Human Antibodies that Slow Erythrocyte Invasion Potentiate Malaria-Neutralizing Antibodies.

The Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) is the leading target for next-generation vaccines against the disease-causing blood-stage of malaria. However, little is known about how human antibodies confer functional immunity against this antigen. We isolated a panel of human monoclonal antibodies (mAbs) against PfRH5 from peripheral blood B cells from vaccinees in the first clinical trial of a PfRH5-based vaccine. We identified a subset of mAbs with neutralizing activity that bind to three distinct sites and another subset of mAbs that are non-functional, or even antagonistic to neutralizing antibodies. We also identify the epitope of a novel group of non-neutralizing antibodies that significantly reduce the speed of red blood cell invasion by the merozoite, thereby potentiating the effect of all neutralizing PfRH5 antibodies as well as synergizing with antibodies targeting other malaria invasion proteins. Our results provide a roadmap for structure-guided vaccine development to maximize antibody efficacy against blood-stage malaria.

Evaluation of a standardised Vi poly-l-lysine ELISA for serology of Vi capsular polysaccharide antibodies.

Typhoid vaccines based on protein-conjugated capsular Vi polysaccharide (TCVs) prevent typhoid in infants and young children. Analysis of the serum anti-Vi IgG response following immunisation against typhoid confirms the immunogenicity of TCVs and forms an important part of the pathway to licensing. Comparative studies could expedite the licencing process, and the availability of a standardised ELISA method alongside the 1st International Standard (IS) 16/138 for anti-typhoid capsular Vi polysaccharide IgG (human) will facilitate this process. To this end, a non-commercial ELISA based on a coat of Vi and poly-l-lysine (Vi-PLL ELISA) was evaluated by 10 laboratories. Eight serum samples, including IS 16/138, were tested in the standardised Vi-PLL ELISA (n = 10), a commercial Vi ELISA (n = 3) and a biotinylated Vi ELISA (n = 1). Valid estimates of potencies relative to IS 16/138 were obtained for all samples in the Vi-PLL ELISA and the commercial ELISA, with good repeatability and reproducibility evident from the study results and concordant estimates obtained by the two ELISA methods. The study demonstrates that the Vi-PLL ELISA can be used in clinical trial studies to determine the immunogenicity of TCVs.

Demonstration of the Blood-Stage Plasmodium falciparum Controlled Human Malaria Infection Model to Assess Efficacy of the P. falciparum Apical Membrane Antigen 1 Vaccine, FMP2.1/AS01.

BACKGROUND: Models of controlled human malaria infection (CHMI) initiated by mosquito bite have been widely used to assess efficacy of preerythrocytic vaccine candidates in small proof-of-concept phase 2a clinical trials. Efficacy testing of blood-stage malaria parasite vaccines, however, has generally relied on larger-scale phase 2b field trials in malaria-endemic populations. We report the use of a blood-stage P. falciparum CHMI model to assess blood-stage vaccine candidates, using their impact on the parasite multiplication rate (PMR) as the primary efficacy end point. METHODS: Fifteen healthy United Kingdom adult volunteers were vaccinated with FMP2.1, a protein vaccine that is based on the 3D7 clone sequence of apical membrane antigen 1 (AMA1) and formulated in Adjuvant System 01 (AS01). Twelve vaccinees and 15 infectivity controls subsequently underwent blood-stage CHMI. Parasitemia was monitored by quantitative real-time polymerase chain reaction (PCR) analysis, and PMR was modeled from these data. RESULTS: FMP2.1/AS01 elicited anti-AMA1 T-cell and serum antibody responses. Analysis of purified immunoglobulin G showed functional growth inhibitory activity against P. falciparum in vitro. There were no vaccine- or CHMI-related safety concerns. All volunteers developed blood-stage parasitemia, with no impact of the vaccine on PMR. CONCLUSIONS: FMP2.1/AS01 demonstrated no efficacy after blood-stage CHMI. However, the model induced highly reproducible infection in all volunteers and will accelerate proof-of-concept testing of future blood-stage vaccine candidates. CLINICAL TRIALS REGISTRATION: NCT02044198.

Increased sample volume and use of quantitative reverse-transcription PCR can improve prediction of liver-to-blood inoculum size in controlled human malaria infection studies.

