Superior antibody immunogenicity of a viral-vectored RH5 blood-stage malaria vaccine in Tanzanian infants as compared to adults.

BACKGROUND: RH5 is a leading blood-stage candidate antigen for a Plasmodium falciparum vaccine; however, its safety and immunogenicity in malaria-endemic populations are unknown. METHODS: A phase 1b, single-center, dose-escalation, age-de-escalation, double-blind, randomized, controlled trial was conducted in Bagamoyo, Tanzania (NCT03435874). Between 12th April and 25th October 2018, 63 healthy adults (18-35 years), young children (1-6 years), and infants (6-11 months) received a priming dose of viral-vectored ChAd63 RH5 or rabies control vaccine. Sixty participants were boosted with modified vaccinia virus Ankara (MVA) RH5 or rabies control vaccine 8 weeks later and completed 6 months of follow-up post priming. Primary outcomes were the number of solicited and unsolicited adverse events post vaccination and the number of serious adverse events over the study period. Secondary outcomes included measures of the anti-RH5 immune response. FINDINGS: Vaccinations were well tolerated, with profiles comparable across groups. No serious adverse events were reported. Vaccination induced RH5-specific cellular and humoral responses. Higher anti-RH5 serum immunoglobulin G (IgG) responses were observed post boost in young children and infants compared to adults. Vaccine-induced antibodies showed growth inhibition activity (GIA) in vitro against P. falciparum blood-stage parasites; their highest levels were observed in infants. CONCLUSIONS: The ChAd63-MVA RH5 vaccine shows acceptable safety and reactogenicity and encouraging immunogenicity in children and infants residing in a malaria-endemic area. The levels of functional GIA observed in RH5-vaccinated infants are the highest reported to date following human vaccination. These data support onward clinical development of RH5-based blood-stage vaccines to protect against clinical malaria in young African infants. FUNDING: Medical Research Council, London, UK.

The impact of Loa loa microfilaraemia on research subject retention during a whole sporozoite malaria vaccine trial in Equatorial Guinea.

Loa loa microfilariae were found on thick blood smears (TBSs) from 8 of 300 (2.7%) residents of Bioko Island, Equatorial Guinea, during a Plasmodium falciparum sporozoite malaria vaccine clinical trial. Only one subject was found to have microfilaraemia on his first exam; parasites were not discovered in the other seven until subsequent TBSs were performed, at times many weeks into the study. All infected individuals were asymptomatic, and were offered treatment with diethylcarbamazine, per national guidelines. L. loa microfilaraemia complicated the enrolment or continued participation of these eight trial subjects, and only one was able to complete all study procedures. If ruling out loiasis is deemed to be important during clinical trials, tests that are more sensitive than TBSs should be performed.

Role of human Pegivirus infections in whole Plasmodium falciparum sporozoite vaccination and controlled human malaria infection in African volunteers.

