Blood-stage Challenge Research Articles

The malaria blood stage antigen PfCyRPA formulated with the TLR-4 agonist adjuvant GLA-SE elicits parasite growth inhibitory antibodies in experimental animals

BackgroundPlasmodium falciparum cysteine-rich protective antigen (PfCyRPA) is an invasion complex protein essential for erythrocyte invasion. In contrast to several previously clinically tested merozoite vaccine candidate antigens, PfCyRPA is not polymorphic, making it a promising candidate antigen for blood stage vaccine development.MethodsMice and rabbits were immunized with vaccine formulations of recombinantly expressed PfCyRPA adjuvanted either with the glucopyranosyl lipid A (GLA) containing adjuvants GLA-LSQ, GLA-SE, GLA-Alum or with Nanoalum. ELISA and indirect immunofluorescence assays (IFA) were used to analyse elicited IgG titers and the P. falciparum growth inhibitory activity was determined with a standardized in vitro [3H]-hypoxanthine incorporation assay.ResultsIn the mouse experiments, the GLA adjuvanted formulations were superior to the Nanoalum formulation with respect to antibody titer development, IFA sero-conversion rates and in vitro parasite growth-inhibitory activity. In rabbits, the highest titers of parasite growth inhibitory antibodies were obtained with the GLA-SE formulation. Comparable mean ELISA IgG endpoint titers were reached in rabbits after three immunizations with GLA-SE adjuvanted PfCyRPA doses of 5, 25 and 100 µg, but with 100 µg of antigen, only two immunizations were required to reach this titer.ConclusionPfCyRPA formulated with the human-compatible adjuvant GLA-SE represents an attractive vaccine candidate for early clinical testing in a controlled P. falciparum blood stage challenge trial.

A single dose of genetically-attenuated malaria blood-stage parasites protects against two Plasmodium species infections

Genetically-growth-attenuated blood-stage parasites were generated inPlasmodium falciparumby targeted deletion of NT1 (Nucleoside Transporter-1) gene, and Pfnt1(-) parasites only grew after providing the culture with supra-physiological concentrations of purines. Genetically-attenuatedP. yoeliint1(-)parasites induced sterile-protection against homologous blood-stage infectious challenge after immunization with single subpatent doses, which remained subpatent even in immune-compromised mice. Here, we showed that immunizations with frozen-stocks of equally-mixedP. bergheiandP. yoelii nt1(-)parasites in single subcutaneous doses, which did not lead to patent blood-stage infection, conferred sterile protection against intravenous infectious blood-stage challenge with wild-type parasites ofP. bergheiANKA andP. yoelii17X-NL strains. This data highlights the possibility that a single subcutaneous sub-patent dose of two species of genetically-growth-attenuated parasites, which can protect humans against twoPlasmodiumspp. infections, could be developed in cultures provided with supra-physiological concentrations of purines, and shipped to endemic areas as frozen-stock doses.

Controlled human malaria infections by mosquito bites induce more severe clinical symptoms than asexual blood-stage challenge infections.

SummaryBackgroundFever and inflammation are a hallmark of clinical Plasmodium falciparum (Pf) malaria induced by circulating asexual parasites. Although clinical manifestations of inflammation are associated with parasite density, this relationship is influenced by a complex network of immune-modulating factors of both human and parasite origin.MethodsIn the Controlled Human Malaria infection (CHMI) model, we compared clinical inflammation in healthy malaria-naïve volunteers infected by either Pf-infected mosquito bites (MB, n=12) or intravenous administration of Pf-infected red blood cells (BS, n=12).FindingsAll volunteers developed patent parasitaemia, but both the incidence and duration of severe adverse events were significantly higher after MB infection. Similarly, clinical laboratory markers of inflammation were significantly increased in the MB-group, as well as serum pro-inflammatory cytokine concentrations including IFN-γ, IL-6, MCP1 and IL-8. Parasite load, as reflected by maximum parasite density and area under the curve, was similar, but median duration of parasitaemia until treatment was longer in the BS-group compared to the MB-group (8 days [range 8 – 8 days] versus 5·5 days [range 3·5 – 12·5 days]). The in vitro response of subsets of peripheral blood mononuclear cells showed attenuated Pf-specific IFNγ production by γδ T-cells in the BS-arm.InterpretationIn conclusion, irrespective the parasite load, Pf-infections by MB induce stronger signs and symptoms of inflammation compared to CHMI by BS infection. The pathophysiological basis remains speculative but may relate to induced immune tolerance.FundingThe trial was supported by PATH's Malaria Vaccine Initiative; the current analyses were supported by the AMMODO Science Award 2019 (TB).