BACKGROUND: Controlled human malaria infection (CHMI) studies increasingly rely on nucleic acid test (NAT) methods to detect and quantify parasites in the blood of infected participants. The lower limits of detection and quantification vary amongst the assays used throughout the world, which may affect the ability of mathematical models to accurately estimate the liver-to-blood inoculum (LBI) values that are used to judge the efficacy of pre-erythrocytic vaccine and drug candidates. METHODS: Samples were collected around the time of onset of pre-patent parasitaemia from subjects who enrolled in two different CHMI clinical trials. Blood samples were tested for Plasmodium falciparum 18S rRNA and/or rDNA targets by different NAT methods and results were compared. Methods included an ultrasensitive, large volume modification of an established quantitative reverse transcription PCR (qRT-PCR) assay that achieves detection of as little as one parasite/mL of whole blood. RESULTS: Large volume qRT-PCR at the University of Washington was the most sensitive test and generated quantifiable data more often than any other NAT methodology. Standard quantitative PCR (qPCR) performed at the University of Oxford and standard volume qRT-PCR performed at the University of Washington were less sensitive than the large volume qRT-PCR, especially at 6.5 days after CHMI. In these trials, the proportion of participants for whom LBI could be accurately quantified using parasite density value greater than or equal to the lower limit of quantification was increased. A greater improvement would be expected in trials in which numerous subjects receive a lower LBI or low dose challenge. CONCLUSIONS: Standard qPCR and qRT-PCR methods with analytical sensitivities of ~20 parasites/mL probably suffice for most CHMI purposes, but the newly developed large volume qRT-PCR may be able to answer specific questions when more analytical sensitivity is required.

Assessment of immune interference, antagonism, and diversion following human immunization with biallelic blood-stage malaria viral-vectored vaccines and controlled malaria infection.

Overcoming antigenic variation is one of the major challenges in the development of an effective vaccine against Plasmodium falciparum, a causative agent of human malaria. Inclusion of multiple Ag variants in subunit vaccine candidates is one strategy that has aimed to overcome this problem for the leading blood-stage malaria vaccine targets, that is, merozoite surface protein 1 (MSP1) and apical membrane Ag 1 (AMA1). However, previous studies, utilizing malaria Ags, have concluded that inclusion of multiple allelic variants, encoding altered peptide ligands, in such a vaccine may be detrimental to both the priming and in vivo restimulation of Ag-experienced T cells. In this study, we analyze the T cell responses to two alleles of MSP1 and AMA1 induced by vaccination of malaria-naive adult volunteers with bivalent viral-vectored vaccine candidates. We show a significant bias to the 3D7/MAD20 allele compared with the Wellcome allele for the 33 kDa region of MSP1, but not for the 19 kDa fragment or the AMA1 Ag. Although this bias could be caused by "immune interference" at priming, the data do not support a significant role for "immune antagonism" during memory T cell restimulation, despite observation of the latter at a minimal epitope level in vitro. A lack of class I HLA epitopes in the Wellcome allele that are recognized by vaccinated volunteers may in fact contribute to the observed bias. We also show that controlled infection with 3D7 strain P. falciparum parasites neither boosts existing 3D7-specific T cell responses nor appears to "immune divert" cellular responses toward the Wellcome allele.

Combining viral vectored and protein-in-adjuvant vaccines against the blood-stage malaria antigen AMA1: report on a phase 1a clinical trial.

The development of effective vaccines against difficult disease targets will require the identification of new subunit vaccination strategies that can induce and maintain effective immune responses in humans. Here we report on a phase 1a clinical trial using the AMA1 antigen from the blood-stage Plasmodium falciparum malaria parasite delivered either as recombinant protein formulated with Alhydrogel adjuvant with and without CPG 7909, or using recombinant vectored vaccines--chimpanzee adenovirus ChAd63 and the orthopoxvirus MVA. A variety of promising "mixed-modality" regimens were tested. All volunteers were primed with ChAd63, and then subsequently boosted with MVA and/or protein-in-adjuvant using either an 8- or 16-week prime-boost interval. We report on the safety of these regimens, as well as the T cell, B cell, and serum antibody responses. Notably, IgG antibody responses primed by ChAd63 were comparably boosted by AMA1 protein vaccine, irrespective of whether CPG 7909 was included in the Alhydrogel adjuvant. The ability to improve the potency of a relatively weak aluminium-based adjuvant in humans, by previously priming with an adenoviral vaccine vector encoding the same antigen, thus offers a novel vaccination strategy for difficult or neglected disease targets when access to more potent adjuvants is not possible.

