Virus-like particles (VLPs) represent a significant advance in the development of subunit vaccines, combining high safety and efficacy. Their particulate nature and dense repetitive subunit ...organization makes them ideal scaffolds for display of vaccine antigens. Traditional approaches for VLP-based antigen display require labor-intensive trial-and-error optimization, and often fail to generate dense antigen display. Here we utilize the split-intein (SpyTag/SpyCatcher) conjugation system to generate stable isopeptide bound antigen-VLP complexes by simply mixing of the antigen and VLP components.
Genetic fusion of SpyTag or SpyCatcher to the N-terminus and/or C-terminus of the Acinetobacter phage AP205 capsid protein resulted in formation of stable, nonaggregated VLPs expressing one SpyCatcher, one SpyTag or two SpyTags per capsid protein. Mixing of spy-VLPs with eleven different vaccine antigens fused to SpyCatcher or SpyTag resulted in formation of antigen-VLP complexes with coupling efficiencies (% occupancy of total VLP binding sites) ranging from 22-88 %. In mice, spy-VLP vaccines presenting the malaria proteins Pfs25 or VAR2CSA markedly increased antibody titer, affinity, longevity and functional efficacy compared to corresponding vaccines employing monomeric proteins. The spy-VLP vaccines also effectively broke B cell self-tolerance and induced potent and durable antibody responses upon vaccination with cancer or allergy-associated self-antigens (PD-L1, CTLA-4 and IL-5).
The spy-VLP system constitutes a versatile and rapid method to develop highly immunogenic VLP-based vaccines. Our data provide proof-of-concept for the technology's ability to present complex vaccine antigens to the immune system and elicit robust functional antibody responses as well as to efficiently break B cell self-tolerance. The spy-VLP-system may serve as a generic tool for the cost-effective development of effective VLP-vaccines against both infectious- and non-communicable diseases and could facilitate rapid and unbiased screening of vaccine candidate antigens.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Plasmodium falciparum (Pf) is a major cause of human malaria and is transmitted by infected Anopheles mosquitoes. The initial asymptomatic infection is characterized by parasite invasion of ...hepatocytes, followed by massive replication generating schizonts with blood‐infective merozoites. Hepatocytes can be categorized by their zonal location and metabolic functions within a liver lobule. To understand specific host conditions that affect infectivity, we studied Pf parasite liver stage development in relation to the metabolic heterogeneity of fresh human hepatocytes. We found selective preference of different Pf strains for a minority of hepatocytes, which are characterized by the particular presence of glutamine synthetase (hGS). Schizont growth is significantly enhanced by hGS uptake early in development, showcasing a novel import system. In conclusion, Pf development is strongly determined by the differential metabolic status in hepatocyte subtypes. These findings underscore the importance of detailed understanding of hepatocyte host‐Pf interactions and may delineate novel pathways for intervention strategies.
SYNOPSIS
In vitro studies of the liver stage of malaria parasite Plasmodium falciparum (Pf) are hampered by low rates of infection and development in cultured hepatocytes. Here, particular human hepatocyte subtypes are shown to be differentially permissive for development of Pf schizonts, suggesting new avenues for in vitro infection model improvements to study the biology of this complex parasite life‐cycle stage.
Two distinct Pf strains NF135 and NF175 show selective preference for a hepatocyte subtype that specifically expresses glutamine synthetase.
Widely used standard Pf strain NF54 shows an apparent reduced preference for glutamine synthetase‐expressing hepatocytes and concomitantly relatively diminished intra‐hepatic development.
Host glutamine synthetase is taken up by intra‐hepatic parasites during early development.
Degree of glutamine synthetase expression/uptake correlates with larger schizont size.
Development of P. falciparum parasites benefits from infection of a glutamine synthase‐expressing subpopulation of human hepatocytes, suggesting avenues for in vitro infection model improvement and better understanding of host‐parasite interactions.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Recent evidence suggests that certain vaccines, including Bacillus-Calmette Guérin (BCG), can induce changes in the innate immune system with non-specific memory characteristics, termed 'trained ...immunity'. Here we present the results of a randomised, controlled phase 1 clinical trial in 20 healthy male and female volunteers to evaluate the induction of immunity and protective efficacy of the anti-tuberculosis BCG vaccine against a controlled human malaria infection. After malaria challenge infection, BCG vaccinated volunteers present with earlier and more severe clinical adverse events, and have significantly earlier expression of NK cell activation markers and a trend towards earlier phenotypic monocyte activation. Furthermore, parasitemia in BCG vaccinated volunteers is inversely correlated with increased phenotypic NK cell and monocyte activation. The combined data demonstrate that BCG vaccination alters the clinical and immunological response to malaria, and form an impetus to further explore its potential in strategies for clinical malaria vaccine development.
