Evaluation of the precision of the Plasmodium knowlesi growth inhibition assay for Plasmodium vivax Duffy-binding protein-based malaria vaccine development.

Recent data indicate increasing disease burden and importance of Plasmodium vivax (Pv) malaria. A robust assay will be essential for blood-stage Pv vaccine development. Results of the in vitro growth inhibition assay (GIA) with transgenic P. knowlesi (Pk) parasites expressing the Pv Duffy-binding protein region II (PvDBPII) correlate with in vivo protection in the first PvDBPII controlled human malaria infection (CHMI) trials, making the PkGIA an ideal selection tool once the precision of the assay is defined. To determine the precision in percentage of inhibition in GIA (%GIA) and in GIA50 (antibody concentration that gave 50 %GIA), ten GIAs with transgenic Pk parasites were conducted with four different anti-PvDBPII human monoclonal antibodies (mAbs) at concentrations of 0.016 to 2 mg/mL, and three GIAs with eighty anti-PvDBPII human polyclonal antibodies (pAbs) at 10 mg/mL. A significant assay-to-assay variation was observed, and the analysis revealed a standard deviation (SD) of 13.1 in the mAb and 5.94 in the pAb dataset for %GIA, with a LogGIA50 SD of 0.299 (for mAbs). Moreover, the ninety-five percent confidence interval (95 %CI) for %GIA or GIA50 in repeat assays was calculated in this investigation. The error range determined in this study will help researchers to compare PkGIA results from different assays and studies appropriately, thus supporting the development of future blood-stage malaria vaccine candidates, specifically second-generation PvDBPII-based formulations.

PvDBPII elicits multiple antibody-mediated mechanisms that reduce growth in a Plasmodium vivax challenge trial.

The receptor-binding domain, region II, of the Plasmodium vivax Duffy binding protein (PvDBPII) binds the Duffy antigen on the reticulocyte surface to mediate invasion. A heterologous vaccine challenge trial recently showed that a delayed dosing regimen with recombinant PvDBPII SalI variant formulated with adjuvant Matrix-MTM reduced the in vivo parasite multiplication rate (PMR) in immunized volunteers challenged with the Thai P. vivax isolate PvW1. Here, we describe extensive analysis of the polyfunctional antibody responses elicited by PvDBPII immunization and identify immune correlates for PMR reduction. A classification algorithm identified antibody features that significantly contribute to PMR reduction. These included antibody titre, receptor-binding inhibitory titre, dissociation constant of the PvDBPII-antibody interaction, complement C1q and Fc gamma receptor binding and specific IgG subclasses. These data suggest that multiple immune mechanisms elicited by PvDBPII immunization are likely to be associated with protection and the immune correlates identified could guide the development of an effective vaccine for P. vivax malaria. Importantly, all the polyfunctional antibody features that correlated with protection cross-reacted with both PvDBPII SalI and PvW1 variants, suggesting that immunization with PvDBPII should protect against diverse P. vivax isolates.

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.

A systematic analysis of the human immune response to Plasmodium vivax.

BACKGROUNDThe biology of Plasmodium vivax is markedly different from that of P. falciparum; how this shapes the immune response to infection remains unclear. To address this shortfall, we inoculated human volunteers with a clonal field isolate of P. vivax and tracked their response through infection and convalescence.METHODSParticipants were injected intravenously with blood-stage parasites and infection dynamics were tracked in real time by quantitative PCR. Whole blood samples were used for high dimensional protein analysis, RNA sequencing, and cytometry by time of flight, and temporal changes in the host response to P. vivax were quantified by linear regression. Comparative analyses with P. falciparum were then undertaken using analogous data sets derived from prior controlled human malaria infection studies.RESULTSP. vivax rapidly induced a type I inflammatory response that coincided with hallmark features of clinical malaria. This acute-phase response shared remarkable overlap with that induced by P. falciparum but was significantly elevated (at RNA and protein levels), leading to an increased incidence of pyrexia. In contrast, T cell activation and terminal differentiation were significantly increased in volunteers infected with P. falciparum. Heterogeneous CD4+ T cells were found to dominate this adaptive response and phenotypic analysis revealed unexpected features normally associated with cytotoxicity and autoinflammatory disease.CONCLUSIONP. vivax triggers increased systemic interferon signaling (cf P. falciparum), which likely explains its reduced pyrogenic threshold. In contrast, P. falciparum drives T cell activation far in excess of P. vivax, which may partially explain why falciparum malaria more frequently causes severe disease.TRIAL REGISTRATIONClinicalTrials.gov NCT03797989.FUNDINGThe European Union's Horizon 2020 Research and Innovation programme, the Wellcome Trust, and the Royal Society.

