RESUMO
Discovering potent human monoclonal antibodies (mAbs) targeting the Plasmodium falciparum circumsporozoite protein (PfCSP) on sporozoites (SPZ) and elucidating their mechanisms of neutralization will facilitate translation for passive prophylaxis and aid next-generation vaccine development. Here, we isolated a neutralizing human mAb, L9 that preferentially bound NVDP minor repeats of PfCSP with high affinity while cross-reacting with NANP major repeats. L9 was more potent than six published neutralizing human PfCSP mAbs at mediating protection against mosquito bite challenge in mice. Isothermal titration calorimetry and multiphoton microscopy showed that L9 and the other most protective mAbs bound PfCSP with two binding events and mediated protection by killing SPZ in the liver and by preventing their egress from sinusoids and traversal of hepatocytes. This study defines the subdominant PfCSP minor repeats as neutralizing epitopes, identifies an in vitro biophysical correlate of SPZ neutralization, and demonstrates that the liver is an important site for antibodies to prevent malaria.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antiprotozoários/imunologia , Antimaláricos/imunologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Esporozoítos/imunologia , Adolescente , Adulto , Animais , Linhagem Celular , Linhagem Celular Tumoral , Epitopos/imunologia , Feminino , Células HEK293 , Hepatócitos/imunologia , Hepatócitos/parasitologia , Humanos , Fígado/imunologia , Fígado/parasitologia , Malária/imunologia , Malária/parasitologia , Vacinas Antimaláricas/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Adulto JovemRESUMO
SUMMARYMalaria remains one of the biggest health problems in the world. While significant reductions in malaria morbidity and mortality had been achieved from 2000 to 2015, the favorable trend has stalled, rather significant increases in malaria cases are seen in multiple areas. In 2022, there were 249 million estimated cases, and 608,000 malaria-related deaths, mostly in infants and children aged under 5 years, globally. Therefore, in addition to the expansion of existing anti-malarial control measures, it is critical to develop new tools, such as vaccines and monoclonal antibodies (mAbs), to fight malaria. In the last 2 years, the first and second malaria vaccines, both targeting Plasmodium falciparum circumsporozoite proteins (PfCSP), have been recommended by the World Health Organization to prevent P. falciparum malaria in children living in moderate to high transmission areas. While the approval of the two malaria vaccines is a considerable milestone in vaccine development, they have much room for improvement in efficacy and durability. In addition to the two approved vaccines, recent clinical trials with mAbs against PfCSP, blood-stage vaccines against P. falciparum or P. vivax, and transmission-blocking vaccine or mAb against P. falciparum have shown promising results. This review summarizes the development of the anti-PfCSP vaccines and mAbs, and recent topics in the blood- and transmission-blocking-stage vaccine candidates and mAbs. We further discuss issues of the current vaccines and the directions for the development of next-generation vaccines.
Assuntos
Anticorpos Monoclonais , Vacinas Antimaláricas , Vacinas Antimaláricas/imunologia , Humanos , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Plasmodium falciparum/imunologia , Malária/prevenção & controle , Malária/imunologia , Malária Falciparum/prevenção & controle , Malária Falciparum/imunologia , Anticorpos Antiprotozoários/imunologia , Proteínas de Protozoários/imunologia , Ensaios Clínicos como AssuntoRESUMO
Antibodies targeting the human malaria parasite Plasmodium falciparum circumsporozoite protein (PfCSP) can prevent infection and disease. PfCSP contains multiple central repeating NANP motifs; some of the most potent anti-infective antibodies against malaria bind to these repeats. Multiple antibodies can bind the repeating epitopes concurrently by engaging into homotypic Fab-Fab interactions, which results in the ordering of the otherwise largely disordered central repeat into a spiral. Here, we characterize IGHV3-33/IGKV1-5-encoded monoclonal antibody (mAb) 850 elicited by immunization of transgenic mice with human immunoglobulin loci. mAb 850 binds repeating NANP motifs with picomolar affinity, potently inhibits Plasmodium falciparum (Pf) in vitro and, when passively administered in a mouse challenge model, reduces liver burden to a similar extent as some of the most potent anti-PfCSP mAbs yet described. Like other IGHV3-33/IGKV1-5-encoded anti-NANP antibodies, mAb 850 primarily utilizes its HCDR3 and germline-encoded aromatic residues to recognize its core NANP motif. Biophysical and cryo-electron microscopy analyses reveal that up to 19 copies of Fab 850 can bind the PfCSP repeat simultaneously, and extensive homotypic interactions are observed between densely-packed PfCSP-bound Fabs to indirectly improve affinity to the antigen. Together, our study expands on the molecular understanding of repeat-induced homotypic interactions in the B cell response against PfCSP for potently protective mAbs against Pf infection.
