Multidose Priming and Delayed Boosting Improve Plasmodium falciparum Sporozoite Vaccine Efficacy Against Heterologous P. falciparum Controlled Human Malaria Infection.

BACKGROUND: A live-attenuated Plasmodium falciparum sporozoite (SPZ) vaccine (PfSPZ Vaccine) has shown up to 100% protection against controlled human malaria infection (CHMI) using homologous parasites (same P. falciparum strain as in the vaccine). Using a more stringent CHMI, with heterologous parasites (different P. falciparum strain), we assessed the impact of higher PfSPZ doses, a novel multi-dose prime regimen, and a delayed vaccine boost upon vaccine efficacy (VE). METHODS: We immunized 4 groups that each contained 15 healthy, malaria-naive adults. Group 1 received 5 doses of 4.5 x 105 PfSPZ (Days 1, 3, 5, and 7; Week 16). Groups 2, 3, and 4 received 3 doses (Weeks 0, 8, and 16), with Group 2 receiving 9.0 × 105/doses; Group 3 receiving 18.0 × 105/doses; and Group 4 receiving 27.0 × 105 for dose 1 and 9.0 × 105 for doses 2 and 3. VE was assessed by heterologous CHMI after 12 or 24 weeks. Volunteers not protected at 12 weeks were boosted prior to repeat CHMI at 24 weeks. RESULTS: At 12-week CHMI, 6/15 (40%) participants in Group 1 (P = .04) and 3/15 (20%) participants in Group 2 remained aparasitemic, as compared to 0/8 controls. At 24-week CHMI, 3/13 (23%) participants in Group 3 and 3/14 (21%) participants in Group 4 remained aparasitemic, versus 0/8 controls (Groups 2-4, VE not significant). Postboost, 9/14 (64%) participants versus 0/8 controls remained aparasitemic (3/6 in Group 1, P = .025; 6/8 in Group 2, P = .002). CONCLUSIONS: Administering 4 stacked priming injections (multi-dose priming) resulted in 40% VE against heterologous CHMI, while dose escalation of PfSPZ using single-dose priming was not significantly protective. Boosting unprotected subjects improved VE at 24 weeks, to 64%. CLINICAL TRIALS REGISTRATION: NCT02601716.

Mosquito bite immunization with radiation-attenuated Plasmodium falciparum sporozoites: safety, tolerability, protective efficacy and humoral immunogenicity.

BACKGROUND: In this phase 1 clinical trial, healthy adult, malaria-naïve subjects were immunized with radiation-attenuated Plasmodium falciparum sporozoites (PfRAS) by mosquito bite and then underwent controlled human malaria infection (CHMI). The PfRAS model for immunization against malaria had previously induced >90 % sterile protection against homologous CHMI. This study was to further explore the safety, tolerability and protective efficacy of the PfRAS model and to provide biological specimens to characterize protective immune responses and identify protective antigens in support of malaria vaccine development. METHODS: Fifty-seven subjects were screened, 41 enrolled and 30 received at least one immunization. The true-immunized subjects received PfRAS via mosquito bite and the mock-immunized subjects received mosquito bites from irradiated uninfected mosquitoes. Sera and peripheral blood mononuclear cells (PBMCs) were collected before and after PfRAS immunizations. RESULTS: Immunization with PfRAS was generally safe and well tolerated, and repeated immunization via mosquito bite did not appear to increase the risk or severity of AEs. Local adverse events (AEs) of true-immunized and mock-immunized groups consisted of erythaema, papules, swelling, and induration and were consistent with reactions from mosquito bites seen in nature. Two subjects, one true- and one mock-immunized, developed large local reactions that completely resolved, were likely a result of mosquito salivary antigens, and were withdrawn from further participation as a safety precaution. Systemic AEs were generally rare and mild, consisting of headache, myalgia, nausea, and low-grade fevers. Two true-immunized subjects experienced fever, malaise, myalgia, nausea, and rigours approximately 16 h after immunization. These symptoms likely resulted from pre-formed antibodies interacting with mosquito salivary antigens. Ten subjects immunized with PfRAS underwent CHMI and five subjects (50 %) were sterilely protected and there was a significant delay to parasitaemia in the other five subjects. All ten subjects developed humoral immune responses to whole sporozoites and to the circumsporozoite protein prior to CHMI, although the differences between protected and non-protected subjects were not statistically significant for this small sample size. CONCLUSIONS: The protective efficacy of this clinical trial (50 %) was notably less than previously reported (>90 %). This may be related to differences in host genetics or the inherent variability in mosquito biting behavior and numbers of sporozoites injected. Differences in trial procedures, such as the use of leukapheresis prior to CHMI and of a longer interval between the final immunization and CHMI in these subjects compared to earlier trials, may also have reduced protective efficacy. This trial has been retrospectively registered at ISRCTN ID 17372582, May 31, 2016.