Vaccination with Plasmodium vivax Duffy-binding protein inhibits parasite growth during controlled human malaria infection.

There are no licensed vaccines against Plasmodium vivax. We conducted two phase 1/2a clinical trials to assess two vaccines targeting P. vivax Duffy-binding protein region II (PvDBPII). Recombinant viral vaccines using chimpanzee adenovirus 63 (ChAd63) and modified vaccinia virus Ankara (MVA) vectors as well as a protein and adjuvant formulation (PvDBPII/Matrix-M) were tested in both a standard and a delayed dosing regimen. Volunteers underwent controlled human malaria infection (CHMI) after their last vaccination, alongside unvaccinated controls. Efficacy was assessed by comparisons of parasite multiplication rates in the blood. PvDBPII/Matrix-M, given in a delayed dosing regimen, elicited the highest antibody responses and reduced the mean parasite multiplication rate after CHMI by 51% (n = 6) compared with unvaccinated controls (n = 13), whereas no other vaccine or regimen affected parasite growth. Both viral-vectored and protein vaccines were well tolerated and elicited expected, short-lived adverse events. Together, these results support further clinical evaluation of the PvDBPII/Matrix-M P. vivax vaccine.

Assessment of precision in growth inhibition assay (GIA) using human anti-PfRH5 antibodies.

BACKGROUND: For blood-stage malaria vaccine development, the in vitro growth inhibition assay (GIA) has been widely used to evaluate functionality of vaccine-induced antibodies (Ab), and Plasmodium falciparum reticulocyte-binding protein homolog 5 (RH5) is a leading blood-stage antigen. However, precision, also called "error of assay (EoA)", in GIA readouts and the source of EoA has not been evaluated systematically. METHODS: In the Main GIA experiment, 4 different cultures of P. falciparum 3D7 parasites were prepared with red blood cells (RBC) collected from 4 different donors. For each culture, 7 different anti-RH5 Ab (either monoclonal or polyclonal Ab) were tested by GIA at two concentrations on three different days (168 data points). To evaluate sources of EoA in % inhibition in GIA (%GIA), a linear model fit was conducted including donor (source of RBC) and day of GIA as independent variables. In addition, 180 human anti-RH5 polyclonal Ab were tested in a Clinical GIA experiment, where each Ab was tested at multiple concentrations in at least 3 independent GIAs using different RBCs (5,093 data points). The standard deviation (sd) in %GIA and in GIA50 (Ab concentration that gave 50%GIA) readouts, and impact of repeat assays on 95% confidence interval (95%CI) of these readouts was estimated. RESULTS: The Main GIA experiment revealed that the RBC donor effect was much larger than the day effect, and an obvious donor effect was also observed in the Clinical GIA experiment. Both %GIA and log-transformed GIA50 data reasonably fit a constant sd model, and sd of %GIA and log-transformed GIA50 measurements were calculated as 7.54 and 0.206, respectively. Taking the average of three repeat assays (using three different RBCs) reduces the 95%CI width in %GIA or in GIA50 measurements by ~ half compared to a single assay. CONCLUSIONS: The RBC donor effect (donor-to-donor variance on the same day) in GIA was much bigger than the day effect (day-to-day variance using the same donor's RBC) at least for the RH5 Ab evaluated in this study; thus, future GIA studies should consider the donor effect. In addition, the 95%CI for %GIA and GIA50 shown here help when comparing GIA results from different samples/groups/studies; therefore, this study supports future malaria blood-stage vaccine development.

Delayed boosting improves human antigen-specific Ig and B cell responses to the RH5.1/AS01B malaria vaccine.