Abstract
The malaria parasite
Plasmodium falciparum
replicates inside erythrocytes in the blood of infected humans. During each replication cycle, a small proportion of parasites commits to sexual ...development and differentiates into gametocytes, which are essential for parasite transmission via the mosquito vector. Detailed molecular investigation of gametocyte biology and transmission has been hampered by difficulties in generating large numbers of these highly specialised cells. Here, we engineer
P. falciparum
NF54 inducible gametocyte producer (iGP) lines for the routine mass production of synchronous gametocytes via conditional overexpression of the sexual commitment factor GDV1. NF54/iGP lines consistently achieve sexual commitment rates of 75% and produce viable gametocytes that are transmissible by mosquitoes. We also demonstrate that further genetic engineering of NF54/iGP parasites is a valuable tool for the targeted exploration of gametocyte biology. In summary, we believe the iGP approach developed here will greatly expedite basic and applied malaria transmission stage research.
The
Plasmodium falciparum Pf
s230 and
Pf
s48/45 proteins are expressed during transmission from man to mosquito and are leading candidates for a malaria transmission blocking vaccine. Individually ...they generate transmission blocking (TB) antibodies in rodent models. Whether the single protein vaccines are suitable to use in field settings will primarily depend on their potency to elicit functional antibodies. We hypothesized that a combination of both proteins will be more potent than each protein individually. Therefore we designed chimeric proteins composed of fragments of both
Pf
s230 and
Pf
s48/45 as well as single protein fragments, and expressed these in
Lactoccus lactis
. Both the individual
Pf
s230 and
Pf
s48/45 fragments and chimeras elicited high levels of functional antibodies in mice. Importantly, one of the chimeric proteins elicited over threefold higher transmission blocking antibody responses than the single antigens alone. Furthermore the immunogenicity of one of the chimeras could be enhanced through coupling to a virus-like particle (VLP). Altogether these data support further clinical development of these novel constructs.
Long-lasting and sterile homologous protection against malaria can be achieved by the exposure of malaria-naive volunteers under chemoprophylaxis to
-infected mosquitoes (chemoprophylaxis and ...sporozoite CPS immunization). While CPS-induced antibodies neutralize sporozoite infectivity
and
, antibody-mediated effector mechanisms are still poorly understood. Here, we investigated whether complement contributes to CPS-induced preerythrocytic immunity. Sera collected before and after CPS immunization in the presence of active or inactive complement were assessed for the recognition of homologous NF54 and heterologous NF135.C10 sporozoites, complement fixation, sporozoite lysis, and possible subsequent effects on
sporozoite infectivity in human hepatocytes. CPS immunization induced sporozoite-specific IgM (
< 0.0001) and IgG (
= 0.001) antibodies with complement-fixing capacities (
< 0.0001). Sporozoite lysis (
= 0.017), traversal (
< 0.0001), and hepatocyte invasion inhibition (
< 0.0001) by CPS-induced antibodies were strongly enhanced in the presence of active complement. Complement-mediated invasion inhibition in the presence of CPS-induced antibodies negatively correlated with cumulative parasitemia during CPS immunizations (
= 0.013). While IgG antibodies similarly recognized homologous and heterologous sporozoites, IgM binding to heterologous sporozoites was reduced (
= 0.023). Although CPS-induced antibodies did not differ in their abilities to fix complement, lyse sporozoites, or inhibit the traversal of homologous and heterologous sporozoites, heterologous sporozoite invasion was more strongly inhibited in the presence of active complement (
= 0.008). These findings demonstrate that CPS-induced antibodies have complement-fixing activity, thereby significantly further enhancing the functional inhibition of homologous and heterologous sporozoite infectivity
The combined data highlight the importance of complement as an additional immune effector mechanism in preerythrocytic immunity after whole-parasite immunization against
malaria.