Ex vivo tetramer staining and cell surface phenotyping for early activation markers CD38 and HLA-DR to enumerate and characterize malaria antigen-specific CD8+ T-cells induced in human volunteers immunized with a Plasmodium falciparum adenovirus-vectored malaria vaccine expressing AMA1.

BACKGROUND: Malaria is responsible for up to a 600,000 deaths per year; conveying an urgent need for the development of a malaria vaccine. Studies with whole sporozoite vaccines in mice and non-human primates have shown that sporozoite-induced CD8+ T cells targeting liver stage antigens can mediate sterile protection. There is a need for a direct method to identify and phenotype malaria vaccine-induced CD8+ T cells in humans. METHODS: Fluorochrome-labelled tetramers consisting of appropriate MHC class I molecules in complex with predicted binding peptides derived from Plasmodium falciparum AMA-1 were used to label ex vivo AMA-1 epitope specific CD8+ T cells from research subjects responding strongly to immunization with the NMRC-M3V-Ad-PfCA (adenovirus-vectored) malaria vaccine. The identification of these CD8+ T cells on the basis of their expression of early activation markers was also investigated. RESULTS: Analyses by flow cytometry demonstrated that two of the six tetramers tested: TLDEMRHFY: HLA-A*01:01 and NEVVVKEEY: HLA-B*18:01, labelled tetramer-specific CD8+ T cells from two HLA-A*01:01 volunteers and one HLA-B*18:01 volunteer, respectively. By contrast, post-immune CD8+ T cells from all six of the immunized volunteers exhibited enhanced expression of the CD38 and HLA-DRhi early activation markers. For the three volunteers with positive tetramer staining, the early activation phenotype positive cells included essentially all of the tetramer positive, malaria epitope- specific CD8+ T cells suggesting that the early activation phenotype could identify all malaria vaccine-induced CD8+ T cells without prior knowledge of their exact epitope specificity. CONCLUSIONS: The results demonstrated that class I tetramers can identify ex vivo malaria vaccine antigen-specific CD8+ T cells and could therefore be used to determine their frequency, cell surface phenotype and transcription factor usage. The results also demonstrated that vaccine antigen-specific CD8+ T cells could be identified by activation markers without prior knowledge of their antigen-specificity, using a subunit vaccine for proof-of-concept. Whether, whole parasite or adjuvanted protein vaccines will also induce {CD38 and HLA-DRhi}+ CD8+ T cell populations reflective of the antigen-specific response will the subject of future investigations.

IMRAS-Immunization with radiation-attenuated Plasmodium falciparum sporozoites by mosquito bite: Cellular immunity to sporozoites, CSP, AMA1, TRAP and CelTOS.

