Innate immune activation restricts priming and protective efficacy of the radiation-attenuated PfSPZ malaria vaccine.

A systems analysis was conducted to determine the potential molecular mechanisms underlying differential immunogenicity and protective efficacy results of a clinical trial of the radiation-attenuated whole sporozoite PfSPZ Vaccine in African infants. Innate immune activation and myeloid signatures at pre-vaccination baseline correlated with protection from Pf parasitemia in placebo controls. These same signatures were associated with susceptibility to parasitemia among infants who received the highest and most protective PfSPZ Vaccine dose. Machine learning identified spliceosome, proteosome, and resting dendritic cell signatures as pre-vaccination features predictive of protection after highest-dose PfSPZ vaccination, whereas baseline CSP-specific IgG predicted non-protection. Pre-vaccination innate inflammatory and myeloid signatures were associated with higher sporozoite-specific IgG Ab response but undetectable PfSPZ-specific CD8+ T-cell responses post-vaccination. Consistent with these human data, innate stimulation in vivo conferred protection against infection by sporozoite injection in malaria-naïve mice while diminishing the CD8+ T-cell response to radiation-attenuated sporozoites. These data suggest a dichotomous role of innate stimulation for malaria protection and induction of protective immunity of whole-sporozoite malaria vaccines. The uncoupling of vaccine-induced protective immunity achieved by Abs from more protective CD8+ T cell responses suggest that PfSPZ Vaccine efficacy in malaria-endemic settings may be constrained by opposing antigen presentation pathways.

A replication competent Plasmodium falciparum parasite completely attenuated by dual gene deletion.

Vaccination with infectious Plasmodium falciparum (Pf) sporozoites (SPZ) administered with antimalarial drugs (PfSPZ-CVac), confers superior sterilizing protection against infection when compared to vaccination with replication-deficient, radiation-attenuated PfSPZ. However, the requirement for drug administration constitutes a major limitation for PfSPZ-CVac. To obviate this limitation, we generated late liver stage-arresting replication competent (LARC) parasites by deletion of the Mei2 and LINUP genes (mei2-/linup- or LARC2). We show that Plasmodium yoelii (Py) LARC2 sporozoites did not cause breakthrough blood stage infections and engendered durable sterilizing immunity against various infectious sporozoite challenges in diverse strains of mice. We next genetically engineered a PfLARC2 parasite strain that was devoid of extraneous DNA and produced cryopreserved PfSPZ-LARC2. PfSPZ-LARC2 liver stages replicated robustly in liver-humanized mice but displayed severe defects in late liver stage differentiation and did not form liver stage merozoites. This resulted in complete abrogation of parasite transition to viable blood stage infection. Therefore, PfSPZ-LARC2 is the next-generation vaccine strain expected to unite the safety profile of radiation-attenuated PfSPZ with the superior protective efficacy of PfSPZ-CVac.

Implementation of a Randomized, Placebo-Controlled Trial of Live Attenuated Malaria Sporozoite Vaccines in an Indonesian Military Study Population.

Malaria eradication efforts prioritize safe and efficient vaccination strategies, although none with high-level efficacy against malaria infection are yet available. Among several vaccine candidates, Sanaria® PfSPZ Vaccine and Sanaria PfSPZ-CVac are, respectively, live radiation- and chemo-attenuated sporozoite vaccines designed to prevent infection with Plasmodium falciparum, the leading cause of malaria-related morbidity and mortality. We are conducting a randomized normal saline placebo-controlled trial called IDSPZV1 that will analyze the safety, tolerability, immunogenicity, and efficacy of PfSPZ Vaccine and PfSPZ-CVac administered pre-deployment to malaria-naive Indonesian soldiers assigned to temporary duties in a high malaria transmission area. We describe the manifold challenges of enrolling and immunizing 345 soldier participants at their home base in western Indonesia before their nearly 6,000-km voyage to eastern Indonesia, where they are being monitored for incident P. falciparum and Plasmodium vivax malaria cases during 9 months of exposure. The unique regulatory, ethical, and operational complexities of this trial demonstrate the importance of thorough planning, frequent communication, and close follow-up with stakeholders. Effective engagement with the military community and the ability to adapt to unanticipated events have proven key to the success of this trial.

