Immune responses associated with protection induced by chemoattenuated PfSPZ vaccine in malaria-naive Europeans.

Vaccination of malaria-naive volunteers with a high dose of Plasmodium falciparum sporozoites chemoattenuated by chloroquine (CQ) (PfSPZ-CVac [CQ]) has previously demonstrated full protection against controlled human malaria infection (CHMI). However, lower doses of PfSPZ-CVac [CQ] resulted in incomplete protection. This provides the opportunity to understand the immune mechanisms needed for better vaccine-induced protection by comparing individuals who were protected with those not protected. Using mass cytometry, we characterized immune cell composition and responses of malaria-naive European volunteers who received either lower doses of PfSPZ-CVac [CQ], resulting in 50% protection irrespective of the dose, or a placebo vaccination, with everyone becoming infected following CHMI. Clusters of CD4+ and γδ T cells associated with protection were identified, consistent with their known role in malaria immunity. Additionally, EMRA CD8+ T cells and CD56+CD8+ T cell clusters were associated with protection. In a cohort from a malaria-endemic area in Gabon, these CD8+ T cell clusters were also associated with parasitemia control in individuals with lifelong exposure to malaria. Upon stimulation with P. falciparum-infected erythrocytes, CD4+, γδ, and EMRA CD8+ T cells produced IFN-γ and/or TNF, indicating their ability to mediate responses that eliminate malaria parasites.

A paradigm for Africa-centric vaccine development in Equatorial Guinea.

The Equatorial Guinea Malaria Vaccine Initiative (EGMVI) highlights how long-term African government and international energy industry investment, plus novel partnerships, can enable clinical development of vaccines in Africa, for Africa. We review achievements and challenges of this pioneering, award-winning, public-private partnership which offers a model for future Africa-centric clinical research and development (R&D).

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.

Safety and protective efficacy of PfSPZ Vaccine administered to HIV-negative and -positive Tanzanian adults.

BACKGROUNDSanaria PfSPZ Vaccine, composed of attenuated Plasmodium falciparum (Pf) sporozoites (SPZ), protects against malaria. We conducted this clinical trial to assess the safety and efficacy of PfSPZ Vaccine in HIV-positive (HIV+) individuals, since the HIV-infection status of participants in mass vaccination programs may be unknown.METHODSThis randomized, double-blind, placebo-controlled trial enrolled 18- to 45-year-old HIV-negative (HIV-) and well-controlled HIV+ Tanzanians (HIV viral load <40 copies/mL, CD4 counts >500 cells/µL). Participants received 5 doses of PfSPZ Vaccine or normal saline (NS) over 28 days, followed by controlled human malaria infection (CHMI) 3 weeks later.RESULTSThere were no solicited adverse events in the 9 HIV- and 12 HIV+ participants. After CHMI, 6 of 6 NS controls, 1 of 5 HIV- vaccinees, and 4 of 4 HIV+ vaccinees were Pf positive by quantitative PCR (qPCR). After immunization, anti-Pf circumsporozoite protein (anti-PfCSP) (isotype and IgG subclass) and anti-PfSPZ antibodies, anti-PfSPZ CD4+ T cell responses, and Vδ2+ γδ CD3+ T cells were nonsignificantly higher in HIV- than in HIV+ vaccinees. Sera from HIV- vaccinees had significantly higher inhibition of PfSPZ invasion of hepatocytes in vitro and antibody-dependent complement deposition (ADCD) and Fcγ3B binding by anti-PfCSP and ADCD by anti-cell-traversal protein for ookinetes and SPZ (anti-PfCelTOS) antibodies.CONCLUSIONSPfSPZ Vaccine was safe and well tolerated in HIV+ vaccinees, but not protective. Vaccine efficacy was 80% in HIV- vaccinees (P = 0.012), whose sera had significantly higher inhibition of PfSPZ invasion of hepatocytes and enrichment of multifunctional PfCSP antibodies. A more potent PfSPZ vaccine or regimen is needed to protect those living with HIV against Pf infection in Africa.TRIAL REGISTRATIONClinicalTrials.gov NCT03420053.FUNDINGEquatorial Guinea Malaria Vaccine Initiative (EGMVI), made up of the Government of Equatorial Guinea Ministries of Mines and Hydrocarbons, and Health and Social Welfare, Marathon Equatorial Guinea Production Limited, Noble Energy, Atlantic Methanol Production Company, and EG LNG; Swiss government, through ESKAS scholarship grant no. 2016.0056; Intramural Research Program of the National Institute of Allergy and Infectious Diseases, NIH; NIH grant 1U01AI155354-01.