Analysis of human B-cell responses following ChAd63-MVA MSP1 and AMA1 immunization and controlled malaria infection.

Acquisition of non-sterilizing natural immunity to Plasmodium falciparum malaria has been shown in low transmission areas following multiple exposures. However, conflicting data from endemic areas suggest that the parasite may interfere with the induction of effective B-cell responses. To date, the impact of blood-stage parasite exposure on antigen-specific B cells has not been reported following controlled human malaria infection (CHMI). Here we analysed human B-cell responses in a series of Phase I/IIa clinical trials, which include CHMI, using candidate virus-vectored vaccines encoding two blood-stage antigens: merozoite surface protein 1 (MSP1) and apical membrane antigen 1 (AMA1). Previously vaccinated volunteers show boosting of pre-existing antigen-specific memory B-cell (mBC) responses following CHMI. In contrast, unvaccinated malaria-naive control volunteers developed an mBC response against MSP1 but not AMA1. Serum IgG correlated with the mBC response after booster vaccination but this relationship was less well maintained following CHMI. A significant reduction in peripheral MSP1-specific mBC was observed at the point of diagnosis of blood-stage infection. This was coincident with a reduction in peripheral blood B-cell subsets expressing CXCR3 and elevated serum levels of interferon-γ and CXCL9, suggesting migration away from the periphery. These CHMI data confirm that mBC and antibody responses can be induced and boosted by blood-stage parasite exposure, in support of epidemiological studies on low-level parasite exposure.

ChAd63-MVA-vectored blood-stage malaria vaccines targeting MSP1 and AMA1: assessment of efficacy against mosquito bite challenge in humans.

The induction of cellular immunity, in conjunction with antibodies, may be essential for vaccines to protect against blood-stage infection with the human malaria parasite Plasmodium falciparum. We have shown that prime-boost delivery of P. falciparum blood-stage antigens by chimpanzee adenovirus 63 (ChAd63) followed by the attenuated orthopoxvirus MVA is safe and immunogenic in healthy adults. Here, we report on vaccine efficacy against controlled human malaria infection delivered by mosquito bites. The blood-stage malaria vaccines were administered alone, or together (MSP1+AMA1), or with a pre-erythrocytic malaria vaccine candidate (MSP1+ME-TRAP). In this first human use of coadministered ChAd63-MVA regimes, we demonstrate immune interference whereby responses against merozoite surface protein 1 (MSP1) are dominant over apical membrane antigen 1 (AMA1) and ME-TRAP. We also show that induction of strong cellular immunity against MSP1 and AMA1 is safe, but does not impact on parasite growth rates in the blood. In a subset of vaccinated volunteers, a delay in time to diagnosis was observed and sterilizing protection was observed in one volunteer coimmunized with MSP1+AMA1-results consistent with vaccine-induced pre-erythrocytic, rather than blood-stage, immunity. These data call into question the utility of T cell-inducing blood-stage malaria vaccines and suggest that the focus should remain on high-titer antibody induction against susceptible antigen targets.

Clinical assessment of a recombinant simian adenovirus ChAd63: a potent new vaccine vector.

BACKGROUND: Vaccine development in human Plasmodium falciparum malaria has been hampered by the exceptionally high levels of CD8(+) T cells required for efficacy. Use of potently immunogenic human adenoviruses as vaccine vectors could overcome this problem, but these are limited by preexisting immunity to human adenoviruses. METHODS: From 2007 to 2010, we undertook a phase I dose and route finding study of a new malaria vaccine, a replication-incompetent chimpanzee adenovirus 63 (ChAd63) encoding the preerythrocytic insert multiple epitope thrombospondin-related adhesion protein (ME-TRAP; n = 54 vaccinees) administered alone (n = 28) or with a modified vaccinia virus Ankara (MVA) ME-TRAP booster immunization 8 weeks later (n = 26). We observed an excellent safety profile. High levels of TRAP antigen-specific CD8(+) and CD4(+) T cells, as detected by interferon γ enzyme-linked immunospot assay and flow cytometry, were induced by intramuscular ChAd63 ME-TRAP immunization at doses of 5 × 10(10) viral particles and above. Subsequent administration of MVA ME-TRAP boosted responses to exceptionally high levels, and responses were maintained for up to 30 months postvaccination. CONCLUSIONS: The ChAd63 chimpanzee adenovirus vector appears safe and highly immunogenic, providing a viable alternative to human adenoviruses as vaccine vectors for human use. CLINICAL TRIALS REGISTRATION: NCT00890019.