Ultra-low volume intradermal administration of radiation-attenuated sporozoites with the glycolipid adjuvant 7DW8-5 completely protects mice against malaria.

Radiation-attenuated sporozoite (RAS) vaccines can completely prevent blood stage Plasmodium infection by inducing liver-resident memory CD8+ T cells to target parasites in the liver. Such T cells can be induced by 'Prime-and-trap' vaccination, which here combines DNA priming against the P. yoelii circumsporozoite protein (CSP) with a subsequent intravenous (IV) dose of liver-homing RAS to "trap" the activated and expanding T cells in the liver. Prime-and-trap confers durable protection in mice, and efforts are underway to translate this vaccine strategy to the clinic. However, it is unclear whether the RAS trapping dose must be strictly administered by the IV route. Here we show that intradermal (ID) RAS administration can be as effective as IV administration if RAS are co-administrated with the glycolipid adjuvant 7DW8-5 in an ultra-low inoculation volume. In mice, the co-administration of RAS and 7DW8-5 in ultra-low ID volumes (2.5 µL) was completely protective and dose sparing compared to standard volumes (10-50 µL) and induced protective levels of CSP-specific CD8+ T cells in the liver. Our finding that adjuvants and ultra-low volumes are required for ID RAS efficacy may explain why prior reports about higher volumes of unadjuvanted ID RAS proved less effective than IV RAS. The ID route may offer significant translational advantages over the IV route and could improve sporozoite vaccine development.

Ultra-low volume intradermal administration of radiation-attenuated sporozoites with the glycolipid adjuvant 7DW8-5 completely protects mice against malaria.

Malaria is caused by Plasmodium parasites and was responsible for over 247 million infections and 619,000 deaths in 2021. Radiation-attenuated sporozoite (RAS) vaccines can completely prevent blood stage infection by inducing protective liver-resident memory CD8+ T cells. Such T cells can be induced by 'prime-and-trap' vaccination, which here combines DNA priming against the P. yoelii circumsporozoite protein (CSP) with a subsequent intravenous (IV) dose of liver-homing RAS to "trap" the activated and expanding T cells in the liver. Prime-and-trap confers durable protection in mice, and efforts are underway to translate this vaccine strategy to the clinic. However, it is unclear whether the RAS trapping dose must be strictly administered by the IV route. Here we show that intradermal (ID) RAS administration can be as effective as IV administration if RAS are co-administrated with the glycolipid adjuvant 7DW8-5 in an ultra-low inoculation volume. In mice, the co-administration of RAS and 7DW8-5 in ultra-low ID volumes (2.5 µL) was completely protective and dose sparing compared to standard volumes (10-50 µL) and induced protective levels of CSP-specific CD8+ T cells in the liver. Our finding that adjuvants and ultra-low volumes are required for ID RAS efficacy may explain why prior reports about higher volumes of unadjuvanted ID RAS proved less effective. The ID route may offer significant translational advantages over the IV route and could improve sporozoite vaccine development.

Sporozoite immunization: innovative translational science to support the fight against malaria.

INTRODUCTION: Malaria, a devastating febrile illness caused by protozoan parasites, sickened 247,000,000 people in 2021 and killed 619,000, mostly children and pregnant women in sub-Saharan Africa. A highly effective vaccine is urgently needed, especially for Plasmodium falciparum (Pf), the deadliest human malaria parasite. AREAS COVERED: Sporozoites (SPZ), the parasite stage transmitted by Anopheles mosquitoes to humans, are the only vaccine immunogen achieving >90% efficacy against Pf infection. This review describes >30 clinical trials of PfSPZ vaccines in the U.S.A., Europe, Africa, and Asia, based on first-hand knowledge of the trials and PubMed searches of 'sporozoites,' 'malaria,' and 'vaccines.' EXPERT OPINION: First generation (radiation-attenuated) PfSPZ vaccines are safe, well tolerated, 80-100% efficacious against homologous controlled human malaria infection (CHMI) and provide 18-19 months protection without boosting in Africa. Second generation chemo-attenuated PfSPZ are more potent, 100% efficacious against stringent heterologous (variant strain) CHMI, but require a co-administered drug, raising safety concerns. Third generation, late liver stage-arresting, replication competent (LARC), genetically-attenuated PfSPZ are expected to be both safe and highly efficacious. Overall, PfSPZ vaccines meet safety, tolerability, and efficacy requirements for protecting pregnant women and travelers exposed to Pf in Africa, with licensure for these populations possible within 5 years. Protecting children and mass vaccination programs to block transmission and eliminate malaria are long-term objectives.