Piloting delivery of PfSPZ vaccines for malaria through a cryogenic vaccine cold chain to travel and military medicine clinics.

BACKGROUND: PfSPZ vaccines comprising Plasmodium falciparum (Pf) sporozoites (SPZ) have demonstrated > 90% protection against variant Pf malaria infections for at least 12 weeks; they are the only vaccines with the level of efficacy necessary to protect travellers. PfSPZ are eukaryotic cells stabilized by cryopreservation and distributed using a cryogenic (below -150 °C) cold chain. The Ebola vaccine and mRNA vaccines against SARS-CoV-2 pioneered uptake of vaccines requiring non-standard ultra-low temperature cold chains. The cryogenic cold chain using liquid nitrogen (LN2) vapour phase (LNVP) cryoshippers, is simpler, more efficient than -80, -20 or 2-8 °C cold chains, and does not use electricity. This study was conducted to evaluate implementation and integration of a cryogenically distributed vaccine at travel and military immunization clinics. METHODS: We conducted sequential 28-day studies evaluating vaccine shipping, storage, maintenance and accession at two US military and two civilian travel health/immunization clinics. In each clinic, personnel were trained in equipment use, procurement and handling of LN2, temperature monitoring and inventory record keeping by in-person or video instruction. RESULTS: Sites required 2-4 h/person for two persons to assimilate and develop the expertise to manage vaccine storage and LNVP operations. LN2 for recharging cryoshippers was delivered every 1-2 weeks. Vaccine ordering, receipt, storage and inventory control was conducted effectively. Simulated single dose vaccine cryovial retrieval and thawing were performed successfully in different travel clinic settings. Continuous temperature monitoring at each site was maintained with only one short excursion above -150 °C (-145 °C) through shipping, use and reverse logistics. Staff, during and at study conclusion, provided feedback that has been incorporated into our models for cold chain logistics. CONCLUSIONS: These studies demonstrated that the training in delivery, storage, administration and integration of PfSPZ vaccines can be successfully managed in different immunization clinic settings for travellers and military personnel.

Plasmodium knowlesi in pig-tailed macaques: a potential new model for malaria vaccine research.

BACKGROUND: Plasmodium knowlesi is an established experimental model for basic and pre-clinical malaria vaccine research. Historically, rhesus macaques have been the most common host for malaria vaccine studies with P. knowlesi parasites. However, rhesus are not natural hosts for P. knowlesi, and there is interest in identifying alternative hosts for vaccine research. The study team previously reported that pig-tailed macaques (PTM), a natural host for P. knowlesi, could be challenged with cryopreserved P. knowlesi sporozoites (PkSPZ), with time to blood stage infection equivalent to in rhesus. Here, additional exploratory studies were performed to evaluate PTM as potential hosts for malaria vaccine studies. The aim was to further characterize the parasitological and veterinary health outcomes after PkSPZ challenge in this macaque species. METHODS: Malaria-naïve PTM were intravenously challenged with 2.5 × 103 PkSPZ and monitored for blood stage infection by Plasmodium 18S rRNA RT-PCR and thin blood smears. Disease signs were evaluated by daily observations, complete blood counts, serum chemistry tests, and veterinary examinations. After anti-malarial drug treatment, a subset of animals was re-challenged and monitored as above. Whole blood gene expression analysis was performed on selected animals to assess host response to infection. RESULTS: In naïve animals, the kinetics of P. knowlesi blood stage replication was reproducible, with parasite burden rising linearly during an initial acute phase of infection from 6 to 11 days post-challenge, before plateauing and transitioning into a chronic low-grade infection. After re-challenge, infections were again reproducible, but with lower blood stage parasite densities. Clinical signs of disease were absent or mild and anti-malarial treatment was not needed until the pre-defined study day. Whole blood gene expression analysis identified immunological changes associated with acute and chronic phases of infection, and further differences between initial challenge versus re-challenge. CONCLUSIONS: The ability to challenge PTM with PkSPZ and achieve reliable blood stage infections indicate this model has significant potential for malaria vaccine studies. Blood stage P. knowlesi infection in PTM is characterized by low parasite burdens and a benign disease course, in contrast with the virulent P. knowlesi disease course commonly reported in rhesus macaques. These findings identify new opportunities for malaria vaccine research using this natural host-parasite combination.