Whole-Killed Blood-Stage Vaccine: Is It Worthwhile to Further Develop It to Control Malaria?

Major challenges have been encountered regarding the development of highly efficient subunit malaria vaccines, and so whole-parasite vaccines have regained attention in recent years. The whole-killed blood-stage vaccine (WKV) is advantageous as it can be easily manufactured and efficiently induced protective immunity against a blood-stage challenge, as well as inducing cross-stage protection against both the liver and sexual-stages. However, it necessitates a high dose of parasitized red blood cell (pRBC) lysate for immunization, and this raises concerns regarding its safety and low immunogenicity. Knowledge of the major components of WKV that can induce or evade the host immune response, and the development of appropriate human-compatible adjuvants will greatly help to optimize the WKV. Therefore, we argue that the further development of the WKV is worthwhile to control and potentially eradicate malaria worldwide.

ELQ-331 as a prototype for extremely durable chemoprotection against malaria

BackgroundThe potential benefits of long-acting injectable chemoprotection (LAI-C) against malaria have been recently recognized, prompting a call for suitable candidate drugs to help meet this need. On the basis of its known pharmacodynamic and pharmacokinetic profiles after oral dosing, ELQ-331, a prodrug of the parasite mitochondrial electron transport inhibitor ELQ-300, was selected for study of pharmacokinetics and efficacy as LAI-C in mice.MethodsFour trials were conducted in which mice were injected with a single intramuscular dose of ELQ-331 or other ELQ-300 prodrugs in sesame oil with 1.2% benzyl alcohol; the ELQ-300 content of the doses ranged from 2.5 to 30 mg/kg. Initial blood stage challenges with Plasmodium yoelii were used to establish the model, but the definitive study measure of efficacy was outcome after sporozoite challenge with a luciferase-expressing P. yoelii, assessed by whole-body live animal imaging. Snapshot determinations of plasma ELQ-300 concentration ([ELQ-300]) were made after all prodrug injections; after the highest dose of ELQ-331 (equivalent to 30 mg/kg ELQ-300), both [ELQ-331] and [ELQ-300] were measured at a series of timepoints from 6 h to 5½ months after injection.ResultsA single intramuscular injection of ELQ-331 outperformed four other ELQ-300 prodrugs and, at a dose equivalent to 30 mg/kg ELQ-300, protected mice against challenge with P. yoelii sporozoites for at least 4½ months. Pharmacokinetic evaluation revealed rapid and essentially complete conversion of ELQ-331 to ELQ-300, a rapidly achieved (< 6 h) and sustained (4–5 months) effective plasma ELQ-300 concentration, maximum ELQ-300 concentrations far below the estimated threshold for toxicity, and a distinctive ELQ-300 concentration versus time profile. Pharmacokinetic modeling indicates a high-capacity, slow-exchange tissue compartment which serves to accumulate and then slowly redistribute ELQ-300 into blood, and this property facilitates an extremely long period during which ELQ-300 concentration is sustained above a minimum fully-protective threshold (60–80 nM).ConclusionsExtrapolation of these results to humans predicts that ELQ-331 should be capable of meeting and far-exceeding currently published duration-of-effect goals for anti-malarial LAI-C. Furthermore, the distinctive pharmacokinetic profile of ELQ-300 after treatment with ELQ-331 may facilitate durable protection and enable protection for far longer than 3 months. These findings suggest that ELQ-331 warrants consideration as a leading prototype for LAI-C.