BACKGROUND: Diverse vaccination outcomes and protection levels among different populations pose a serious challenge to the development of an effective malaria vaccine. Co-infections are among many factors associated with immune dysfunction and sub-optimal vaccination outcomes. Chronic, asymptomatic viral infections can contribute to the modulation of vaccine efficacy through various mechanisms. Human Pegivirus-1 (HPgV-1) persists in immune cells thereby potentially modulating immune responses. We investigated whether Pegivirus infection influences vaccine-induced responses and protection in African volunteers undergoing whole P. falciparum sporozoites-based malaria vaccination and controlled human malaria infections (CHMI). METHODS: HPgV-1 prevalence was quantified by RT-qPCR in plasma samples of 96 individuals before, post vaccination with PfSPZ Vaccine and after CHMI in cohorts from Tanzania and Equatorial Guinea. The impact of HPgV-1 infection was evaluated on (1) systemic cytokine and chemokine levels measured by Luminex, (2) PfCSP-specific antibody titers quantified by ELISA, (3) asexual blood-stage parasitemia pre-patent periods and parasite multiplication rates, (4) HPgV-1 RNA levels upon asexual blood-stage parasitemia induced by CHMI. RESULTS: The prevalence of HPgV-1 was 29.2% (28/96) and sequence analysis of the 5' UTR and E2 regions revealed the predominance of genotypes 1, 2 and 5. HPgV-1 infection was associated with elevated systemic levels of IL-2 and IL-17A. Comparable vaccine-induced anti-PfCSP antibody titers, asexual blood-stage multiplication rates and pre-patent periods were observed in HPgV-1 positive and negative individuals. However, a tendency for higher protection levels was detected in the HPgV-1 positive group (62.5%) compared to the negative one (51.6%) following CHMI. HPgV-1 viremia levels were not significantly altered after CHMI. CONCLUSIONS: HPgV-1 infection did not alter PfSPZ Vaccine elicited levels of PfCSP-specific antibody responses and parasite multiplication rates. Ongoing HPgV-1 infection appears to improve to some degree protection against CHMI in PfSPZ-vaccinated individuals. This is likely through modulation of immune system activation and systemic cytokines as higher levels of IL-2 and IL17A were observed in HPgV-1 infected individuals. CHMI is safe and well tolerated in HPgV-1 infected individuals. Identification of cell types and mechanisms of both silent and productive infection in individuals will help to unravel the biology of this widely present but largely under-researched virus.

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.

The Equatoguinean Malaria Vaccine Initiative: From the Launching of a Clinical Research Platform to Malaria Elimination Planning in Central West Africa.

Fifteen years of investment in malaria control on Bioko Island, Equatorial Guinea (EG), dramatically reduced malaria-associated morbidity and mortality, but the impact has plateaued. To progress toward elimination, EG is investing in the development of a malaria vaccine. We assessed the unique public-private partnership that has had such a significant impact on malaria on Bioko Island and now added a major effort on malaria vaccine development. As part of a $79M commitment, the EG government (75%) and three American energy companies (25%) have invested since 2012 greater than $55M in the Equatoguinean Malaria Vaccine Initiative (EGMVI) to support clinical development of Sanaria® PfSPZ vaccines (Sanaria Inc., Rockville, MD). In turn, the vaccine development program is building human capital and physical capacity. The EGMVI established regulatory and ethical oversight to ensure compliance with the International Conference on Harmonization and Good Clinical Practices for the first importation of investigational product, ethical approval, and conduct of a clinical trial in Equatoguinean history. The EGMVI has completed three vaccine trials in EG, two vaccine trials in Tanzania, and a malaria incidence study, and initiated preparations for a 2,100-volunteer clinical trial. Personnel are training for advanced degrees abroad and have been trained in Good Clinical Practices and protocol-specific methods. A new facility has established the foundation for a national research institute. Biomedical research and development within this visionary, ambitious public-private partnership is fostering major improvements in EG. The EGMVI plans to use a PfSPZ Vaccine alongside standard malaria control interventions to eliminate Pf malaria from Bioko, becoming a potential model for elimination campaigns elsewhere.

Antigen-stimulated PBMC transcriptional protective signatures for malaria immunization.

Identifying immune correlates of protection and mechanisms of immunity accelerates and streamlines the development of vaccines. RTS,S/AS01E, the most clinically advanced malaria vaccine, has moderate efficacy in African children. In contrast, immunization with sporozoites under antimalarial chemoprophylaxis (CPS immunization) can provide 100% sterile protection in naïve adults. We used systems biology approaches to identifying correlates of vaccine-induced immunity based on transcriptomes of peripheral blood mononuclear cells from individuals immunized with RTS,S/AS01E or chemoattenuated sporozoites stimulated with parasite antigens in vitro. Specifically, we used samples of individuals from two age cohorts and three African countries participating in an RTS,S/AS01E pediatric phase 3 trial and malaria-naïve individuals participating in a CPS trial. We identified both preimmunization and postimmunization transcriptomic signatures correlating with protection. Signatures were validated in independent children and infants from the RTS,S/AS01E phase 3 trial and individuals from an independent CPS trial with high accuracies (>70%). Transcription modules revealed interferon, NF-κB, Toll-like receptor (TLR), and monocyte-related signatures associated with protection. Preimmunization signatures suggest that priming the immune system before vaccination could potentially improve vaccine immunogenicity and efficacy. Last, signatures of protection could be useful to determine efficacy in clinical trials, accelerating vaccine candidate testing. Nevertheless, signatures should be tested more extensively across multiple cohorts and trials to demonstrate their universal predictive capacity.