BACKGROUND: Immunization with radiation-attenuated sporozoites (RAS) by mosquito bites provides >90% sterile protection against Plasmodium falciparum malaria in humans. We conducted a clinical trial based on data from previous RAS clinical trials that suggested that 800-1200 infected bites should induce ~50% protective vaccine efficacy (VE) against controlled human malaria infection (CHMI) administered three weeks after the final immunization. Two cohorts were immunized separately. VE was 55% in Cohort 1 but 90% in Cohort 2, the cohort that received a higher first dose and a reduced (fractional) fifth dose. Immune responses were better boosted by the fractional fifth dose in Cohort 2 and suggested the importance of the fractional fifth dose for increased protection in Cohort 2 responses. Three protected subjects were later boosted and were protected suggesting that protection could be extended to at least 67 weeks. METHODS: The ex vivo FluoroSpot assay was used to measure peripheral IFN-γ, IL2, and IFN-γ+IL2 responses to PfNF54 sporozoites and malaria antigens CSP, AMA1, TRAP, and CelTOS using pools of synthetic overlapping 15mer peptides spanning each antigen. RESULTS: There was no correlation between IFN-γ, IL2, and IFN-γ+IL2 responses to sporozoites and protection, but fold-increases between post-4th and post-5th responses greater than 1.0 occurred mostly in protected subjects. IFN-γ and IL2 responses to TRAP, CelTOS and CSP occurred only in protected subjects. Peripheral IFN-γ, IL2, and IFN-γ+IL2 responses were short-lived and low by 27 weeks post-CHMI but were restored by boosting. CONCLUSIONS: These studies highlight the importance of vaccine dose and schedule for vaccine efficacy, and suggest that CSP, TRAP, AMA1 and CelTOS may be targets of protective immunity. The correlation between fold-increases in responses and protection should be explored in other vaccine trials. TRIAL REGISTRATION: ClinicalTrials.gov NCT01994525.

A three-antigen Plasmodium falciparum DNA prime-Adenovirus boost malaria vaccine regimen is superior to a two-antigen regimen and protects against controlled human malaria infection in healthy malaria-naïve adults.

BACKGROUND: A DNA-prime/human adenovirus serotype 5 (HuAd5) boost vaccine encoding Plasmodium falciparum (Pf) circumsporozoite protein (PfCSP) and Pf apical membrane antigen-1 (PfAMA1), elicited protection in 4/15 (27%) of subjects against controlled human malaria infection (CHMI) that was statistically associated with CD8+ T cell responses. Subjects with high level pre-existing immunity to HuAd5 were not protected, suggesting an adverse effect on vaccine efficacy (VE). We replaced HuAd5 with chimpanzee adenovirus 63 (ChAd63), and repeated the study, assessing both the two-antigen (CSP, AMA1 = CA) vaccine, and a novel three-antigen (CSP, AMA1, ME-TRAP = CAT) vaccine that included a third pre-erythrocytic stage antigen [malaria multiple epitopes (ME) fused to the Pf thrombospondin-related adhesive protein (TRAP)] to potentially enhance protection. METHODOLOGY: This was an open label, randomized Phase 1 trial, assessing safety, tolerability, and VE against CHMI in healthy, malaria naïve adults. Forty subjects (20 each group) were to receive three monthly CA or CAT DNA priming immunizations, followed by corresponding ChAd63 boost four months later. Four weeks after the boost, immunized subjects and 12 infectivity controls underwent CHMI by mosquito bite using the Pf3D7 strain. VE was assessed by determining the differences in time to parasitemia as detected by thick blood smears up to 28-days post CHMI and utilizing the log rank test, and by calculating the risk ratio of each treatment group and subtracting from 1, with significance calculated by the Cochran-Mantel-Haenszel method. RESULTS: In both groups, systemic adverse events (AEs) were significantly higher after the ChAd63 boost than DNA immunizations. Eleven of 12 infectivity controls developed parasitemia (mean 11.7 days). In the CA group, 15 of 16 (93.8%) immunized subjects developed parasitemia (mean 12.0 days). In the CAT group, 11 of 16 (63.8%) immunized subjects developed parasitemia (mean 13.0 days), indicating significant protection by log rank test compared to infectivity controls (p = 0.0406) and the CA group (p = 0.0229). VE (1 minus the risk ratio) in the CAT group was 25% compared to -2% in the CA group. The CA and CAT vaccines induced robust humoral (ELISA antibodies against CSP, AMA1 and TRAP, and IFA responses against sporozoites and Pf3D7 blood stages), and cellular responses (IFN-γ FluoroSpot responses to CSP, AMA1 and TRAP) that were not associated with protection. CONCLUSIONS: This study demonstrated that the ChAd63 CAT vaccine exhibited significant protective efficacy, and confirmed protection was afforded by adding a third antigen (T) to a two-antigen (CA) formulation to achieve increased VE. Although the ChAd63-CAT vaccine was associated with increased frequencies of systemic AEs compared to the CA vaccine and, historically, compared to the HuAd5 vectored malaria vaccine encoding CSP and AMA1, they were transient and associated with increased vector dosing.