Assuntos
Vacinas Antimaláricas , Malária Falciparum , Malária , Humanos , Camundongos , Animais , Plasmodium falciparum , Microscopia Crioeletrônica , Malária Falciparum/parasitologia , Proteínas de Protozoários , Malária/parasitologia , Camundongos Transgênicos , Anticorpos Monoclonais , Anticorpos AntiprotozoáriosRESUMO
Potent and durable vaccine responses will be required for control of malaria caused by Plasmodium falciparum (Pf). RTS,S/AS01 is the first, and to date, the only vaccine that has demonstrated significant reduction of clinical and severe malaria in endemic cohorts in Phase 3 trials. Although the vaccine is protective, efficacy declines over time with kinetics paralleling the decline in antibody responses to the Pf circumsporozoite protein (PfCSP). Although most attention has focused on antibodies to repeat motifs on PfCSP, antibodies to other regions may play a role in protection. Here, we expressed and characterized seven monoclonal antibodies to the C-terminal domain of CSP (ctCSP) from volunteers immunized with RTS,S/AS01. Competition and crystal structure studies indicated that the antibodies target two different sites on opposite faces of ctCSP. One site contains a polymorphic region (denoted α-ctCSP) and has been previously characterized, whereas the second is a previously undescribed site on the conserved ß-sheet face of the ctCSP (denoted ß-ctCSP). Antibodies to the ß-ctCSP site exhibited broad reactivity with a diverse panel of ctCSP peptides whose sequences were derived from field isolates of P. falciparum whereas antibodies to the α-ctCSP site showed very limited cross reactivity. Importantly, an antibody to the ß-site demonstrated inhibition activity against malaria infection in a murine model. This study identifies a previously unidentified conserved epitope on CSP that could be targeted by prophylactic antibodies and exploited in structure-based vaccine design.
Assuntos
Vacinas Antimaláricas , Malária Falciparum , Malária , Animais , Anticorpos Antiprotozoários , Epitopos , Humanos , Malária Falciparum/prevenção & controle , Camundongos , Plasmodium falciparum , Proteínas de Protozoários/genéticaRESUMO
In the acidic lysosome-like digestive vacuole, Plasmodium parasites crystallize heme from hemoglobin into hemozoin, or malaria pigment. Upon release of progeny merozoites, the residual hemozoin is phagocytized by macrophages principally in the liver and spleen where the heme crystals can persist for months to years, as heme oxygenase does not readily degrade the crystal. Previous studies demonstrated hemozoin modulation of monocytes and macrophages. Hemozoin modulates immune function activity of monocytes/macrophages. Here, we used purified/washed hemozoin (W-Hz) isolated from murine Plasmodium berghei infections and intravenously (i.v.) injected it back into naive mice. We characterized the modulating effect of W-Hz on liver-stage replication. Purified washed hemozoin decreases P. berghei liver levels both at 1 week and 1 month after i.v. injection in a dose and time dependent fashion. The injected hemozoin fully protected in nine out of 10 mice given a 50 sporozoite inoculum, and in 10 out of 10 mice against 2,000 sporozoites when they were infected an hour or a day after hemozoin inoculation. DNase treatment at the hemozoin reversed the observed liver load reduction. The liver load reduction was similar in mature B- and T-cell-deficient RAG-1 knockout (KO) mice suggesting an innate immune protection mechanism. This work indicates a role for residual hemozoin in down modulation of Plasmodium liver stages.