Safety and Immunogenicity of Radiation-Attenuated PfSPZ Vaccine in Equatoguinean Infants, Children, and Adults.

The radiation-attenuated Plasmodium falciparum sporozoites (PfSPZ) Vaccine has demonstrated safety and immunogenicity in 5-month-old to 50-year-old Africans in multiple trials. Except for one, each trial has restricted enrollment to either infants and children or adults < 50 years old. This trial was conducted in Equatorial Guinea and assessed the safety, tolerability, and immunogenicity of three direct venous inoculations of 1.8 × 106 or 2.7 × 106 PfSPZ, of PfSPZ Vaccine, or normal saline administered at 8-week intervals in a randomized, double-blind, placebo-controlled trial stratified by age (6-11 months and 1-5, 6-10, 11-17, 18-35, and 36-61 years). All doses were successfully administered. In all, 192/207 injections (93%) in those aged 6-61 years were rated as causing no or mild pain. There were no significant differences in solicited adverse events (AEs) between vaccinees and controls in any age group (P ≥ 0.17). There were no significant differences between vaccinees and controls with respect to the rates or severity of unsolicited AEs or laboratory abnormalities. Development of antibodies to P. falciparum circumsporozoite protein occurred in 67/69 vaccinees (97%) and 0/15 controls. Median antibody levels were highest in infants and 1-5-year-olds and declined progressively with age. Antibody responses in children were greater than in adults protected against controlled human malaria infection. Robust immunogenicity, combined with a benign AE profile, indicates children are an ideal target for immunization with PfSPZ Vaccine.

Clonal evolution and TCR specificity of the human TFH cell response to Plasmodium falciparum CSP.

T follicular helper (TFH) cells play a crucial role in the development of long-lived, high-quality B cell responses after infection and vaccination. However, little is known about how antigen-specific TFH cells clonally evolve in response to complex pathogens and what guides the targeting of different epitopes. Here, we assessed the cell phenotype, clonal dynamics, and T cell receptor (TCR) specificity of human circulating TFH (cTFH) cells during successive malaria immunizations with radiation-attenuated Plasmodium falciparum (Pf) sporozoites. Repeated parasite exposures induced a dynamic, polyclonal cTFH response with high frequency of cells specific to a small number of epitopes in Pf circumsporozoite protein (PfCSP), the primary sporozoite surface protein and well-defined vaccine target. Human leukocyte antigen (HLA) restrictions and differences in TCR generation probability were associated with differences in the epitope targeting frequency and indicated the potential of amino acids 311 to 333 in the Th2R/T* region as a T cell supertope. But most of vaccine-induced anti-amino acid 311 to 333 TCRs, including convergent TCRs with high sequence similarity, failed to tolerate natural polymorphisms in their target peptide sequence, thus demonstrating that the TFH cell response was limited to the vaccine strain. These data suggest that the high parasite diversity in endemic areas will limit boosting of the vaccine-induced TFH cell response by natural infections. Our findings may guide the further design of PfCSP-based malaria vaccines able to induce potent T helper cell responses for broad, long-lasting antibody responses.

A First for Human Vaccinology: GMP Compliant Radiation Attenuation of Plasmodium falciparum Sporozoites for Production of a Vaccine Against Malaria.