Malaria vaccine candidate based on Duffy-binding protein elicits strain transcending functional antibodies in a Phase I trial

Reticulocyte invasion by Plasmodium vivax requires interaction of the Duffy-binding protein (PvDBP) with host Duffy antigen receptor for chemokines (DARCs). The binding domain of PvDBP maps to a cysteine-rich region referred to as region II (PvDBPII). Blocking this interaction offers a potential path to prevent P. vivax blood-stage growth and P. vivax malaria. This forms the rationale for development of a vaccine based on PvDBPII. Here we report results of a Phase I randomized trial to evaluate the safety and immunogenicity of recombinant PvDBPII formulated with glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE). Thirty-six malaria-naive, healthy Indian male subjects aged 18–45 years were assigned into three cohorts corresponding to doses of 10, 25 and 50 µg of PvDBPII formulated with 5 µg of GLA-SE. Each cohort included nine PvDBPII/GLA-SE vaccinees and three hepatitis B control vaccine recipients. Each subject received the assigned vaccine intramuscularly on days 0, 28 and 56, and was followed up till day 180. No serious AE was reported and PvDBPII/GLA-SE was well-tolerated and safe. Analysis by ELISA showed that all three doses of PvDBPII elicited antigen-specific binding-inhibitory antibodies. The 50 µg dose elicited antibodies against PvDBPII that had the highest binding-inhibitory titres and were most persistent. Importantly, the antibody responses were strain transcending and blocked receptor binding of diverse PvDBP alleles. These results support further clinical development of PvDBPII/GLA-SE to evaluate efficacy against sporozoite or blood-stage challenge in controlled human malaria infection (CHMI) models and against natural P. vivax challenge in malaria endemic areas.

A Plasmodium Parasite with Complete Late Liver Stage Arrest Protects against Preerythrocytic and Erythrocytic Stage Infection in Mice.

Genetically attenuated malaria parasites (GAP) that arrest during liver stage development are powerful immunogens and afford complete and durable protection against sporozoite infection. Late liver stage-arresting GAP provide superior protection against sporozoite challenge in mice compared to early live stage-arresting attenuated parasites. However, very few late liver stage-arresting GAP have been generated to date. Therefore, identification of additional loci that are critical for late liver stage development and can be used to generate novel late liver stage-arresting GAPs is of importance. We further explored genetic attenuation in Plasmodium yoelii by combining two gene deletions, PlasMei2 and liver-specific protein 2 (LISP2), that each cause late liver stage arrest with various degrees of infrequent breakthrough to blood stage infection. The dual gene deletion resulted in a synthetic lethal phenotype that caused complete attenuation in a highly susceptible mouse strain. P. yoeliiplasmei2-lisp2- arrested late in liver stage development and did not persist in livers beyond 3 days after infection. Immunization with this GAP elicited robust protective antibody responses in outbred and inbred mice against sporozoites, liver stages, and blood stages as well as eliciting protective liver-resident T cells. The immunization afforded protection against both sporozoite challenge and blood stage challenge. These findings provide evidence that completely attenuated late liver stage-arresting GAP are achievable via the synthetic lethal approach and might enable a path forward for the creation of a completely attenuated late liver stage-arresting P. falciparum GAP.

A Plasmodium vivax Plasmid DNA- and Adenovirus-Vectored Malaria Vaccine Encoding Blood-Stage Antigens AMA1 and MSP142 in a Prime/Boost Heterologous Immunization Regimen Partially Protects Aotus Monkeys against Blood-Stage Challenge.

Malaria is caused by parasites of the genus Plasmodium, which are transmitted to humans by the bites of Anopheles mosquitoes. After the elimination of Plasmodium falciparum, it is predicted that Plasmodium vivax will remain an important cause of morbidity and mortality outside Africa, stressing the importance of developing a vaccine against P. vivax malaria. In this study, we assessed the immunogenicity and protective efficacy of two P. vivax antigens, apical membrane antigen 1 (AMA1) and the 42-kDa C-terminal fragment of merozoite surface protein 1 (MSP142) in a plasmid recombinant DNA prime/adenoviral (Ad) vector boost regimen in Aotus monkeys. Groups of 4 to 5 monkeys were immunized with plasmid DNA alone, Ad alone, prime/boost regimens with each antigen, prime/boost regimens with both antigens, and empty vector controls and then subjected to blood-stage challenge. The heterologous immunization regimen with the antigen pair was more protective than either antigen alone or both antigens delivered with a single vaccine platform, on the basis of their ability to induce the longest prepatent period and the longest time to the peak level of parasitemia, the lowest peak and mean levels of parasitemia, the smallest area under the parasitemia curve, and the highest self-cure rate. Overall, prechallenge MSP142 antibody titers strongly correlated with a decreased parasite burden. Nevertheless, a significant proportion of immunized animals developed anemia. In conclusion, the P. vivax plasmid DNA/Ad serotype 5 vaccine encoding blood-stage parasite antigens AMA1 and MSP142 in a heterologous prime/boost immunization regimen provided significant protection against blood-stage challenge in Aotus monkeys, indicating the suitability of these antigens and this regimen for further development.