Two members of 6-cysteine (6-cys) protein family, P48/45 and P230, are important for gamete fertility in rodent and human malaria parasites and are leading transmission blocking vaccine antigens. ...Rodent and human parasites encode a paralog of P230, called P230p. While P230 is expressed in male and female parasites, P230p is expressed only in male gametocytes and gametes. In rodent malaria parasites this protein is dispensable throughout the complete life-cycle; however, its function in P. falciparum is unknown. Using CRISPR/Cas9 methodology we disrupted the gene encoding Pfp230p resulting in P. falciparum mutants (PfΔp230p) lacking P230p expression. The PfΔp230p mutants produced normal numbers of male and female gametocytes, which retained expression of P48/45 and P230. Upon activation male PfΔp230p gametocytes undergo exflagellation and form male gametes. However, male gametes are unable to attach to red blood cells resulting in the absence of characteristic exflagellation centres in vitro. In the absence of P230p, zygote formation as well as oocyst and sporozoite development were strongly reduced (>98%) in mosquitoes. These observations demonstrate that P230p, like P230 and P48/45, has a vital role in P. falciparum male fertility and zygote formation and warrants further investigation as a potential transmission blocking vaccine candidate.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Background. Single low-dose primaquine (PQ) reduces Plasmodium falciparum infectivity before it impacts gametocyte density. Here, we examined the effect of PQ on gametocyte sex ratio as a possible ...explanation for this early sterilizing effect. Methods. Quantitative reverse-transcription polymerase chain reaction assays were developed to quantify female gametocytes (targeting Pfs25 messenger RNA mRNA) and male gametocytes (targeting Pf3D7_1469900 mRNA) in 2 randomized trials in Kenya and Mali, comparing dihydroartemisinin-piperaquine (DP) alone to DP with PQ. Gametocyte sex ratio was examined in relation to time since treatment and infectivity to mosquitoes. Results. In Kenya, the median proportion of male gametocytes was 0.33 at baseline. Seven days after treatment, gametocyte density was significantly reduced in the DP-PQ arm relative to the DP arm (females: 0.05% interquartile range {IQR}, 0.0–0.7% of baseline; males: 3.4% IQR, 0.4%–32.9% of baseline; P < .001). Twenty-four hours after treatment, gametocyte sex ratio became male-biased and was not significantly different between the DP and DP-PQ groups. In Mali, there was no significant difference in sex ratio between the DP and DP-PQ groups (>0.125 mg/kg) 48 hours after treatment, and gametocyte sex ratio was not associated with mosquito infection rates. Conclusions. The early sterilizing effects of PQ may not be explained by the preferential clearance of male gametocytes and may be due to an effect on gametocyte fitness.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Summary Background We have shown that immunity to infection with Plasmodium falciparum can be induced experimentally in malaria-naive volunteers through immunisation by bites of infected mosquitoes ...while simultaneously preventing disease with chloroquine prophylaxis. This immunity was associated with parasite-specific production of interferon γ and interleukin 2 by pluripotent effector memory cells in vitro. We aim to explore the persistence of protection and immune responses in the same volunteers. Methods In an open-label study at the Radboud University Nijmegen Medical Centre (Nijmegen, Netherlands), from November to December, 2009, we rechallenged previously immune volunteers (28 months after immunisation) with the bites of five mosquitoes infected with P falciparum . Newly recruited malaria-naive volunteers served as infection controls. Our primary outcome was the detection of blood-stage parasitaemia by microscopy. We assessed the kinetics of parasitaemia with real-time quantitative PCR (rtPCR) and recorded clinical signs and symptoms. In-vitro production of interferon γ and interleukin 2 by effector memory T cells was studied after stimulation with sporozoites and red blood cells infected with P falciparum . Differences in cellular immune responses between the study groups were assessed with the Mann-Whitney test. This study is registered with ClinicalTrials.gov , number NCT00757887. Findings Four of six immune volunteers were microscopically negative after rechallenge. rtPCR-based detection of blood-stage parasites in these individuals was negative throughout follow-up. Patent parasitaemia was delayed in the remaining two immunised volunteers. In-vitro assays showed the long-term persistence of parasite-specific pluripotent effector memory T-cell responses in protected volunteers. The four protected volunteers reported several mild to moderate adverse events, of which the most commonly reported symptom was headache (one to three episodes per volunteer). The two patients with delayed patency had adverse events similar to those in the control group. Interpretation Artificially induced immunity lasts longer than generally recorded after natural exposure; providing a new avenue of research into the mechanisms of malaria immunity. Funding Dioraphte Foundation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Pfs230 is essential for Plasmodium falciparum transmission to mosquitoes and is the protein targeted by the most advanced malaria-transmission-blocking vaccine candidate. Prior understanding of ...functional epitopes on Pfs230 is based on two monoclonal antibodies (mAbs) with moderate transmission-reducing activity (TRA), elicited from subunit immunization. Here, we screened the B cell repertoire of two naturally exposed individuals possessing serum TRA and identified five potent mAbs from sixteen Pfs230 domain-1-specific mAbs. Structures of three potent and three low-activity antibodies bound to Pfs230 domain 1 revealed four distinct epitopes. Highly potent mAbs from natural infection recognized a common conformational epitope that is highly conserved across P. falciparum field isolates, while antibodies with negligible TRA derived from natural infection or immunization recognized three distinct sites. Our study provides molecular blueprints describing P. falciparum TRA, informed by contrasting potent and non-functional epitopes elicited by natural exposure and vaccination.
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•Isolated mAbs directed to Pfs230 elicited in naturally exposed individuals•Highly potent transmission-reducing mAbs identified that target Pfs230 domain 1•Structural delineation reveals potent and non-functional epitopes on Pfs230 domain 1
Pfs230 domain 1 represents the most clinically advanced transmission-blocking vaccine candidate, but how human antibodies acquired after natural exposure recognize the protein is unknown. Ivanochko et al. report a panel of human mAbs targeting Pfs230 D1, including the most potent mAbs reported to date, and contrast structure-activity relationships against this malarial antigen to reveal the determinants of transmission-reducing activity.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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