Modifications to vaccine delivery that increase serum antibody longevity are of great interest for maximizing efficacy. We have previously shown that a delayed fractional (DFx) dosing schedule (0-1-6 month) - using AS01B-adjuvanted RH5.1 malaria antigen - substantially improves serum IgG durability as compared with monthly dosing (0-1-2 month; NCT02927145). However, the underlying mechanism and whether there are wider immunological changes with DFx dosing were unclear. Here, PfRH5-specific Ig and B cell responses were analyzed in depth through standardized ELISAs, flow cytometry, systems serology, and single-cell RNA-Seq (scRNA-Seq). Data indicate that DFx dosing increases the magnitude and durability of circulating PfRH5-specific B cells and serum IgG1. At the peak antibody magnitude, DFx dosing was distinguished by a systems serology feature set comprising increased FcRn binding, IgG avidity, and proportion of G2B and G2S2F IgG Fc glycans, alongside decreased IgG3, antibody-dependent complement deposition, and proportion of G1S1F IgG Fc glycan. Concomitantly, scRNA-Seq data show a higher CDR3 percentage of mutation from germline and decreased plasma cell gene expression in circulating PfRH5-specific B cells. Our data, therefore, reveal a profound impact of DFx dosing on the humoral response and suggest plausible mechanisms that could enhance antibody longevity, including improved FcRn binding by serum Ig and a potential shift in the underlying cellular response from circulating short-lived plasma cells to nonperipheral long-lived plasma cells.

Analyses of human vaccine-specific circulating and bone marrow-resident B cell populations reveal benefit of delayed vaccine booster dosing with blood-stage malaria antigens.

We have previously reported primary endpoints of a clinical trial testing two vaccine platforms for the delivery of Plasmodium vivax malaria DBPRII: viral vectors (ChAd63, MVA), and protein/adjuvant (PvDBPII with 50µg Matrix-M™ adjuvant). Delayed boosting was necessitated due to trial halts during the pandemic and provides an opportunity to investigate the impact of dosing regimens. Here, using flow cytometry - including agnostic definition of B cell populations with the clustering tool CITRUS - we report enhanced induction of DBPRII-specific plasma cell and memory B cell responses in protein/adjuvant versus viral vector vaccinees. Within protein/adjuvant groups, delayed boosting further improved B cell immunogenicity compared to a monthly boosting regimen. Consistent with this, delayed boosting also drove more durable anti-DBPRII serum IgG. In an independent vaccine clinical trial with the P. falciparum malaria RH5.1 protein/adjuvant (50µg Matrix-M™) vaccine candidate, we similarly observed enhanced circulating B cell responses in vaccinees receiving a delayed final booster. Notably, a higher frequency of vaccine-specific (putatively long-lived) plasma cells was detected in the bone marrow of these delayed boosting vaccinees by ELISPOT and correlated strongly with serum IgG. Finally, following controlled human malaria infection with P. vivax parasites in the DBPRII trial, in vivo growth inhibition was observed to correlate with DBPRII-specific B cell and serum IgG responses. In contrast, the CD4+ and CD8+ T cell responses were impacted by vaccine platform but not dosing regimen and did not correlate with in vivo growth inhibition in a challenge model. Taken together, our DBPRII and RH5 data suggest an opportunity for protein/adjuvant dosing regimen optimisation in the context of rational vaccine development against pathogens where protection is antibody-mediated.

Repeat controlled human malaria infection of healthy UK adults with blood-stage Plasmodium falciparum: Safety and parasite growth dynamics.

In endemic settings it is known that natural malaria immunity is gradually acquired following repeated exposures. Here we sought to assess whether similar acquisition of blood-stage malaria immunity would occur following repeated parasite exposure by controlled human malaria infection (CHMI). We report the findings of repeat homologous blood-stage Plasmodium falciparum (3D7 clone) CHMI studies VAC063C (ClinicalTrials.gov NCT03906474) and VAC063 (ClinicalTrials.gov NCT02927145). In total, 24 healthy, unvaccinated, malaria-naïve UK adult participants underwent primary CHMI followed by drug treatment. Ten of these then underwent secondary CHMI in the same manner, and then six of these underwent a final tertiary CHMI. As with primary CHMI, malaria symptoms were common following secondary and tertiary infection, however, most resolved within a few days of treatment and there were no long term sequelae or serious adverse events related to CHMI. Despite detectable induction and boosting of anti-merozoite serum IgG antibody responses following each round of CHMI, there was no clear evidence of anti-parasite immunity (manifest as reduced parasite growth in vivo) conferred by repeated challenge with the homologous parasite in the majority of volunteers. However, three volunteers showed some variation in parasite growth dynamics in vivo following repeat CHMI that were either modest or short-lived. We also observed no major differences in clinical symptoms or laboratory markers of infection across the primary, secondary and tertiary challenges. However, there was a trend to more severe pyrexia after primary CHMI and the absence of a detectable transaminitis post-treatment following secondary and tertiary infection. We hypothesize that this could represent the initial induction of clinical immunity. Repeat homologous blood-stage CHMI is thus safe and provides a model with the potential to further the understanding of naturally acquired immunity to blood-stage infection in a highly controlled setting. Clinical Trial Registration: ClinicalTrials.gov, identifier NCT03906474, NCT02927145.