Ionizing radiation (UV, X-ray and É£) administered at an appropriate dose to pathogenic organisms can prevent replication while preserving metabolic activity. We have established the GMP process for attenuation by ionizing radiation of the Plasmodium falciparum (Pf) sporozoites (SPZ) in Sanaria® PfSPZ Vaccine, a protective vaccine against malaria. Mosquitoes raised and infected aseptically with Pf were transferred into infected mosquito transport containers (IMTC) and É£-irradiated using a 60Co source. PfSPZ were then extracted, purified, vialed, and cryopreserved. To establish the appropriate radiation conditions, the irradiation field inside the IMTCs was mapped using radiochromic film and alanine transfer dosimeters. Dosimeters were irradiated for times calculated to provide 120-170 Gy at the minimum dose location inside the IMTC and regression analysis was used to determine the time required to achieve a lower 95% confidence interval for 150 Gy. A formula incorporating the half-life of 60Co was then used to construct tables of irradiation times for each calendar day. From the mapping studies, formulae were derived to estimate the minimum and maximum doses of irradiation received inside the IMTC from a reference dosimeter mounted on the outside wall. For PfSPZ Vaccine manufacture a dose of 150 Gy was targeted for each irradiation event, a dose known to completely attenuate PfSPZ. The reference dosimeters were processed by the National Institute of Standards and Technology. There have been 587 irradiation events to produce PfSPZ Vaccine during 13 years which generated multiple lots released for pre-clinical studies and clinical trials. The estimated doses at the minimum dose location (mean 154.3 ± 1.77 Gy; range 150.0-159.3 Gy), and maximum dose location (mean 166.3 ± 3.65 Gy, range 155.7 to 175.3 Gy), in IMTCs were normally distributed. Overall dose uniformity was 1.078 ± 0.012. There was no siginifcant change in measured dose over 13 years. As of January 2022, 21 clinical trials of PfSPZ Vaccine have been conducted, with 1,740 volunteers aged 5 months to 61 years receiving 5,648 doses of PfSPZ Vaccine totalling >5.3 billion PfSPZ administered. There have been no breakthrough infections, confirming the consistency and robustness of the radiation attenuation process.

Heterologous protection against malaria by a simple chemoattenuated PfSPZ vaccine regimen in a randomized trial.

Immunization with Plasmodium falciparum (Pf) sporozoites under chemoprophylaxis (PfSPZ-CVac) is the most efficacious approach to malaria vaccination. Implementation is hampered by a complex chemoprophylaxis regimen and missing evidence for efficacy against heterologous infection. We report the results of a double-blinded, randomized, placebo-controlled trial of a simplified, condensed immunization regimen in malaria-naive volunteers (EudraCT-Nr: 2018-004523-36). Participants are immunized by direct venous inoculation of 1.1 × 105 aseptic, purified, cryopreserved PfSPZ (PfSPZ Challenge) of the PfNF54 strain or normal saline (placebo) on days 1, 6 and 29, with simultaneous oral administration of 10 mg/kg chloroquine base. Primary endpoints are vaccine efficacy tested by controlled human malaria infection (CHMI) using the highly divergent, heterologous strain Pf7G8 and safety. Twelve weeks following immunization, 10/13 participants in the vaccine group are sterilely protected against heterologous CHMI, while (5/5) participants receiving placebo develop parasitemia (risk difference: 77%, p = 0.004, Boschloo's test). Immunization is well tolerated with self-limiting grade 1-2 headaches, pyrexia and fatigue that diminish with each vaccination. Immunization induces 18-fold higher anti-Pf circumsporozoite protein (PfCSP) antibody levels in protected than in unprotected vaccinees (p = 0.028). In addition anti-PfMSP2 antibodies are strongly protection-associated by protein microarray assessment. This PfSPZ-CVac regimen is highly efficacious, simple, safe, well tolerated and highly immunogenic.

Immunogenicity and Protective Efficacy of Radiation-Attenuated and Chemo-Attenuated PfSPZ Vaccines in Equatoguinean Adults.