Whole-Killed Blood-Stage Vaccine-Induced Immunity Suppresses the Development of Malaria Parasites in Mosquitoes.

As a malaria transmission-blocking vaccine alone does not confer a direct benefit to the recipient, it is necessary to develop a vaccine that not only blocks malaria transmission but also protects vaccinated individuals. In this study we observed that a whole-killed blood-stage vaccine (WKV) not only conferred protection against the blood-stage challenge but also markedly inhibited the transmission of different strains of the malaria parasite. Although the parasitemia is much lower in WKV-immunized mice challenged with malaria parasites, the gametocytemia is comparable between control and immunized mice during the early stages of infection. The depletion of CD4+ T cells prior to the adoptive transfer of parasites into WKV-immunized mice has no effect on the development of the malaria parasite in the mosquito, but the adoptive transfer of the serum from the immunized mice into the parasite-inoculated mice remarkably suppresses the development of malaria parasites in mosquitoes. Furthermore, immunized mice challenged with the malaria parasite generate higher levels of parasite-specific Abs and the inflammatory cytokines MCP-1 and IFN-γ. However, the adoptive transfer of parasite-specific IgG or the depletion of MCP-1, but not IFN-γ, to some extent is closely associated with the suppression of malaria parasite development in mosquitoes. These data strongly suggest that WKV-induced immune responses confer protection against the mosquito stage, which is largely dependent on malaria parasite-specific Abs and MCP-1. This finding sheds new light on blocking malaria transmission through the immunization of individuals with the WKV.

Chemically Attenuated Blood-Stage Plasmodium yoelii Parasites Induce Long-Lived and Strain-Transcending Protection.

The development of a vaccine is essential for the elimination of malaria. However, despite many years of effort, a successful vaccine has not been achieved. Most subunit vaccine candidates tested in clinical trials have provided limited efficacy, and thus attenuated whole-parasite vaccines are now receiving close scrutiny. Here, we test chemically attenuated Plasmodium yoelii 17X and demonstrate significant protection following homologous and heterologous blood-stage challenge. Protection against blood-stage infection persisted for at least 9 months. Activation of both CD4+ and CD8+ T cells was shown after vaccination; however, in vivo studies demonstrated a pivotal role for both CD4+ T cells and B cells since the absence of either cell type led to loss of vaccine-induced protection. In spite of significant activation of circulating CD8+ T cells, liver-stage immunity was not evident. Neither did vaccine-induced CD8+ T cells contribute to blood-stage protection; rather, these cells contributed to pathogenesis, since all vaccinated mice depleted of both CD4+ and CD8+ T cells survived a challenge infection. This study provides critical insight into whole-parasite vaccine-induced immunity and strong support for testing whole-parasite vaccines in humans.

Mechanisms of stage-transcending protection following immunization of mice with late liver stage-arresting genetically attenuated malaria parasites.

Malaria, caused by Plasmodium parasite infection, continues to be one of the leading causes of worldwide morbidity and mortality. Development of an effective vaccine has been encumbered by the complex life cycle of the parasite that has distinct pre-erythrocytic and erythrocytic stages of infection in the mammalian host. Historically, malaria vaccine development efforts have targeted each stage in isolation. An ideal vaccine, however, would target multiple life cycle stages with multiple arms of the immune system and be capable of eliminating initial infection in the liver, the subsequent blood stage infection, and would prevent further parasite transmission. We have previously shown that immunization of mice with Plasmodium yoelii genetically attenuated parasites (GAP) that arrest late in liver stage development elicits stage-transcending protection against both a sporozoite challenge and a direct blood stage challenge. Here, we show that this immunization strategy engenders both T- and B-cell responses that are essential for stage-transcending protection, but the relative importance of each is determined by the host genetic background. Furthermore, potent anti-blood stage antibodies elicited after GAP immunization rely heavily on FC-mediated functions including complement fixation and FC receptor binding. These protective antibodies recognize the merozoite surface but do not appear to recognize the immunodominant merozoite surface protein-1. The antigen(s) targeted by stage-transcending immunity are present in both the late liver stages and blood stage parasites. The data clearly show that GAP-engendered protective immune responses can target shared antigens of pre-erythrocytic and erythrocytic parasite life cycle stages. As such, this model constitutes a powerful tool to identify novel, protective and stage-transcending T and B cell targets for incorporation into a multi-stage subunit vaccine.