Impact of a blood-stage vaccine on Plasmodium vivax malaria.

Background: There are no licensed vaccines against Plasmodium vivax , the most common cause of malaria outside of Africa. Methods: We conducted two Phase I/IIa clinical trials to assess the safety, immunogenicity and efficacy of two vaccines targeting region II of P. vivax Duffy-binding protein (PvDBPII). Recombinant viral vaccines (using ChAd63 and MVA vectors) were administered at 0, 2 months or in a delayed dosing regimen (0, 17, 19 months), whilst a protein/adjuvant formulation (PvDBPII/Matrix-M™) was administered monthly (0, 1, 2 months) or in a delayed dosing regimen (0, 1, 14 months). Delayed regimens were due to trial halts during the COVID-19 pandemic. Volunteers underwent heterologous controlled human malaria infection (CHMI) with blood-stage P. vivax parasites at 2-4 weeks following their last vaccination, alongside unvaccinated controls. Efficacy was assessed by comparison of parasite multiplication rate (PMR) in blood post-CHMI, modelled from parasitemia measured by quantitative polymerase-chain-reaction (qPCR). Results: Thirty-two volunteers were enrolled and vaccinated (n=16 for each vaccine). No safety concerns were identified. PvDBPII/Matrix-M™, given in the delayed dosing regimen, elicited the highest antibody responses and reduced the mean PMR following CHMI by 51% (range 36-66%; n=6) compared to unvaccinated controls (n=13). No other vaccine or regimen impacted parasite growth. In vivo growth inhibition of blood-stage P. vivax correlated with functional antibody readouts of vaccine immunogenicity. Conclusions: Vaccination of malaria-naïve adults with a delayed booster regimen of PvDBPII/ Matrix-M™ significantly reduces the growth of blood-stage P. vivax . Funded by the European Commission and Wellcome Trust; VAC069, VAC071 and VAC079 ClinicalTrials.gov numbers NCT03797989 , NCT04009096 and NCT04201431 .

RH5.1-CyRPA-Ripr antigen combination vaccine shows little improvement over RH5.1 in a preclinical setting.

Background: RH5 is the leading vaccine candidate for the Plasmodium falciparum blood stage and has shown impact on parasite growth in the blood in a human clinical trial. RH5 binds to Ripr and CyRPA at the apical end of the invasive merozoite form, and this complex, designated RCR, is essential for entry into human erythrocytes. RH5 has advanced to human clinical trials, and the impact on parasite growth in the blood was encouraging but modest. This study assessed the potential of a protein-in-adjuvant blood stage malaria vaccine based on a combination of RH5, Ripr and CyRPA to provide improved neutralizing activity against P. falciparum in vitro. Methods: Mice were immunized with the individual RCR antigens to down select the best performing adjuvant formulation and rats were immunized with the individual RCR antigens to select the correct antigen dose. A second cohort of rats were immunized with single, double and triple antigen combinations to assess immunogenicity and parasite neutralizing activity in growth inhibition assays. Results: The DPX® platform was identified as the best performing formulation in potentiating P. falciparum inhibitory antibody responses to these antigens. The three antigens derived from RH5, Ripr and CyRPA proteins formulated with DPX induced highly inhibitory parasite neutralising antibodies. Notably, RH5 either as a single antigen or in combination with Ripr and/or CyRPA, induced inhibitory antibodies that outperformed CyRPA, Ripr. Conclusion: An RCR combination vaccine may not induce substantially improved protective immunity as compared with RH5 as a single immunogen in a clinical setting and leaves the development pathway open for other antigens to be combined with RH5 as a next generation malaria vaccine.