Development of a metabolically active, non-replicating sporozoite vaccine to prevent Plasmodium falciparum malaria.

Immunization of volunteers by the bite of mosquitoes carrying radiation-attenuated Plasmodium falciparum sporozoites protects greater than 90% of such volunteers against malaria, if adequate numbers of immunizing biting sessions and sporozoite-infected mosquitoes are used. Nonetheless, until recently it was considered impossible to develop, license and commercialize a live, whole parasite P. falciparum sporozoite (PfSPZ) vaccine. In 2003 Sanaria scientists reappraised the potential impact of a metabolically active, non-replicating PfSPZ vaccine, and outlined the challenges to producing such a vaccine. Six years later, significant progress has been made in overcoming these challenges. This progress has enabled the manufacture and release of multiple clinical lots of a 1(st) generation metabolically active, non-replicating PfSPZ vaccine, the Sanaria PfSPZ Vaccine, submission of a successful Investigational New Drug application to the US Food and Drug Administration, and initiation of safety, immunogenicity and protective efficacy studies in volunteers in MD, US. Efforts are now focused on how best to achieve submission of a successful Biologics License Application and introduce the vaccine to the primary target population of African children in the shortest possible period of time. This will require implementation of a systematic, efficient clinical development plan. Short term challenges include optimizing the (1) efficiency and scale up of the manufacturing process and quality control assays, (2) dosage regimen and method of administration, (3) potency of the vaccine, and (4) logistics of delivering the vaccine to those who need it most, and finalizing the methods for vaccine stabilization and attenuation. A medium term goal is to design and build a facility for manufacturing highly potent and stable vaccine for pivotal Phase 3 studies and commercial launch.

IMRAS-A clinical trial of mosquito-bite immunization with live, radiation-attenuated P. falciparum sporozoites: Impact of immunization parameters on protective efficacy and generation of a repository of immunologic reagents.

BACKGROUND: Immunization with radiation-attenuated sporozoites (RAS) by mosquito bite provides >90% sterile protection against Plasmodium falciparum (Pf) malaria in humans. RAS invade hepatocytes but do not replicate. CD8+ T cells recognizing parasite-derived peptides on the surface of infected hepatocytes are likely the primary protective mechanism. We conducted a randomized clinical trial of RAS immunization to assess safety, to achieve 50% vaccine efficacy (VE) against controlled human malaria infection (CHMI), and to generate reagents from protected and non-protected subjects for future identification of protective immune mechanisms and antigens. METHODS: Two cohorts (Cohort 1 and Cohort 2) of healthy, malaria-naïve, non-pregnant adults age 18-50 received five monthly immunizations with infected (true-immunized, n = 21) or non-infected (mock-immunized, n = 5) mosquito bites and underwent homologous CHMI at 3 weeks. Immunization parameters were selected for 50% protection based on prior clinical data. Leukapheresis was done to collect plasma and peripheral blood mononuclear cells. RESULTS: Adverse event rates were similar in true- and mock-immunized subjects. Two true- and two mock-immunized subjects developed large local reactions likely caused by mosquito salivary gland antigens. In Cohort 1, 11 subjects received 810-1235 infected bites; 6/11 (55%) were protected against CHMI vs. 0/3 mock-immunized and 0/6 infectivity controls (VE 55%). In Cohort 2, 10 subjects received 839-1131 infected bites with a higher first dose and a reduced fifth dose; 9/10 (90%) were protected vs. 0/2 mock-immunized and 0/6 controls (VE 90%). Three/3 (100%) protected subjects administered three booster immunizations were protected against repeat CHMI vs. 0/6 controls (VE 100%). Cohort 2 uniquely showed a significant rise in IFN-γ responses after the third and fifth immunizations and higher antibody responses to CSP. CONCLUSIONS: PfRAS were generally safe and well tolerated. Cohort 2 had a higher first dose, reduced final dose, higher antibody responses to CSP and significant rise of IFN-γ responses after the third and fifth immunizations. Whether any of these factors contributed to increased protection in Cohort 2 requires further investigation. A cryobank of sera and cells from protected and non-protected individuals was generated for future immunological studies and antigen discovery. TRIAL REGISTRATION: ClinicalTrials.gov NCT01994525.