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.

Preliminary studies on the immunogenicity of a prime-and-trap malaria vaccine in nonhuman primates.

Development of next-generation vaccines against Plasmodium falciparum (Pf) is a priority. Many malaria vaccines target the pre-erythrocytic sporozoite (SPZ) and liver stages. These include subunit vaccines based on the Pf circumsporozoite protein (CSP) and attenuated PfSPZ vaccines. However, these strategies require 3-4 doses and have not achieved optimal efficacy against field-transmitted malaria. Prime-and-trap is a recently developed two-step heterologous vaccine strategy that combines priming with DNA encoding CSP followed by a single dose of attenuated SPZ. This strategy aims to induce CD8+ T cells that can eliminate parasites in the liver. Prior data has demonstrated that prime-and-trap with P. yoelii CSP and PySPZ was immunogenic and protective in mice. Here we report preliminary data on the immunogenicity of PfCSP prime and PfSPZ trap vaccine in rhesus macaques. This vaccine induced PfCSP-specific antibodies and T cell responses in all animals. However, response magnitude differed between individuals, suggesting further study is required.

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.

Plasmodium 18S Ribosomal RNA Biomarker Clearance After Food and Drug Administration-Approved Antimalarial Treatment in Controlled Human Malaria Infection Trials.

Background: Sensitive molecular assays, such as quantitative reverse-transcription polymerase chain reaction (qRT-PCR) of Plasmodium 18S ribosomal RNA (rRNA), are increasingly the primary method of detecting infections in controlled human malaria infection (CHMI) trials. However, thick blood smears (TBSs) remain the main method for confirming clearance of parasites after curative treatment, in part owing to uncertainty regarding biomarker clearance rates. Methods: For this analysis, 18S rRNA qRT-PCR data were compiled from 127 Plasmodium falciparum-infected participants treated with chloroquine or atovaquone-proguanil in 6 CHMI studies conducted in Seattle, Washington, over the past decade. A survival analysis approach was used to compare biomarker and TBS clearance times among studies. The effect of the parasite density at which treatment was initiated on clearance time was estimated using linear regression. Results: The median time to biomarker clearance was 3 days (interquartile range, 3-5 days), while the median time to TBS clearance was 1 day (1-2 days). Time to biomarker clearance increased with the parasite density at which treatment was initiated. Parasite density did not have a significant effect on TBS clearance. Conclusions: The Plasmodium 18S rRNA biomarker clears quickly and can be relied on to confirm the adequacy of Food and Drug Administration-approved treatments in CHMI studies at nonendemic sites.

The Dantu blood group prevents parasite growth in vivo: Evidence from a controlled human malaria infection study.

Background: The long co-evolution of Homo sapiens and Plasmodium falciparum has resulted in the selection of numerous human genetic variants that confer an advantage against severe malaria and death. One such variant is the Dantu blood group antigen, which is associated with 74% protection against severe and complicated P. falciparum malaria infections in homozygous individuals, similar to that provided by the sickle haemoglobin allele (HbS). Recent in vitro studies suggest that Dantu exerts this protection by increasing the surface tension of red blood cells, thereby impeding the ability of P. falciparum merozoites to invade them and reducing parasite multiplication. However, no studies have yet explored this hypothesis in vivo. Methods: We investigated the effect of Dantu on early phase P. falciparum (Pf) infections in a controlled human malaria infection (CHMI) study. 141 sickle-negative Kenyan adults were inoculated with 3.2 × 103 aseptic, purified, cryopreserved Pf sporozoites (PfSPZ Challenge) then monitored for blood-stage parasitaemia for 21 days by quantitative polymerase chain reaction (qPCR)analysis of the 18S ribosomal RNA P. falciparum gene. The primary endpoint was blood-stage P. falciparum parasitaemia of ≥500/µl while the secondary endpoint was the receipt of antimalarial treatment in the presence of parasitaemia of any density. On study completion, all participants were genotyped both for Dantu and for four other polymorphisms that are associated with protection against severe falciparum malaria: α+-thalassaemia, blood group O, G6PD deficiency, and the rs4951074 allele in the red cell calcium transporter ATP2B4. Results: The primary endpoint was reached in 25/111 (22.5%) non-Dantu subjects in comparison to 0/27 (0%) Dantu heterozygotes and 0/3 (0.0%) Dantu homozygotes (p=0.01). Similarly, 49/111 (44.1%) non-Dantu subjects reached the secondary endpoint in comparison to only 7/27 (25.9%) and 0/3 (0.0%) Dantu heterozygotes and homozygotes, respectively (p=0.021). No significant impacts on either outcome were seen for any of the other genetic variants under study. Conclusions: This study reveals, for the first time, that the Dantu blood group is associated with high-level protection against early, non-clinical, P. falciparum malaria infections in vivo. Learning more about the mechanisms involved could potentially lead to new approaches to the prevention or treatment of the disease. Our study illustrates the power of CHMI with PfSPZ Challenge for directly testing the protective impact of genotypes previously identified using other methods. Funding: The Kenya CHMI study was supported by an award from Wellcome (grant number 107499). SK was supported by a Training Fellowship (216444/Z/19/Z), TNW by a Senior Research Fellowship (202800/Z/16/Z), JCR by an Investigator Award (220266/Z/20/Z), and core support to the KEMRI-Wellcome Trust Research Programme in Kilifi, Kenya (203077), all from Wellcome. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. For the purpose of Open Access, the authors have applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission. Clinical trial number: NCT02739763.