PD-1 deficiency enhances humoral immunity of malaria infection treatment vaccine.

Malaria infection treatment vaccine (ITV) is a promising strategy to induce homologous and heterologous protective immunity against the blood stage of the parasite. However, the underlying mechanism of protection remains largely unknown. Here, we found that a malaria-specific antibody (Ab) could mediate the protective immunity of ITV-immunized mice. Interestingly, PD-1 deficiency greatly elevated the levels of both malaria-specific total IgG and subclass IgG2a and enhanced the protective efficacy of ITV-immunized mice against the blood-stage challenge. A serum adoptive-transfer assay demonstrated that the increased Ab level contributed to the enhanced protective efficacy of the immunized PD-1-deficient mice. Further study showed that PD-1 deficiency could also promote the expansion of germinal center (GC) B cells and malaria parasite-specific TFH cells in the spleens of ITV-immunized mice. These results suggest that PD-1 deficiency improves the protective efficacy of ITV-immunized mice by promoting the generation of malaria parasite-specific Ab and the expansion of GC B cells. The results of this study provide new evidence to support the negative function of PD-1 on humoral immunity and will guide the design of a more effective malaria vaccine.

Towards a multi-antigen multi-stage malaria vaccine

A highly effective malaria vaccine is a major goal of global health research and will likely require a multi-stage product. Oxford researchers are developing the concept of a highly effective multi-stage P. falciparum vaccine to the point of proof-of-concept phase II testing in Europe, prior to trials in malaria-endemic areas. Remarkable recent advances in vaccine design for all four stages of the P. falciparum parasite’s life-cycle allow testing of a multi-stage multi-component vaccine for the first time, with strong chances of success. These advances are i) the availability of a new vectored prime-boost vaccination regime based on the chimpanzee adenovirus technology that has been found to induce exceptionally potent CD8+ T cell responses and high titre antibodies against multiple malaria antigens; ii) the development of an improved virus-like particle (VLP) version of the leading partially protective RTS,S sporozoite vaccine candidate, termed R21, that lacks the excess of HBsAg in RTS,S; iii) the identification, using a vector technology screen, of the blood-stage antigen RH5 as the first antigen to induce potent strain-transcending neutralization of blood-stage parasites in in vitro growth inhibition assays; and iv) the demonstration that antibodies against a mosquito-stage antigens that induce 100% transmission blocking against field isolates of P. falciparum in Africa are inducible by a new nanoparticle vaccine candidate. In parallel similar approaches using vectors and VLPs are underway to target the pre-erythrocytic stages of P. vivax, including the hypnozoite, and a phase I trial of the vivax blood-stage vaccine candidate, PvRII, is nearing completion. We are aiming to undertake phase I/II clinical trials to assess the P. falciparum pre-erythrocytic, blood-stage and mosquito-stage components individually, and then together, using state-of-the art immunomonitoring, key functional assays of vaccine-induced immunogenicity, and sporozoite and blood-stage parasite challenges to measure efficacy prior to field testing. An update on this programme will be presented. A viral vectored prime-boost regime has recently shown high efficacy against malaria infection in East Africa and the first combination trial of RTS,S/AS01 with these vectors has been completed. The prospects for achieving high efficacy with such combination approaches now appear very good.

Microneedle-mediated immunization of an adenovirus-based malaria vaccine enhances antigen-specific antibody immunity and reduces anti-vector responses compared to the intradermal route.