Protection against Plasmodium falciparum malaria by PfSPZ Vaccine.

BACKGROUND: A radiation-attenuated Plasmodium falciparum (Pf) sporozoite (SPZ) malaria vaccine, PfSPZ Vaccine, protected 6 of 6 subjects (100%) against homologous Pf (same strain as in the vaccine) controlled human malaria infection (CHMI) 3 weeks after 5 doses administered intravenously. The next step was to assess protective efficacy against heterologous Pf (different from Pf in the vaccine), after fewer doses, and at 24 weeks. METHODS: The trial assessed tolerability, safety, immunogenicity, and protective efficacy of direct venous inoculation (DVI) of 3 or 5 doses of PfSPZ Vaccine in non-immune subjects. RESULTS: Three weeks after final immunization, 5 doses of 2.7 × 105 PfSPZ protected 12 of 13 recipients (92.3% [95% CI: 48.0, 99.8]) against homologous CHMI and 4 of 5 (80.0% [10.4, 99.5]) against heterologous CHMI; 3 doses of 4.5 × 105 PfSPZ protected 13 of 15 (86.7% [35.9, 98.3]) against homologous CHMI. Twenty-four weeks after final immunization, the 5-dose regimen protected 7 of 10 (70.0% [17.3, 93.3]) against homologous and 1 of 10 (10.0% [-35.8, 45.6]) against heterologous CHMI; the 3-dose regimen protected 8 of 14 (57.1% [21.5, 76.6]) against homologous CHMI. All 22 controls developed Pf parasitemia. PfSPZ Vaccine was well tolerated, safe, and easy to administer. No antibody or T cell responses correlated with protection. CONCLUSIONS: We have demonstrated for the first time to our knowledge that PfSPZ Vaccine can protect against a 3-week heterologous CHMI in a limited group of malaria-naive adult subjects. A 3-dose regimen protected against both 3-week and 24-week homologous CHMI (87% and 57%, respectively) in this population. These results provide a foundation for developing an optimized immunization regimen for preventing malaria. TRIAL REGISTRATION: ClinicalTrials.gov NCT02215707. FUNDING: Support was provided through the US Army Medical Research and Development Command, Military Infectious Diseases Research Program, and the Naval Medical Research Center's Advanced Medical Development Program.

Human adenovirus 5-vectored Plasmodium falciparum NMRC-M3V-Ad-PfCA vaccine encoding CSP and AMA1 is safe, well-tolerated and immunogenic but does not protect against controlled human malaria infection.