The exception that proves the rule: Virulence gene expression at the onset of Plasmodium falciparum blood stage infections.

Controlled human malaria infections (CHMI) are a valuable tool to study parasite gene expression in vivo under defined conditions. In previous studies, virulence gene expression was analyzed in samples from volunteers infected with the Plasmodium falciparum (Pf) NF54 isolate, which is of African origin. Here, we provide an in-depth investigation of parasite virulence gene expression in malaria-naïve European volunteers undergoing CHMI with the genetically distinct Pf 7G8 clone, originating in Brazil. Differential expression of var genes, encoding major virulence factors of Pf, PfEMP1s, was assessed in ex vivo parasite samples as well as in parasites from the in vitro cell bank culture that was used to generate the sporozoites (SPZ) for CHMI (Sanaria PfSPZ Challenge (7G8)). We report broad activation of mainly B-type subtelomeric located var genes at the onset of a 7G8 blood stage infection in naïve volunteers, mirroring the NF54 expression study and suggesting that the expression of virulence-associated genes is generally reset during transmission from the mosquito to the human host. However, in 7G8 parasites, we additionally detected a continuously expressed single C-type variant, Pf7G8_040025600, that was most highly expressed in both pre-mosquito cell bank and volunteer samples, suggesting that 7G8, unlike NF54, maintains expression of some previously expressed var variants during transmission. This suggests that in a new host, the parasite may preferentially express the variants that previously allowed successful infection and transmission. Trial registration: ClinicalTrials.gov - NCT02704533; 2018-004523-36.

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.

Molecular and functional properties of human Plasmodium falciparum CSP C-terminus antibodies.

Human monoclonal antibodies (mAbs) against the central repeat and junction domain of Plasmodium falciparum circumsporozoite protein (PfCSP) have been studied extensively to guide malaria vaccine design compared to antibodies against the PfCSP C terminus. Here, we describe the molecular characteristics and protective potential of 73 germline and mutated human mAbs against the highly immunogenic PfCSP C-terminal domain. Two mAbs recognized linear epitopes in the C-terminal linker with sequence similarity to repeat and junction motifs, whereas all others targeted conformational epitopes in the α-thrombospondin repeat (α-TSR) domain. Specificity for the polymorphic Th2R/Th3R but not the conserved RII+/CS.T3 region in the α-TSR was associated with IGHV3-21/IGVL3-21 or IGLV3-1 gene usage. Although the C terminus specific mAbs showed signs of more efficient affinity maturation and class-switching compared to anti-repeat mAbs, live sporozoite binding and inhibitory activity was limited to a single C-linker reactive mAb with cross-reactivity to the central repeat and junction. The data provide novel insights in the human anti-C-linker and anti-α-TSR antibody response that support exclusion of the PfCSP C terminus from malaria vaccine designs.

A randomized controlled trial showing safety and efficacy of a whole sporozoite vaccine against endemic malaria.