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.

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

We are using controlled human malaria infection (CHMI) by direct venous inoculation (DVI) of cryopreserved, infectious Plasmodium falciparum (Pf) sporozoites (SPZ) (PfSPZ Challenge) to try to reduce time and costs of developing PfSPZ Vaccine to prevent malaria in Africa. Immunization with five doses at 0, 4, 8, 12, and 20 weeks of 2.7 × 105 PfSPZ of PfSPZ Vaccine gave 65% vaccine efficacy (VE) at 24 weeks against mosquito bite CHMI in U.S. adults and 52% (time to event) or 29% (proportional) VE over 24 weeks against naturally transmitted Pf in Malian adults. We assessed the identical regimen in Tanzanians for VE against PfSPZ Challenge. Twenty- to thirty-year-old men were randomized to receive five doses normal saline or PfSPZ Vaccine in a double-blind trial. Vaccine efficacy was assessed 3 and 24 weeks later. Adverse events were similar in vaccinees and controls. Antibody responses to Pf circumsporozoite protein were significantly lower than in malaria-naïve Americans, but significantly higher than in Malians. All 18 controls developed Pf parasitemia after CHMI. Four of 20 (20%) vaccinees remained uninfected after 3 week CHMI (P = 0.015 by time to event, P = 0.543 by proportional analysis) and all four (100%) were uninfected after repeat 24 week CHMI (P = 0.005 by proportional, P = 0.004 by time to event analysis). Plasmodium falciparum SPZ Vaccine was safe, well tolerated, and induced durable VE in four subjects. Controlled human malaria infection by DVI of PfSPZ Challenge appeared more stringent over 24 weeks than mosquito bite CHMI in United States or natural exposure in Malian adults, thereby providing a rigorous test of VE in Africa.

RTS,S/AS01E Malaria Vaccine Induces Memory and Polyfunctional T Cell Responses in a Pediatric African Phase III Trial.

Comprehensive assessment of cellular responses to the RTS,S/AS01E vaccine is needed to understand potential correlates and ultimately mechanisms of protection against malaria disease. Cellular responses recognizing the RTS,S/AS01E-containing circumsporozoite protein (CSP) and Hepatitis B surface antigen (HBsAg) were assessed before and 1 month after primary vaccination by intracellular cytokine staining and 16-color flow cytometry in 105 RTS,S/AS01-vaccinated and 74 rabies-vaccinated participants (controls) in a pediatric phase III trial in Africa. RTS,S/AS01E-vaccinated children had significantly higher frequencies of CSP- and HBsAg-specific CD4+ T cells producing IL-2, TNF-α, and CD40L and HBsAg-specific CD4+ T producing IFN-γ and IL-17 than baseline and the control group. Vaccine-induced responses were identified in both central and effector memory (EM) compartments. EM CD4+ T cells expressing IL-4 and IL-21 were detected recognizing both vaccine antigens. Consistently higher response rates to both antigens in RTS,S/AS01E-vaccinated than comparator-vaccinated children were observed. RTS,S/AS01E induced polyfunctional CSP- and HBsAg-specific CD4+ T cells, with a greater degree of polyfunctionality in HBsAg responses. In conclusion, RTS,S/AS01E vaccine induces T cells of higher functional heterogeneity and polyfunctionality than previously characterized. Responses detected in memory CD4+ T cell compartments may provide correlates of RTS,S/AS01-induced immunity and duration of protection in future correlates of immunity studies.