Plasmodium falciparum sporozoite (PfSPZ) Vaccine (radiation-attenuated, aseptic, purified, cryopreserved PfSPZ) and PfSPZ-CVac (infectious, aseptic, purified, cryopreserved PfSPZ administered to subjects taking weekly chloroquine chemoprophylaxis) have shown vaccine efficacies (VEs) of 100% against homologous controlled human malaria infection (CHMI) in nonimmune adults. Plasmodium falciparum sporozoite-CVac has never been assessed against CHMI in African vaccinees. We assessed the safety, immunogenicity, and VE against homologous CHMI of three doses of 2.7 × 106 PfSPZ of PfSPZ Vaccine at 8-week intervals and three doses of 1.0 × 105 PfSPZ of PfSPZ-CVac at 4-week intervals with each arm randomized, double-blind, placebo-controlled, and conducted in parallel. There were no differences in solicited adverse events between vaccinees and normal saline controls, or between PfSPZ Vaccine and PfSPZ-CVac recipients during the 6 days after administration of investigational product. However, from days 7-13, PfSPZ-CVac recipients had significantly more AEs, probably because of Pf parasitemia. Antibody responses were 2.9 times higher in PfSPZ Vaccine recipients than PfSPZ-CVac recipients at time of CHMI. Vaccine efficacy at a median of 14 weeks after last PfSPZ-CVac dose was 55% (8 of 13, P = 0.051) and at a median of 15 weeks after last PfSPZ Vaccine dose was 27% (5 of 15, P = 0.32). The higher VE in PfSPZ-CVac recipients of 55% with a 27-fold lower dose was likely a result of later stage parasite maturation in the liver, leading to induction of cellular immunity against a greater quantity and broader array of antigens.

Proteome-wide analysis of a malaria vaccine study reveals personalized humoral immune profiles in Tanzanian adults.

Tanzanian adult male volunteers were immunized by direct venous inoculation with radiation-attenuated, aseptic, purified, cryopreserved Plasmodium falciparum (Pf) sporozoites (PfSPZ Vaccine) and protective efficacy assessed by homologous controlled human malaria infection (CHMI). Serum immunoglobulin G (IgG) responses were analyzed longitudinally using a Pf protein microarray covering 91% of the proteome, providing first insights into naturally acquired and PfSPZ Vaccine-induced whole parasite antibody profiles in malaria pre-exposed Africans. Immunoreactivity was identified against 2239 functionally diverse Pf proteins, showing a wide breadth of humoral response. Antibody-based immune 'fingerprints' in these individuals indicated a strong person-specific immune response at baseline, with little changes in the overall humoral immunoreactivity pattern measured after immunization. The moderate increase in immunogenicity following immunization and the extensive and variable breadth of humoral immune response observed in the volunteers at baseline suggest that pre-exposure reduces vaccine-induced antigen reactivity in unanticipated ways.

A double-blind, placebo-controlled phase 1/2a trial of the genetically attenuated malaria vaccine PfSPZ-GA1.

Immunization with attenuated Plasmodium sporozoites can induce protection against malaria infection, as shown by Plasmodium falciparum (Pf) sporozoites attenuated by radiation in multiple clinical trials. As alternative attenuation strategy with a more homogeneous population of Pf sporozoites (PfSPZ), genetically engineered Plasmodium berghei sporozoites (SPZ) lacking the genes b9 and slarp induced sterile protection against malaria in mice. Consequently, PfSPZ-GA1 Vaccine, a Pf identical double knockout (Pf∆b9∆slarp), was generated as a genetically attenuated malaria parasite vaccine and tested for safety, immunogenicity, and preliminary efficacy in malaria-naïve Dutch volunteers. Dose-escalation immunizations up to 9.0 × 105 PfSPZ of PfSPZ-GA1 Vaccine were well tolerated without breakthrough blood-stage infection. Subsequently, groups of volunteers were immunized three times by direct venous inoculation with cryopreserved PfSPZ-GA1 Vaccine (9.0 × 105 or 4.5 × 105 PfSPZ, N = 13 each), PfSPZ Vaccine (radiation-attenuated PfSPZ, 4.5 × 105 PfSPZ, N = 13), or normal saline placebo at 8-week intervals, followed by exposure to mosquito bite controlled human malaria infection (CHMI). After CHMI, 3 of 25 volunteers from both PfSPZ-GA1 groups were sterilely protected, and the remaining 17 of 22 showed a patency ≥9 days (median patency in controls, 7 days; range, 7 to 9). All volunteers in the PfSPZ Vaccine control group developed parasitemia (median patency, 9 days; range, 7 to 12). Immunized groups exhibited a significant, dose-related increase in anti-Pf circumsporozoite protein (CSP) antibodies and Pf-specific interferon-γ (IFN-γ)-producing T cells. Although no definite conclusion can be drawn on the potential strength of protective efficacy of PfSPZ-GA1 Vaccine, the favorable safety profile and induced immune responses by PfSPZ-GA1 Vaccine warrant further clinical evaluation.