Complementary measurement of nontyphoidal Salmonella-specific IgG and IgA antibodies in oral fluid and serum.

Objectives: Immuno-epidemiological studies of orally acquired, enteric pathogens such as nontyphoidal Salmonella (NTS) often focus on serological measures of immunity, ignoring potentially relevant oral mucosal responses. In this study we sought to assess the levels and detectability of both oral fluid and serum IgG and IgA to NTS antigens, in endemic and non-endemic populations. Methods: IgG and IgA antibodies specific for Salmonella Typhimurium and Salmonella Enteritidis O antigen and phase 1 flagellin were assessed using Enzyme Linked Immunosorbent Assay (ELISA). Paired oral fluid and serum samples were collected from groups of 50 UK adults, Kenyan adults and Kenyan infants. Additionally, oral fluid alone was collected from 304 Kenyan individuals across a range of ages. Results: Antigen-specific IgG and IgA was detectable in the oral fluid of both adults and infants. Oral fluid antibody increased with age, peaking in adulthood for both IgG and IgA but a separate peak was also observed for IgA in infants. Oral fluid and serum responses correlated for IgG but not IgA. Despite standardised collection the relationship between oral fluid volume and antibody levels varied with age and country of origin. Conclusions: Measurement of NTS-specific oral fluid antibody can be used to complement measurement of serum antibody. For IgA in particular, oral fluid may offer insights into how protective immunity to NTS changes as individuals transition with age, from maternal to acquired systemic and mucosal immunity. This may prove useful in helping to guide future vaccine design.

Phase Ia clinical evaluation of the Plasmodium falciparum blood-stage antigen MSP1 in ChAd63 and MVA vaccine vectors.

Efficacy trials of antibody-inducing protein-in-adjuvant vaccines targeting the blood-stage Plasmodium falciparum malaria parasite have so far shown disappointing results. The induction of cell-mediated responses in conjunction with antibody responses is thought to be one alternative strategy that could achieve protective efficacy in humans. Here, we prepared chimpanzee adenovirus 63 (ChAd63) and modified vaccinia virus Ankara (MVA) replication-deficient vectors encoding the well-studied P. falciparum blood-stage malaria antigen merozoite surface protein 1 (MSP1). A phase Ia clinical trial was conducted in healthy adults of a ChAd63-MVA MSP1 heterologous prime-boost immunization regime. The vaccine was safe and generally well tolerated. Fewer systemic adverse events (AEs) were observed following ChAd63 MSP1 than MVA MSP1 administration. Exceptionally strong T-cell responses were induced, and these displayed a mixed of CD4(+) and CD8(+) phenotype. Substantial MSP1-specific serum immunoglobulin G (IgG) antibody responses were also induced, which were capable of recognizing native parasite antigen, but these did not reach titers sufficient to neutralize P. falciparum parasites in vitro. This viral vectored vaccine regime is thus a leading approach for the induction of strong cellular and humoral immunogenicity against difficult disease targets in humans. Further studies are required to assess whether this strategy can achieve protective efficacy against blood-stage malaria infection.

Induction of Functional Specific Antibodies, IgG-Secreting Plasmablasts and Memory B Cells Following BCG Vaccination.

Tuberculosis (TB) is a major global health problem and the only currently-licensed vaccine, BCG, is inadequate. Many TB vaccine candidates are designed to be given as a boost to BCG; an understanding of the BCG-induced immune response is therefore critical, and the opportunity to relate this to circumstances where BCG does confer protection may direct the design of more efficacious vaccines. While the T cell response to BCG vaccination has been well-characterized, there is a paucity of literature on the humoral response. We demonstrate BCG vaccine-mediated induction of specific antibodies in different human populations and macaque species which represent important preclinical models for TB vaccine development. We observe a strong correlation between antibody titers in serum versus plasma with modestly higher titers in serum. We also report for the first time the rapid and transient induction of antibody-secreting plasmablasts following BCG vaccination, together with a robust and durable memory B cell response in humans. Finally, we demonstrate a functional role for BCG vaccine-induced specific antibodies in opsonizing mycobacteria and enhancing macrophage phagocytosis in vitro, which may contribute to the BCG vaccine-mediated control of mycobacterial growth observed. Taken together, our findings indicate that the humoral immune response in the context of BCG vaccination merits further attention to determine whether TB vaccine candidates could benefit from the induction of humoral as well as cellular immunity.