Substantial effort has been placed in developing efficacious recombinant attenuated adenovirus-based vaccines. However induction of immunity to the vector is a significant obstacle to its repeated use. Here we demonstrate that skin-based delivery of an adenovirus-based malaria vaccine, HAdV5-PyMSP142, to mice using silicon microneedles induces equivalent or enhanced antibody responses to the encoded antigen, however it results in decreased anti-vector responses, compared to intradermal delivery. Microneedle-mediated vaccine priming and resultant induction of low anti-vector antibody titres permitted repeated use of the same adenovirus vaccine vector. This resulted in significantly increased antigen-specific antibody responses in these mice compared to ID-treated mice. Boosting with a heterologous vaccine; MVA-PyMSP142 also resulted in significantly greater antibody responses in mice primed with HAdV5-PyMSP142 using MN compared to the ID route. The highest protection against blood-stage malaria challenge was observed when a heterologous route of immunization (MN/ID) was used. Therefore, microneedle-mediated immunization has potential to both overcome some of the logistic obstacles surrounding needle-and-syringe-based immunization as well as to facilitate the repeated use of the same adenovirus vaccine thereby potentially reducing manufacturing costs of multiple vaccines. This could have important benefits in the clinical ease of use of adenovirus-based immunization strategies.

CD8+ T Cells Mediate Robust Stage-Specific Immunity to P. berghei under Chemoprophylaxis and This Protective Environment Is Not Downregulated by the Presence of Blood-Stage Infection

Sterile protection against malaria infection can be achieved by the inoculation of intact sporozoites while treating concomitantly with the 4-aminoquinoline chloroquine. We present an analysis of protective immunity elicited by successive immunization with Plasmodium berghei sporozoites under chemoprophylaxis. Immunization resulted in a protective, stage-specific immune response. Protection appeared to be mediated by CD8+ T cells and was abrogated upon their specific depletion. Adoptive transfer of splenocytes rendered recipient animals resistant to sporozoite infection, but not to blood-stage challenge. Immunization with sporozoites under chemoprophylaxis results in robust immunity, and the presence of blood-stage infection at sporozoite immunization had no downregulating effect on the protective immune response.

CD8 T cell independent immunity after single dose infection-treatment-vaccination (ITV) against Plasmodium yoelii

Sporozoite vaccination of both humans and rodents elicits potent anti-malarial immunity, but the dose of sporozoites and the number of immunizations required varies with vaccination approach. Here we examine the immunological basis for superior protection afforded from single-dose vaccination with virulent sporozoites administered under prophylatic chloroquine-cover, referred to as infection-treatment-vaccination (ITV), compared to the well-studied approach of administering radiation-attenuated Plasmodium sporozoites (RAS). Earlier rodent studies utilizing ITV and RAS vaccination suggested a major role of CD8 T cells in reducing liver parasite burden after sporozoite challenge in a BALB/c mouse model. Consistent with this, we find that in C57Bl/6 mice ITV elicits substantially higher parasite-specific CD8 T cell responses than RAS vaccination and enhances immunity against P. yoelii infection. However, we show ITV-induced CD8 T cells are not necessary for protection following liver-stage sporozoite or blood-stage parasite challenge. Mechanistically, we found protection afforded from single-dose ITV is associated with low grade, transient parasitemia shortly following cessation of chloroquine treatment and generation of potent antibody responses to blood-stage parasites. Collectively, our data show the mechanistic basis for enhanced protective immunity against P. yoelli elicited by ITV in highly susceptible C57Bl/6 mice is independent of CD8 T cells. These studies may be relevant in understanding the potent immunity observed with ITV in humans.

Assessment of antibody-dependent respiratory burst activity from mouse neutrophils on Plasmodium yoelii malaria challenge outcome.

New tools are required to expedite the development of an effective vaccine against the blood-stage infection with the human malaria parasite Plasmodium falciparum. This work describes the assessment of the ADRB assay in a mouse model, characterizing the functional interaction between antimalarial serum antibodies and FcRs upon neutrophils. We describe a reproducible, antigen-specific assay, dependent on functional FcR signaling, and show that ADRB activity is induced equally by IgG1 and IgG2a isotypes and is modulated by blocking FcR function. However, following immunization of mice with the blood-stage vaccine candidate antigen MSP142, no measurable ADRB activity was induced against PEMS and neither was vaccine efficacy modulated against Plasmodium yoelii blood-stage challenge in γ(-/-) mice compared with WT mice. In contrast, following a primary, nonlethal P. yoelii parasite challenge, serum from vaccinated mice and nonimmunized controls showed anti-PEMS ADRB activity. Upon secondary challenge, nonimmunized γ(-/-) mice showed a reduced ability to control blood-stage parasitemia compared with immunized γ(-/-) mice; however, WT mice, depleted of their neutrophils, did not lose their ability to control infection. Thus, whereas neutrophil-induced ADRB against PEMS does not appear to play a role in protection against P. yoelii rodent malaria, induction of ADRB activity after challenge suggests that antigen targets of anti-PEMS ADRB activity remain to be established, as well as further supporting the observation that ADRB activity to P. falciparum arises following repeated natural exposure.