Increase of Dose Associated With Decrease in Protection Against Controlled Human Malaria Infection by PfSPZ Vaccine in Tanzanian Adults.

BACKGROUND: A vaccine would be an ideal tool for reducing malaria's impact. PfSPZ Vaccine (radiation attenuated, aseptic, purified, cryopreserved Plasmodium falciparum [Pf] sporozoites [SPZ]) has been well tolerated and safe in >1526 malaria-naive and experienced 6-month to 65-year-olds in the United States, Europe, and Africa. When vaccine efficacy (VE) of 5 doses of 2.7 × 105 PfSPZ of PfSPZ Vaccine was assessed in adults against controlled human malaria infection (CHMI) in the United States and Tanzania and intense field transmission of heterogeneous Pf in Mali, Tanzanians had the lowest VE (20%). METHODS: To increase VE in Tanzania, we increased PfSPZ/dose (9 × 105 or 1.8 × 106) and decreased numbers of doses to 3 at 8-week intervals in a double blind, placebo-controlled trial. RESULTS: All 22 CHMIs in controls resulted in parasitemia by quantitative polymerase chain reaction. For the 9 × 105 PfSPZ group, VE was 100% (5/5) at 3 or 11 weeks (P < .000l, Barnard test, 2-tailed). For 1.8 × 106 PfSPZ, VE was 33% (2/6) at 7.5 weeks (P = .028). VE of dosage groups (100% vs 33%) was significantly different (P = .022). Volunteers underwent repeat CHMI at 37-40 weeks after last dose. 6/6 and 5/6 volunteers developed parasitemia, but time to first parasitemia was significantly longer than controls in the 9 × 105 PfSPZ group (10.89 vs 7.80 days) (P = .039), indicating a significant reduction in parasites in the liver. Antibody and T-cell responses were higher in the 1.8 × 106 PfSPZ group. CONCLUSIONS: In Tanzania, increasing the dose from 2.7 × 105 to 9 × 105 PfSPZ increased VE from 20% to 100%, but increasing to 1.8 × 106 PfSPZ significantly reduced VE. CLINICAL TRIALS REGISTRATION: NCT02613520.

Safety and Differential Antibody and T-Cell Responses to the Plasmodium falciparum Sporozoite Malaria Vaccine, PfSPZ Vaccine, by Age in Tanzanian Adults, Adolescents, Children, and Infants.

In 2016, there were more cases and deaths caused by malaria globally than in 2015. An effective vaccine would be an ideal additional tool for reducing malaria's impact. Sanaria® PfSPZ Vaccine, composed of radiation-attenuated, aseptic, purified, cryopreserved Plasmodium falciparum (Pf) sporozoites (SPZ) has been well tolerated and safe in malaria-naïve and experienced adults in the United States and Mali and protective against controlled human malaria infection with Pf in the United States and field transmission of Pf in Mali, but had not been assessed in younger age groups. We, therefore, evaluated PfSPZ Vaccine in 93 Tanzanians aged 45 years to 6 months in a randomized, double-blind, normal saline placebo-controlled trial. There were no significant differences in adverse events between vaccinees and controls or between dosage regimens. Because all age groups received three doses of 9.0 × 105 PfSPZ of PfSPZ Vaccine, immune responses were compared at this dosage. Median antibody responses against Pf circumsporozoite protein and PfSPZ were highest in infants and lowest in adults. T-cell responses were highest in 6-10-year olds after one dose and 1-5-year olds after three doses; infants had no significant positive T-cell responses. The safety data were used to support initiation of trials in > 300 infants in Kenya and Equatorial Guinea. Because PfSPZ Vaccine-induced protection is thought to be mediated by T cells, the T-cell data suggest PfSPZ Vaccine may be more protective in children than in adults, whereas infants may not be immunologically mature enough to respond to the PfSPZ Vaccine immunization regimen assessed.