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.

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.

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.

Therapeutic Monoclonal Antibodies for Ebola Virus Infection Derived from Vaccinated Humans.

We describe therapeutic monoclonal antibodies isolated from human volunteers vaccinated with recombinant adenovirus expressing Ebola virus glycoprotein (EBOV GP) and boosted with modified vaccinia virus Ankara. Among 82 antibodies isolated from peripheral blood B cells, almost half neutralized GP pseudotyped influenza virus. The antibody response was diverse in gene usage and epitope recognition. Although close to germline in sequence, neutralizing antibodies with binding affinities in the nano- to pico-molar range, similar to "affinity matured" antibodies from convalescent donors, were found. They recognized the mucin-like domain, glycan cap, receptor binding region, and the base of the glycoprotein. A cross-reactive cocktail of four antibodies, targeting the latter three non-overlapping epitopes, given on day 3 of EBOV infection, completely protected guinea pigs. This study highlights the value of experimental vaccine trials as a rich source of therapeutic human monoclonal antibodies.

Structural basis for inhibition of Plasmodium vivax invasion by a broadly neutralizing vaccine-induced human antibody.

The most widespread form of malaria is caused by Plasmodium vivax. To replicate, this parasite must invade immature red blood cells through a process requiring interaction of the P. vivax Duffy binding protein (PvDBP) with its human receptor, the Duffy antigen receptor for chemokines. Naturally acquired antibodies that inhibit this interaction associate with clinical immunity, suggesting PvDBP as a leading candidate for inclusion in a vaccine to prevent malaria due to P. vivax. Here, we isolated a panel of monoclonal antibodies from human volunteers immunized in a clinical vaccine trial of PvDBP. We screened their ability to prevent PvDBP from binding to the Duffy antigen receptor for chemokines, and their capacity to block red blood cell invasion by a transgenic Plasmodium knowlesi parasite genetically modified to express PvDBP and to prevent reticulocyte invasion by multiple clinical isolates of P. vivax. This identified a broadly neutralizing human monoclonal antibody that inhibited invasion of all tested strains of P. vivax. Finally, we determined the structure of a complex of this antibody bound to PvDBP, indicating the molecular basis for inhibition. These findings will guide future vaccine design strategies and open up possibilities for testing the prophylactic use of such an antibody.

Human vaccination against Plasmodium vivax Duffy-binding protein induces strain-transcending antibodies.

BACKGROUND: Plasmodium vivax is the most widespread human malaria geographically; however, no effective vaccine exists. Red blood cell invasion by the P. vivax merozoite depends on an interaction between the Duffy antigen receptor for chemokines (DARC) and region II of the parasite's Duffy-binding protein (PvDBP_RII). Naturally acquired binding-inhibitory antibodies against this interaction associate with clinical immunity, but it is unknown whether these responses can be induced by human vaccination. METHODS: Safety and immunogenicity of replication-deficient chimpanzee adenovirus serotype 63 (ChAd63) and modified vaccinia virus Ankara (MVA) viral vectored vaccines targeting PvDBP_RII (Salvador I strain) were assessed in an open-label dose-escalation phase Ia study in 24 healthy UK adults. Vaccines were delivered by the intramuscular route in a ChAd63-MVA heterologous prime-boost regimen using an 8-week interval. RESULTS: Both vaccines were well tolerated and demonstrated a favorable safety profile in malaria-naive adults. PvDBP_RII-specific ex-vivo IFN-γ T cell, antibody-secreting cell, memory B cell, and serum IgG responses were observed after the MVA boost immunization. Vaccine-induced antibodies inhibited the binding of vaccine homologous and heterologous variants of recombinant PvDBP_RII to the DARC receptor, with median 50% binding-inhibition titers greater than 1:100. CONCLUSION: We have demonstrated for the first time to our knowledge that strain-transcending antibodies can be induced against the PvDBP_RII antigen by vaccination in humans. These vaccine candidates warrant further clinical evaluation of efficacy against the blood-stage P. vivax parasite. TRIAL REGISTRATION: Clinicaltrials.gov NCT01816113. FUNDING: Support was provided by the UK Medical Research Council, UK National Institute of Health Research Oxford Biomedical Research Centre, and the Wellcome Trust.

