cell” refers to all other T cells. After the final vaccination, at week 22, the CD4 T-cell responses were above prevaccine responses by 0.17 ± 0.05 and 0.18 ± 0.05% points, respectively. These responses were lower than after the same immunization regimen in malaria-naïve U.S. adults.

46PfRBC-specific CD8 T cells were not significantly above the prevaccine levels, and PfSPZ-specific CD8 T cells were slightly above background (Figure 4C, D); responses were lower than in U.S. adults.,

47In contrast to other PfSPZ Vaccine trials,,– there was negligible change in the frequency of circulating γδ T cells (Figure 4E) or activation as measured by change in expression of the activation markers HLA-DR and CD38 following immunization (Figure 4F). To identify potential explanations for lower cellular immune responses in Tanzanians, we examined frequency of T regulatory (Treg) cells (CD4+Foxp3+CD25+CD127−) expressing the activation marker CD137 (also known as 4-1BB) after stimulation with PfRBC. There was no difference in prevaccine frequency of PfRBC-specific Tregs in the Tanzanians as compared with Americans (Figure 4G). Consistent with CD4 and CD8 T-cell responses, PfRBC-specific Tregs were highest after first immunization (Figure 4H). Last, the prevaccine frequency of total memory T cells relative to total naïve T cells was significantly higher than in Americans (Figure 4I).

48To our knowledge, this was the first assessment of the VE of a malaria vaccine in Africa against CHMI. Plasmodium falciparum SPZ Vaccine was well tolerated and safe but less immunogenic and protective in Tanzanian men than in U.S. volunteers.

49In our studies, all 18 controls became infected. Four of 20 (20%) recipients of five doses of 2.7 × 105 PfSPZ did not become infected after homologous CHMI by DVI 3 weeks after the last immunization. By contrast, 12/13 (92.3%) volunteers in the United States who received five doses of 2.7 × 105 PfSPZ were protected after homologous CHMI by mosquito bite 3 weeks after the last vaccine dose. When the four uninfected Tanzanian volunteers underwent repeat homologous CHMI at 24 weeks after the last dose, all four (100%) were protected. In the United States, Seven of 10 previously protected volunteers were protected when they underwent homologous CHMI at 24 weeks and all five volunteers in the United States who were protected at 21 weeks after the last immunization (four doses of 2.7 × 105 PfSPZ) were protected against repeat mosquito-administered CHMI at 59 weeks. This could be due to boosting by the small numbers of PfSPZ administered during the CHMI, or is more likely due to the fact that in these protected individuals, the protective immune responses induced by immunization were sustained.

50The same exact immunization regimen was assessed for VE against intense field transmission of heterogeneous Pf in Mali. Vaccine efficacy against infection with Pf on TBS was 52% by time to event and 29% by proportional analysis during 24 weeks after the last vaccine dose. This was higher than the VE by proportional analysis against homologous CHMI in Tanzania. In Tanzania, there was a significant delay in the onset of parasitemia after CHMI at 3 and 24 weeks in subjects who received five doses of 2.7 × 105 PfSPZ and were not fully protected (Figure 2B–D). Nonetheless, the proportional analysis suggests that homologous CHMI by DVI of a 100% infectious dose of homologous PfSPZ Challenge is at least as rigorous as a test of VE and potentially more rigorous than intense field transmission of heterogeneous Pf.

51Vaccine-induced antibody and T-cell responses in the Tanzanians were lower than in malaria-naïve Americans who received the exact same dosage regimen. Two weeks after the last dose, the median antibody responses to PfCSP, the major protein on the surface of PfSPZ, were 4.3 times lower in the Tanzanians than those in Americans (P = 0.0012, Student’s t-test, 2-tailed), but significantly higher than in Malians who received the same immunization regimen (P = 0.002) (Figure 3G).

52The T-cell responses were also lower than in Americans, (Figure 4), but this could only be assessed in PBMCs from individuals who received the lower dose (five doses of 1.35 × 105 PfSPZ), not in the individuals who received the higher dose (five doses of 2.7 × 105 PfSPZ), the group that had sustained protection for 24 weeks. Thus, it is possible that had PBMCs from the higher dose group been assessed, responses would have been comparable to the responses in nonimmune Americans. The Tanzanians who were assessed had a significantly higher proportion of total memory T cells compared with total naïve T cells at the baseline than did the Americans. This higher frequency of memory cells compared with naïve cells may explain the lower immunogenicity due to less available naïve cells for expansion during the vaccinations. Moreover, the greater frequency of non-Pf–specific memory T cells may compete for infected cell contacts during pathogen surveillance. These data suggest that PfSPZ Vaccine immunogenicity may be dependent on cumulative history of Pf exposure. Another explanation is that an activated immune microenvironment in the Tanzanians as compared with the Americans reduced immune responses. Helminth infections have been associated with reduced immune responses to malaria; however, the paucity of helminth infections in this population does not support helminth infection as a cause of the reduced immune responses.

53There were no differences between vaccine and NS placebo recipients in regard to vaccine tolerability or AEs; 97.1% of the DVI administrations were rated painless and no volunteer experienced any local AE. Systemic AEs, most commonly headache, were mild, infrequent, and of short duration, with a similar frequency in NS controls as in vaccinees (no statistically significant differences in rates).

54Among the controls, 16 of 18 were positive for Pf by TBS after CHMI. However, all 18 were positive by qPCR. This is consistent with findings in Gabon after CHMI. It is likely that preexisting asexual blood stage immunity limits Pf replication in some individuals. Thus, they never reach the threshold for detection by TBS. In our CHMI studies in Bagamoyo, we now use qPCR to confirm positive TBS, and retrospectively or in real time, assess parasitemia in all volunteers by qPCR.

55We propose that increasing the numbers of PfSPZ per dose and altering intervals between doses will lead to overcoming the downregulation of humoral and cell-mediated immunity most likely because of previous exposure to Pf and thereby increase immune responses to PfSPZ Vaccine and VE. We also hypothesize that immune responses in younger, less malaria-exposed individuals will be of greater magnitude than those in adults.