Poor CD4+ T Cell Immunogenicity Limits Humoral Immunity to P. falciparum Transmission-Blocking Candidate Pfs25 in Humans.

Plasmodium falciparum transmission-blocking vaccines (TBVs) targeting the Pfs25 antigen have shown promise in mice but the same efficacy has never been achieved in humans. We have previously published pre-clinical data related to a TBV candidate Pfs25-IMX313 encoded in viral vectors which was very promising and hence progressed to human clinical trials. The results from the clinical trial of this vaccine were very modest. Here we unravel why, contrary to mice, this vaccine has failed to induce robust antibody (Ab) titres in humans to elicit transmission-blocking activity. We examined Pfs25-specific B cell and T follicular helper (Tfh) cell responses in mice and humans after vaccination with Pfs25-IMX313 encoded by replication-deficient chimpanzee adenovirus serotype 63 (ChAd63) and the attenuated orthopoxvirus modified vaccinia virus Ankara (MVA) delivered in the heterologous prime-boost regimen via intramuscular route. We found that after vaccination, the Pfs25-IMX313 was immunologically suboptimal in humans compared to mice in terms of serum Ab production and antigen-specific B, CD4+ and Tfh cell responses. We identified that the key determinant for the poor anti-Pfs25 Ab formation in humans was the lack of CD4+ T cell recognition of Pfs25-IMX313 derived peptide epitopes. This is supported by correlations established between the ratio of proliferated antigen-specific CD4+/Tfh-like T cells, CXCL13 sera levels, and the corresponding numbers of circulating Pfs25-specific memory B cells, that consequently reflected on antigen-specific IgG sera levels. These correlations can inform the design of next-generation Pfs25-based vaccines for robust and durable blocking of malaria transmission.

Controlled human malaria infection with a clone of Plasmodium vivax with high-quality genome assembly.

Controlled human malaria infection (CHMI) provides a highly informative means to investigate host-pathogen interactions and enable in vivo proof-of-concept efficacy testing of new drugs and vaccines. However, unlike Plasmodium falciparum, well-characterized P. vivax parasites that are safe and suitable for use in modern CHMI models are limited. Here, 2 healthy malaria-naive United Kingdom adults with universal donor blood group were safely infected with a clone of P. vivax from Thailand by mosquito-bite CHMI. Parasitemia developed in both volunteers, and prior to treatment, each volunteer donated blood to produce a cryopreserved stabilate of infected RBCs. Following stringent safety screening, the parasite stabilate from one of these donors (PvW1) was thawed and used to inoculate 6 healthy malaria-naive United Kingdom adults by blood-stage CHMI, at 3 different dilutions. Parasitemia developed in all volunteers, who were then successfully drug treated. PvW1 parasite DNA was isolated and sequenced to produce a high-quality genome assembly by using a hybrid assembly method. We analyzed leading vaccine candidate antigens and multigene families, including the vivax interspersed repeat (VIR) genes, of which we identified 1145 in the PvW1 genome. Our genomic analysis will guide future assessment of candidate vaccines and drugs, as well as experimental medicine studies.

Safety and Immunogenicity of ChAd63/MVA Pfs25-IMX313 in a Phase I First-in-Human Trial.

Background: Transmission blocking vaccines targeting the sexual-stages of the malaria parasite could play a major role to achieve elimination and eradication of malaria. The Plasmodium falciparum Pfs25 protein (Pfs25) is the most clinically advanced candidate sexual-stage antigen. IMX313, a complement inhibitor C4b-binding protein that forms heptamers with the antigen fused to it, improve antibody responses. This is the first time that viral vectors have been used to induce antibodies in humans against an antigen that is expressed only in the mosquito vector. Methods: Clinical trial looking at safety and immunogenicity of two recombinant viral vectored vaccines encoding Pfs25-IMX313 in healthy malaria-naive adults. Replication-deficient chimpanzee adenovirus serotype 63 (ChAd63) and the attenuated orthopoxvirus modified vaccinia virus Ankara (MVA), encoding Pfs25-IMX313, were delivered by the intramuscular route in a heterologous prime-boost regimen using an 8-week interval. Safety data and samples for immunogenicity assays were taken at various time-points. Results: The reactogenicity of the vaccines was similar to that seen in previous trials using the same viral vectors encoding other antigens. The vaccines were immunogenic and induced both antibody and T cell responses against Pfs25, but significant transmission reducing activity (TRA) was not observed in most volunteers by standard membrane feeding assay. Conclusion: Both vaccines were well tolerated and demonstrated a favorable safety profile in malaria-naive adults. However, the transmission reducing activity of the antibodies generated were weak, suggesting the need for an alternative vaccine formulation. Trial Registration: Clinicaltrials.gov NCT02532049.