BACKGROUND: In a prior study, a DNA prime / adenovirus boost vaccine (DNA/Ad) expressing P. falciparum circumsporozoite protein (CSP) and apical membrane antigen-1 (AMA1) (NMRC-M3V-D/Ad-PfCA Vaccine) induced 27% protection against controlled human malaria infection (CHMI). To investigate the contribution of DNA priming, we tested the efficacy of adenovirus vaccine alone (NMRC-M3V-Ad-PfCA ) in a Phase 1 clinical trial. METHODOLOGY/PRINCIPAL FINDINGS: The regimen was a single intramuscular injection with two non-replicating human serotype 5 adenovectors encoding CSP and AMA1, respectively. One x 10 (10) particle units of each construct were combined prior to administration. The regimen was safe and well-tolerated. Four weeks later, 18 study subjects received P. falciparum CHMI administered by mosquito bite. None were fully protected although one showed delayed onset of parasitemia. Antibody responses were low, with geometric mean CSP ELISA titer of 381 (range<50-1626) and AMA1 ELISA of 4.95 µg/mL (range 0.2-38). Summed ex vivo IFN-γ ELISpot responses to overlapping peptides were robust, with geometric mean spot forming cells/million peripheral blood mononuclear cells [sfc/m] for CSP of 273 (range 38-2550) and for AMA1 of 1303 (range 435-4594). CD4+ and CD8+ T cell IFN-γ responses to CSP were positive by flow cytometry in 25% and 56% of the research subjects, respectively, and to AMA1 in 94% and 100%, respectively. SIGNIFICANCE: In contrast to DNA/Ad, Ad alone did not protect against CHMI despite inducing broad, cell-mediated immunity, indicating that DNA priming is required for protection by the adenovirus-vectored vaccine. ClinicalTrials.gov Identifier: NCT00392015.

Adenovirus-5-vectored P. falciparum vaccine expressing CSP and AMA1. Part B: safety, immunogenicity and protective efficacy of the CSP component.

BACKGROUND: A protective malaria vaccine will likely need to elicit both cell-mediated and antibody responses. As adenovirus vaccine vectors induce both these responses in humans, a Phase 1/2a clinical trial was conducted to evaluate the efficacy of an adenovirus serotype 5-vectored malaria vaccine against sporozoite challenge. METHODOLOGY/PRINCIPAL FINDINGS: NMRC-MV-Ad-PfC is an adenovirus vector encoding the Plasmodium falciparum 3D7 circumsporozoite protein (CSP). It is one component of a two-component vaccine NMRC-M3V-Ad-PfCA consisting of one adenovector encoding CSP and one encoding apical membrane antigen-1 (AMA1) that was evaluated for safety and immunogenicity in an earlier study (see companion paper, Sedegah et al). Fourteen Ad5 seropositive or negative adults received two doses of NMRC-MV-Ad-PfC sixteen weeks apart, at 1 x 1010 particle units per dose. The vaccine was safe and well tolerated. All volunteers developed positive ELISpot responses by 28 days after the first immunization (geometric mean 272 spot forming cells/million[sfc/m]) that declined during the following 16 weeks and increased after the second dose to levels that in most cases were less than the initial peak (geometric mean 119 sfc/m). CD8+ predominated over CD4+ responses, as in the first clinical trial. Antibody responses were poor and like ELISpot responses increased after the second immunization but did not exceed the initial peak. Pre-existing neutralizing antibodies (NAb) to Ad5 did not affect the immunogenicity of the first dose, but the fold increase in NAb induced by the first dose was significantly associated with poorer antibody responses after the second dose, while ELISpot responses remained unaffected. When challenged by the bite of P. falciparum-infected mosquitoes, two of 11 volunteers showed a delay in the time to patency compared to infectivity controls, but no volunteers were sterilely protected. SIGNIFICANCE: The NMRC-MV-Ad-PfC vaccine expressing CSP was safe and well tolerated given as two doses, but did not provide sterile protection. TRIAL REGISTRATION: ClinicalTrials.gov NCT00392015.

New World monkey Aotus nancymae as a model for Campylobacter jejuni infection and immunity.

Three groups of six monkeys (Aotus nancymae) each were inoculated intragastrically with increasing doses of Campylobacter jejuni. Infection resulted in fecal colonization (100% of monkeys), dose-related diarrhea, and robust immune responses. Colonization duration and diarrhea rate were reduced upon secondary challenge. A. nancymae may be useful for studying anti-Campylobacter vaccine efficacy.