DSM265 for Plasmodium falciparum chemoprophylaxis: a randomised, double blinded, phase 1 trial with controlled human malaria infection.

BACKGROUND: A drug for causal (ie, pre-erythrocytic) prophylaxis of Plasmodium falciparum malaria with prolonged activity would substantially advance malaria control. DSM265 is an experimental antimalarial that selectively inhibits the parasite dihydroorotate dehydrogenase. DSM265 shows in vitro activity against liver and blood stages of P falciparum. We assessed the prophylactic activity of DSM265 against controlled human malaria infection (CHMI). METHODS: At the Institute of Tropical Medicine, Eberhard Karls University (Tübingen, Germany), healthy, malaria-naive adults were allocated to receive 400 mg DSM265 or placebo either 1 day (cohort 1A) or 7 days (cohort 2) before CHMI by direct venous inoculation (DVI) of 3200 aseptic, purified, cryopreserved P falciparum sporozoites (PfSPZ Challenge; Sanaria Inc, Rockville, MD, USA). An additional group received daily atovaquone-proguanil (250-100 mg) for 9 days, starting 1 day before CHMI (cohort 1B). Allocation to DSM265, atovaquone-proguanil, or placebo was randomised by an interactive web response system. Allocation to cohort 1A and 1B was open-label, within cohorts 1A and 2, allocation to DSM265 and placebo was double-blinded. All treatments were given orally. Volunteers were treated with an antimalarial on day 28, or when parasitaemic, as detected by thick blood smear (TBS) microscopy. The primary efficacy endpoint was time-to-parasitaemia, assessed by TBS. All participants receiving at least one dose of chemoprophylaxis or placebo were considered for safety, those receiving PfSPZ Challenge for efficacy analyses. Log-rank test was used to compare time-to-parasitemia between interventions. The trial was registered with ClinicalTrials.gov, number NCT02450578. FINDINGS: 22 participants were enrolled between Oct 23, 2015, and Jan 18, 2016. Five participants received 400 mg DSM265 and two participants received placebo 1 day before CHMI (cohort 1A), six participants received daily atovaquone-proguanil 1 day before CHMI (cohort 1B), and six participants received 400 mg DSM265 and two participants received placebo 7 days before CHMI (cohort 2). Five of five participants receiving DSM265 1 day before CHMI and six of six in the atovaquone-proguanil cohort were protected, whereas placebo recipients (two of two) developed malaria on days 11 and 14. When given 7 days before CHMI, three of six volunteers receiving DSM265 became TBS positive on days 11, 13, and 24. The remaining three DSM265-treated, TBS-negative participants of cohort 2 developed transient submicroscopic parasitaemia. Both participants receiving placebo 7 days before CHMI became TBS positive on day 11. The only possible DSM265-related adverse event was a moderate transient elevation in serum bilirubin in one participant. INTERPRETATION: A single dose of 400 mg DSM265 was well tolerated and had causal prophylactic activity when given 1 day before CHMI. Future trials are needed to investigate further the use of DSM265 for the prophylaxis of malaria. FUNDING: Global Health Innovative Technology Fund, Wellcome Trust, Bill & Melinda Gates Foundation through Medicines for Malaria Venture, and the German Center for Infection Research.

Sterile protection against human malaria by chemoattenuated PfSPZ vaccine.