Antibodies from malaria-exposed Malians generally interact additively or synergistically with human vaccine-induced RH5 antibodies.

Reticulocyte-binding protein homolog 5 (RH5) is a leading Plasmodium falciparum blood-stage vaccine candidate. Another possible candidate, apical membrane antigen 1 (AMA1), was not efficacious in malaria-endemic populations, likely due to pre-existing antimalarial antibodies that interfered with the activity of vaccine-induced AMA1 antibodies, as judged by in vitro growth inhibition assay (GIA). To determine how pre-existing antibodies interact with vaccine-induced RH5 antibodies, we purify total and RH5-specific immunoglobulin Gs (IgGs) from malaria-exposed Malians and malaria-naive RH5 vaccinees. Infection-induced RH5 antibody titers are much lower than those induced by vaccination, and RH5-specific IgGs show differences in the binding site between the two populations. In GIA, Malian polyclonal IgGs show additive or synergistic interactions with RH5 human monoclonal antibodies and overall additive interactions with vaccine-induced polyclonal RH5 IgGs. These results suggest that pre-existing antibodies will interact favorably with vaccine-induced RH5 antibodies, in contrast to AMA1 antibodies. This study supports RH5 vaccine trials in malaria-endemic regions.

Reduced blood-stage malaria growth and immune correlates in humans following RH5 vaccination.

BACKGROUND: Development of an effective vaccine against the pathogenic blood-stage infection of human malaria has proved challenging, and no candidate vaccine has affected blood-stage parasitemia following controlled human malaria infection (CHMI) with blood-stage Plasmodium falciparum. METHODS: We undertook a phase I/IIa clinical trial in healthy adults in the United Kingdom of the RH5.1 recombinant protein vaccine, targeting the P. falciparum reticulocyte-binding protein homolog 5 (RH5), formulated in AS01B adjuvant. We assessed safety, immunogenicity, and efficacy against blood-stage CHMI. Trial registered at ClinicalTrials.gov, NCT02927145. FINDINGS: The RH5.1/AS01B formulation was administered using a range of RH5.1 protein vaccine doses (2, 10, and 50 µg) and was found to be safe and well tolerated. A regimen using a delayed and fractional third dose, in contrast to three doses given at monthly intervals, led to significantly improved antibody response longevity over ∼2 years of follow-up. Following primary and secondary CHMI of vaccinees with blood-stage P. falciparum, a significant reduction in parasite growth rate was observed, defining a milestone for the blood-stage malaria vaccine field. We show that growth inhibition activity measured in vitro using purified immunoglobulin G (IgG) antibody strongly correlates with in vivo reduction of the parasite growth rate and also identify other antibody feature sets by systems serology, including the plasma anti-RH5 IgA1 response, that are associated with challenge outcome. CONCLUSIONS: Our data provide a new framework to guide rational design and delivery of next-generation vaccines to protect against malaria disease. FUNDING: This study was supported by USAID, UK MRC, Wellcome Trust, NIAID, and the NIHR Oxford-BRC.

Identification and Immune Assessment of T Cell Epitopes in Five Plasmodium falciparum Blood Stage Antigens to Facilitate Vaccine Candidate Selection and Optimization.