Controlled human malaria infection of Tanzanians by intradermal injection of aseptic, purified, cryopreserved Plasmodium falciparum sporozoites.

Controlled human malaria infection (CHMI) by mosquito bite has been used to assess anti-malaria interventions in > 1,500 volunteers since development of methods for infecting mosquitoes by feeding on Plasmodium falciparum (Pf) gametocyte cultures. Such CHMIs have never been used in Africa. Aseptic, purified, cryopreserved Pf sporozoites, PfSPZ Challenge, were used to infect Dutch volunteers by intradermal injection. We conducted a double-blind, placebo-controlled trial to assess safety and infectivity of PfSPZ Challenge in adult male Tanzanians. Volunteers were injected intradermally with 10,000 (N = 12) or 25,000 (N = 12) PfSPZ or normal saline (N = 6). PfSPZ Challenge was well tolerated and safe. Eleven of 12 and 10 of 11 subjects, who received 10,000 and 25,000 PfSPZ respectively, developed parasitemia. In 10,000 versus 25,000 PfSPZ groups geometric mean days from injection to Pf positivity by thick blood film was 15.4 versus 13.5 (P = 0.023). Alpha-thalassemia heterozygosity had no apparent effect on infectivity. PfSPZ Challenge was safe, well tolerated, and infectious.

Child hospitalization due to severe malaria is associated with the ICAM-1Kilifi allele but not adherence patterns of Plasmodium falciparum infected red blood cells to ICAM-1.

This study aimed at determining whether the predisposition of a mutation at position 179 of the ICAM-1 gene to child hospitalization due to malaria was mediated by changes in adherence properties of IRBCs to ICAM-1. ICAM-1 genotypes were determined by nested polymerase chain reaction of isolated DNA from filter blood spots followed by Restriction Fragment Length Polymorphism (RFLP). Plasmodium falciparum adherence assays were done on immobilized purified ICAM-1. Our data indicate that the homozygosity for the ICAM-1(Kilifi) mutation occurs at a frequency of 22.3% in Magugu-Babati, Northern Tanzania. Our results show that there are no differences in IRBC binding profiles across genotypes. We show in this study that homozygosity for the ICAM-1(Kilifi) is associated with child hospitalization (X(2)=14.47, p<0.001). We have further shown that hospitalization was not associated with cytoadherence (X(2)=0.17, p=0.68). We conclude that the ICAM-1(Kilifi) allele occurs at a high frequency in Tanzania and that associations of this allele with higher child hospitalization frequencies is independent of cytoadherence patterns of IRBC isolated from ICAM-1 genotypes, implying that any associations reported to exist between the ICAM-1(Kilifi) mutation and severe malaria are unlikely to be mediated through altered IRBC cytoadherence properties.

CD36 deficiency protects against malarial anaemia in children by reducing Plasmodium falciparum-infected red blood cell adherence to vascular endothelium.

OBJECTIVE: CD36 is a receptor that occurs on the surface of activated immune cells, vascular endothelial cells and participates in phagocytosis and lipid metabolism. CD36 is known to be the major endothelial receptor molecule for field isolates of Plasmodium falciparum. A T1264G mutation in exon X of the gene leads to deficiency of CD36. This study aimed at determining associations between CD36 deficiency, P. falciparum in vitro adherence on purified CD36 and anaemia among children in an endemic area. METHODS: Genotypes were determined by nested polymerase chain reaction of isolated DNA from filter blood spots followed by Restriction Fragment Length Polymorphism (RFLP). Plasmodium falciparum adherence assays were performed on immobilized purified CD36. RESULTS: The data indicate that CD36 is an important cytoadherence receptor that mediates adherence to most P. falciparum field isolates. Our findings also suggest that mutations causing CD36 deficiency may confer significant protection against malarial anaemia (MA) in children (chi(2) = 8.58, P < 0.01). CONCLUSION: That the protective role that CD36 deficiency may confer against MA in children seems to be mediated through reduced cytoadherence of infected red blood cells to vascular endothelium.