A highly protective malaria vaccine would greatly facilitate the prevention and elimination of malaria and containment of drug-resistant parasites. A high level (more than 90%) of protection against malaria in humans has previously been achieved only by immunization with radiation-attenuated Plasmodium falciparum (Pf) sporozoites (PfSPZ) inoculated by mosquitoes; by intravenous injection of aseptic, purified, radiation-attenuated, cryopreserved PfSPZ ('PfSPZ Vaccine'); or by infectious PfSPZ inoculated by mosquitoes to volunteers taking chloroquine or mefloquine (chemoprophylaxis with sporozoites). We assessed immunization by direct venous inoculation of aseptic, purified, cryopreserved, non-irradiated PfSPZ ('PfSPZ Challenge') to malaria-naive, healthy adult volunteers taking chloroquine for antimalarial chemoprophylaxis (vaccine approach denoted as PfSPZ-CVac). Three doses of 5.12 × 104 PfSPZ of PfSPZ Challenge at 28-day intervals were well tolerated and safe, and prevented infection in 9 out of 9 (100%) volunteers who underwent controlled human malaria infection ten weeks after the last dose (group III). Protective efficacy was dependent on dose and regimen. Immunization with 3.2 × 103 (group I) or 1.28 × 104 (group II) PfSPZ protected 3 out of 9 (33%) or 6 out of 9 (67%) volunteers, respectively. Three doses of 5.12 × 104 PfSPZ at five-day intervals protected 5 out of 8 (63%) volunteers. The frequency of Pf-specific polyfunctional CD4 memory T cells was associated with protection. On a 7,455 peptide Pf proteome array, immune sera from at least 5 out of 9 group III vaccinees recognized each of 22 proteins. PfSPZ-CVac is a highly efficacious vaccine candidate; when we are able to optimize the immunization regimen (dose, interval between doses, and drug partner), this vaccine could be used for combination mass drug administration and a mass vaccination program approach to eliminate malaria from geographically defined areas.

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.

Delineation of stage specific expression of Plasmodium falciparum EBA-175 by biologically functional region II monoclonal antibodies.

BACKGROUND: The malaria parasite Plasmodium falciparum EBA-175 binds its receptor sialic acids on glycophorin A when invading erythrocytes. The receptor-binding region (RII) contains two cysteine-rich domains with similar cysteine motifs (F1 and F2). Functional relationships between F1 and F2 domains and characterization of EBA-175 were studied using specific monoclonal antibodies (mAbs) against these domains. METHODS AND FINDINGS: Five mAbs specific for F1 or F2 were generated. Three mAbs specific for F2 potently blocked binding of EBA-175 to erythrocytes, and merozoite invasion of erythrocytes (IC(50) 10 to 100 µg/ml IgG in growth inhibition assays). A mAb specific for F1 blocked EBA-175 binding and merozoite invasion less effectively. The difference observed between the IC(50) of F1 and F2 mAbs was not due to differing association and disassociation rates as determined by surface plasmon resonance. Four of the mAbs recognized conformation-dependent epitopes within F1 or F2. Used in combination, F1 and F2 mAbs blocked the binding of native EBA-175 to erythrocytes and inhibited parasite invasion synergistically in vitro. MAb R217, the most potent, did not recognize sporozoites, 3-day hepatocyte stage parasites, nor rings, trophozoites, gametocytes, retorts, ookinetes, and oocysts but recognized 6-day hepatocyte stage parasites, and schizonts. Even though efficient at blocking binding to erythrocytes and inhibiting invasion into erythrocytes, MAb R217 did not inhibit sporozoite invasion and development in hepatocytes in vitro. CONCLUSIONS: The role of the F1 and F2 domains in erythrocyte invasion and binding was elucidated with mAbs. These mAbs interfere with native EBA-175 binding to erythrocyte in a synergistic fashion. The stage specific expression of EBA-175 showed that the primary focus of activity was the merozoite stage. A recombinant RII protein vaccine consisting of both F1 and F2 domains that could induce synergistic activity should be optimal for induction of antibody responses that interfere with merozoite invasion of erythrocytes.

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.