Human vaccination against RH5 induces neutralizing antimalarial antibodies that inhibit RH5 invasion complex interactions.

The development of a highly effective vaccine remains a key strategic goal to aid the control and eventual eradication of Plasmodium falciparum malaria. In recent years, the reticulocyte-binding protein homolog 5 (RH5) has emerged as the most promising blood-stage P. falciparum candidate antigen to date, capable of conferring protection against stringent challenge in Aotus monkeys. We report on the first clinical trial to our knowledge to assess the RH5 antigen - a dose-escalation phase Ia study in 24 healthy, malaria-naive adult volunteers. We utilized established viral vectors, the replication-deficient chimpanzee adenovirus serotype 63 (ChAd63), and the attenuated orthopoxvirus modified vaccinia virus Ankara (MVA), encoding RH5 from the 3D7 clone of P. falciparum. Vaccines were administered i.m. in a heterologous prime-boost regimen using an 8-week interval and were well tolerated. Vaccine-induced anti-RH5 serum antibodies exhibited cross-strain functional growth inhibition activity (GIA) in vitro, targeted linear and conformational epitopes within RH5, and inhibited key interactions within the RH5 invasion complex. This is the first time to our knowledge that substantial RH5-specific responses have been induced by immunization in humans, with levels greatly exceeding the serum antibody responses observed in African adults following years of natural malaria exposure. These data support the progression of RH5-based vaccines to human efficacy testing.

Changes in Serological Immunology Measures in UK and Kenyan Adults Post-controlled Human Malaria Infection.

Background: The timing of infection is closely determined in controlled human malaria infection (CHMI) studies, and as such they provide a unique opportunity to dissect changes in immunological responses before and after a single infection. The first Kenyan Challenge Study (KCS) (Pan African Clinical Trial Registry: PACTR20121100033272) was performed in 2013 with the aim of establishing the CHMI model in Kenya. This study used aseptic, cryopreserved, attenuated Plasmodium falciparum sporozoites administered by needle and syringe (PfSPZ Challenge) and was the first to evaluate parasite dynamics post-CHMI in individuals with varying degrees of prior exposure to malaria. Methods: We describe detailed serological and functional immunological responses pre- and post-CHMI for participants in the KCS and compare these with those from malaria-naïve UK volunteers who also underwent CHMI (VAC049) (ClinicalTrials.gov NCT01465048) using PfSPZ Challenge. We assessed antibody responses to three key blood-stage merozoite antigens [merozoite surface protein 1 (MSP1), apical membrane protein 1 (AMA1), and reticulocyte-binding protein homolog 5 (RH5)] and functional activity using two candidate measures of anti-merozoite immunity; the growth inhibition activity (GIA) assay and the antibody-dependent respiratory burst activity (ADRB) assay. Results:Clear serological differences were observed pre- and post-CHMI by ELISA between malaria-naïve UK volunteers in VAC049, and Kenyan volunteers who had prior malaria exposure. Antibodies to AMA1 and schizont extract correlated with parasite multiplication rate (PMR) post-CHMI in KCS. Serum from volunteer 110 in KCS, who demonstrated a dramatically reduced PMR in vivo, had no in vitro GIA prior to CHMI but the highest level of ADRB activity. A significant difference in ADRB activity was seen between KCS volunteers with minimal and definite prior exposure to malaria and significant increases were seen in ADRB activity post-CHMI in Kenyan volunteers. Quinine and atovaquone/proguanil, previously assumed to be removed by IgG purification, were identified as likely giving rise to aberrantly high in vitro GIA results. Conclusions: The ADRB activity assay is a promising functional assay that warrants further investigation as a measure of prior exposure to malaria and predictor of control of parasite growth. The CHMI model can be used to evaluate potential measures of naturally-acquired immunity to malaria.