A highly effective malaria vaccine remains elusive despite decades of research. Plasmodium falciparum sporozoite vaccine (PfSPZ Vaccine), a metabolically active, nonreplicating, whole parasite vaccine demonstrated safety and vaccine efficacy (VE) against endemic P. falciparum for 6 months in Malian adults receiving a five-dose regimen. Safety, immunogenicity, and VE of a three-dose regimen were assessed in adults in Balonghin, Burkina Faso in a two-component study: an open-label dose escalation trial with 32 participants followed by a double-blind, randomized, placebo-controlled trial (RCT) with 80 participants randomized to receive three doses of 2.7 × 106 PfSPZ (N = 39) or normal saline (N = 41) just before malaria season. To clear parasitemia, artesunate monotherapy was administered before first and last vaccinations. Thick blood smear microscopy was performed on samples collected during illness and every 4 weeks for 72 weeks after last vaccinations, including two 6-month malaria transmission seasons. Safety outcomes were assessed in all 80 participants who received at least one dose and VE for 79 participants who received three vaccinations. Myalgia was the only symptom that differed between groups. VE (1 - risk ratio; primary VE endpoint) was 38% at 6 months (P = 0.017) and 15% at 18 months (0.078). VE (1 - hazard ratio) was 48% and 46% at 6 and 18 months (P = 0.061 and 0.018). Two weeks after the last dose, antibodies to P. falciparum circumsporozoite protein and PfSPZ were higher in protected versus unprotected vaccinees. A three-dose regimen of PfSPZ Vaccine demonstrated safety and efficacy against malaria infection in malaria-experienced adults.

In vitro production of infectious Plasmodium falciparum sporozoites.

An effective vaccine is needed for the prevention and elimination of malaria. The only immunogens that have been shown to have a protective efficacy of more than 90% against human malaria are Plasmodium falciparum (Pf) sporozoites (PfSPZ) manufactured in mosquitoes (mPfSPZ)1-7. The ability to produce PfSPZ in vitro (iPfSPZ) without mosquitoes would substantially enhance the production of PfSPZ vaccines and mosquito-stage malaria research, but this ability is lacking. Here we report the production of hundreds of millions of iPfSPZ. iPfSPZ invaded human hepatocytes in culture and developed to mature liver-stage schizonts expressing P. falciparum merozoite surface protein 1 (PfMSP1) in numbers comparable to mPfSPZ. When injected into FRGhuHep mice containing humanized livers, iPfSPZ invaded the human hepatocytes and developed to PfMSP1-expressing late liver stage parasites at 45% the quantity of cryopreserved mPfSPZ. Human blood from FRGhuHep mice infected with iPfSPZ produced asexual and sexual erythrocytic-stage parasites in culture, and gametocytes developed to PfSPZ when fed to mosquitoes, completing the P. falciparum life cycle from infectious gametocyte to infectious gametocyte without mosquitoes or primates.

Increased levels of anti-PfCSP antibodies in post-pubertal females versus males immunized with PfSPZ Vaccine does not translate into increased protective efficacy.

Background: While prior research has shown differences in the risk of malaria infection and sickness between males and females, little is known about sex differences in vaccine-induced immunity to malaria. Identifying such differences could elucidate important aspects of malaria biology and facilitate development of improved approaches to malaria vaccination. Methods: Using a standardized enzyme-linked immunosorbent assay, IgG antibodies to the major surface protein on Plasmodium falciparum (Pf) sporozoites (SPZ), the Pf circumsporozoite protein (PfCSP), were measured before and two weeks after administration of a PfSPZ-based malaria vaccine (PfSPZ Vaccine) to 5-month to 61-year-olds in 11 clinical trials in Germany, the US and five countries in Africa, to determine if there were differences in vaccine elicited antibody response between males and females and if these differences were associated with differential protection against naturally transmitted Pf malaria (Africa) or controlled human malaria infection (Germany, the US and Africa). Results: Females ≥ 11 years of age made significantly higher levels of antibodies to PfCSP than did males in most trials, while there was no indication of such differences in infants or children. Although adult females had higher levels of antibodies, there was no evidence of improved protection compared to males. In 2 of the 7 trials with sufficient data, protected males had significantly higher levels of antibodies than unprotected males, and in 3 other trials protected females had higher levels of antibodies than did unprotected females. Conclusion: Immunization with PfSPZ Vaccine induced higher levels of antibodies in post-pubertal females but showed equivalent protection in males and females. We conclude that the increased antibody levels in post-pubertal females did not contribute substantially to improved protection. We hypothesize that while antibodies to PfCSP (and PfSPZ) may potentially contribute directly to protection, they primarily correlate with other, potentially protective immune mechanisms, such as antibody dependent and antibody independent cellular responses in the liver.