Protection against malaria after immunization by chloroquine prophylaxis and sporozoites is mediated by preerythrocytic immunity

Volunteers immunized under chloroquine chemoprophylaxis with Plasmodium falciparum sporozoites (CPS) develop complete, long-lasting protection against homologous sporozoite challenge. Chloroquine affects neither sporozoites nor liver-stages, but kills only asexual forms in erythrocytes once released from the liver into the circulation. Consequently, CPS immunization exposes the host to antigens from both preerythrocytic and blood stages, and induced immunity might target either of these stages. We therefore explored the life cycle stage specificity of CPS-induced protection. Twenty-five malaria-naïve volunteers were enrolled in a clinical trial, 15 of whom received CPS immunization. Five immunized subjects and five controls received a sporozoite challenge by mosquito bites, whereas nine immunized and five control subjects received an i.v. challenge with P. falciparum-infected erythrocytes. The latter approach completely bypasses preerythrocytic stages, enabling a direct comparison of protection against either life cycle stage. CPS-immunized subjects (13 of 14) developed anticircumsporozoite antibodies, whereas only one volunteer generated minimal titers against typical blood-stage antigens. IgG from CPS-immunized volunteers did not inhibit asexual blood-stage growth in vitro. All CPS-immunized subjects (5 of 5) were protected against sporozoite challenge. In contrast, nine of nine CPS-immunized subjects developed parasitemia after blood-stage challenge, with identical prepatent periods and blood-stage multiplication rates compared with controls. Intravenously challenged CPS-immunized subjects showed earlier fever and increased plasma concentrations of inflammatory markers D-dimer, IFN-γ, and monokine induced by IFN-γ than i.v. challenged controls. The complete lack of protection against blood-stage challenge indicates that CPS-induced protection is mediated by immunity against preerythrocytic stages. However, evidence is presented for immune recognition of P. falciparum-infected erythrocytes, suggesting memory responses unable to generate functional immunity.

Still Seeking an Effective “One–Two” Malaria Vaccine Punch

Still Seeking an Effective “One–Two” Malaria Vaccine Punch

Recombinant Viral-Vectored Vaccines Expressing Plasmodium chabaudi AS Apical Membrane Antigen 1: Mechanisms of Vaccine-Induced Blood-Stage Protection

Apical membrane Ag 1 (AMA1) is one of the leading candidate Ags for inclusion in a subunit vaccine against blood-stage malaria. However, the efficacy of Ab-inducing recombinant AMA1 protein vaccines in phase IIa/b clinical trials remains disappointing. In this article, we describe the development of recombinant human adenovirus serotype 5 and modified vaccinia virus Ankara vectors encoding AMA1 from the Plasmodium chabaudi chabaudi strain AS. These vectors, when used in a heterologous prime-boost regimen in BALB/c mice, are capable of inducing strong transgene-specific humoral and cellular immune responses. We show that this vaccination regimen is protective against a nonlethal P. chabaudi chabaudi strain AS blood-stage challenge, resulting in reduced peak parasitemias. The role of vaccine-induced, AMA1-specific Abs and T cells in mediating the antiparasite effect was investigated by in vivo depletion of CD4(+) T cells and adoptive-transfer studies into naive and immunodeficient mice. Depletion of CD4(+) T cells led to a loss of vaccine-induced protection. Adoptive-transfer studies confirmed that efficacy is mediated by both CD4(+) T cells and Abs functioning in the context of an intact immune system. Unlike previous studies, these results confirm that Ag-specific CD4(+) T cells, induced by a clinically relevant vaccine-delivery platform, can make a significant contribution to vaccine blood-stage efficacy in the P. chabaudi model. Given that cell-mediated immunity may also contribute to parasite control in human malaria, these data support the clinical development of viral-vectored vaccines that induce both T cell and Abs against Plasmodium falciparum blood-stage malaria Ags like AMA1.