The hurdles to effective blood stage malaria vaccine design include immune evasion tactics used by the parasite such as redundant invasion pathways and antigen variation among circulating parasite strains. While blood stage malaria vaccine development primarily focuses on eliciting optimal humoral responses capable of blocking erythrocyte invasion, clinically-tested Plasmodium falciparum (Pf) vaccines have not elicited sterile protection, in part due to the dramatically high levels of antibody needed. Recent development efforts with non-redundant, conserved blood stage antigens suggest both high antibody titer and rapid antibody binding kinetics are important efficacy factors. Based on the central role of helper CD4 T cells in development of strong, protective immune responses, we systematically analyzed the class II epitope content in five leading Pf blood stage antigens (RH5, CyRPA, RIPR, AMA1 and EBA175) using in silico, in vitro, and ex vivo methodologies. We employed in silico T cell epitope analysis to enable identification of 67 HLA-restricted class II epitope clusters predicted to bind a panel of nine HLA-DRB1 alleles. We assessed a subset of these for HLA-DRB1 allele binding in vitro, to verify the in silico predictions. All clusters assessed (40 clusters represented by 46 peptides) bound at least two HLA-DR alleles in vitro. The overall epitope prediction to in vitro HLA-DRB1 allele binding accuracy was 71%. Utilizing the set of RH5 class II epitope clusters (10 clusters represented by 12 peptides), we assessed stimulation of T cells collected from HLA-matched RH5 vaccinees using an IFN-γ T cell recall assay. All clusters demonstrated positive recall responses, with the highest responses - by percentage of responders and response magnitude - associated with clusters located in the N-terminal region of RH5. Finally, a statistically significant correlation between in silico epitope predictions and ex vivo IFN-γ recall response was found when accounting for HLA-DR matches between the epitope predictions and donor HLA phenotypes. This is the first comprehensive analysis of class II epitope content in RH5, CyRPA, RIPR, AMA1 and EBA175 accompanied by in vitro HLA binding validation for all five proteins and ex vivo T cell response confirmation for RH5.

Low Adenovirus Vaccine Doses Administered to Skin Using Microneedle Patches Induce Better Functional Antibody Immunogenicity as Compared to Systemic Injection.

Adenovirus-based vaccines are demonstrating promising clinical potential for multiple infectious diseases, including COVID-19. However, the immunogenicity of the vector itself decreases its effectiveness as a boosting vaccine due to the induction of strong anti-vector neutralizing immunity. Here we determined how dissolvable microneedle patches (DMN) for skin immunization can overcome this issue, using a clinically-relevant adenovirus-based Plasmodium falciparum malaria vaccine, AdHu5-PfRH5, in mice. Incorporation of vaccine into patches significantly enhanced its thermostability compared to the liquid form. Conventional high dose repeated immunization by the intramuscular (IM) route induced low antigen-specific IgG titres and high anti-vector immunity. A low priming dose of vaccine, by the IM route, but more so using DMN patches, induced the most efficacious immune responses, assessed by parasite growth inhibitory activity (GIA) assays. Administration of low dose AdHu5-PfRH5 using patches to the skin, boosted by high dose IM, induced the highest antigen-specific serum IgG response after boosting, the greatest skewing of the antibody response towards the antigen and away from the vector, and the highest efficacy. This study therefore demonstrates that repeated use of the same adenovirus vaccine can be highly immunogenic towards the transgene if a low dose is used to prime the response. It also provides a method of stabilizing adenovirus vaccine, in easy-to-administer dissolvable microneedle patches, permitting storage and distribution out of cold chain.

Protein/AS01B vaccination elicits stronger, more Th2-skewed antigen-specific human T follicular helper cell responses than heterologous viral vectors.

Interactions between B cells and CD4+ T follicular helper (Tfh) cells are key determinants of humoral responses. Using samples from clinical trials performed with the malaria vaccine candidate antigen Plasmodium falciparum merozoite protein (PfRH5), we compare the frequency, phenotype, and gene expression profiles of PfRH5-specific circulating Tfh (cTfh) cells elicited by two leading human vaccine delivery platforms: heterologous viral vector prime boost and protein with AS01B adjuvant. We demonstrate that the protein/AS01B platform induces a higher-magnitude antigen-specific cTfh cell response and that this correlates with peak anti-PfRH5 IgG concentrations, frequency of PfRH5-specific memory B cells, and antibody functionality. Furthermore, our data indicate a greater Th2/Tfh2 skew within the polyfunctional response elicited following vaccination with protein/AS01B as compared to a Th1/Tfh1 skew with viral vectors. These data highlight the impact of vaccine platform on the cTfh cell response driving humoral immunity, associating a high-magnitude, Th2-biased cTfh response with potent antibody production.