Assuntos
Malária , Camundongos , Animais , Camundongos Endogâmicos BALB C , Plasmodium berghei/genética , Esporozoítos , Fígado , Heme/metabolismo , DNA/metabolismoRESUMO
Rare and potent monoclonal antibodies (mAbs) against the Plasmodium falciparum (Pf) circumsporozoite protein (CSP) on infective sporozoites (SPZ) preferentially bind the PfCSP junctional tetrapeptide NPDP or NVDP minor repeats while cross-reacting with NANP central repeats in vitro. The extent to which each of these epitopes is required for protection in vivo is unknown. Here, we assessed whether junction-, minor repeat- and central repeat-preferring human mAbs (CIS43, L9 and 317 respectively) bound and protected against in vivo challenge with transgenic P. berghei (Pb) SPZ expressing either PfCSP with the junction and minor repeats knocked out (KO), or PbCSP with the junction and minor repeats knocked in (KI). In vivo protection studies showed that the junction and minor repeats are necessary and sufficient for CIS43 and L9 to neutralize KO and KI SPZ, respectively. In contrast, 317 required major repeats for in vivo protection. These data establish that human mAbs can prevent malaria infection by targeting three different protective epitopes (NPDP, NVDP, NANP) in the PfCSP repeat region. This report will inform vaccine development and the use of mAbs to passively prevent malaria.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antiprotozoários/imunologia , Epitopos/imunologia , Malária Falciparum/prevenção & controle , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Esporozoítos/imunologia , Animais , Feminino , Fígado/imunologia , Fígado/metabolismo , Fígado/parasitologia , Fígado/patologia , Vacinas Antimaláricas/imunologia , Malária Falciparum/imunologia , Malária Falciparum/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Esporozoítos/crescimento & desenvolvimentoRESUMO
Lasting protection has long been a goal for malaria vaccines. The major surface antigen on Plasmodium falciparum sporozoites, the circumsporozoite protein (PfCSP), has been an attractive target for vaccine development and most protective antibodies studied to date interact with the central NANP repeat region of PfCSP. However, it remains unclear what structural and functional characteristics correlate with better protection by one antibody over another. Binding to the junctional region between the N-terminal domain and central NANP repeats has been proposed to result in superior protection: this region initiates with the only NPDP sequence followed immediately by NANP. Here, we isolated antibodies in Kymab mice immunized with full-length recombinant PfCSP and two protective antibodies were selected for further study with reactivity against the junctional region. X-ray and EM structures of two monoclonal antibodies, mAb667 and mAb668, shed light on their differential affinity and specificity for the junctional region. Importantly, these antibodies also bind to the NANP repeat region with equal or better affinity. A comparison with an NANP-only binding antibody (mAb317) revealed roughly similar but statistically distinct levels of protection against sporozoite challenge in mouse liver burden models, suggesting that junctional antibody protection might relate to the ability to also cross-react with the NANP repeat region. Our findings indicate that additional efforts are necessary to isolate a true junctional antibody with no or much reduced affinity to the NANP region to elucidate the role of the junctional epitope in protection.
Assuntos
Anticorpos Monoclonais Murinos/química , Anticorpos Antiprotozoários/química , Sítios de Ligação de Anticorpos , Epitopos/química , Plasmodium falciparum/química , Proteínas de Protozoários/química , Animais , Anticorpos Monoclonais Murinos/imunologia , Anticorpos Antiprotozoários/imunologia , Epitopos/imunologia , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Relação Estrutura-AtividadeRESUMO
BACKGROUND: New strategies are needed to reduce the incidence of malaria, and promising approaches include the development of vaccines and monoclonal antibodies (mAbs) that target the circumsporozoite protein (CSP). To select the best candidates and speed development, it is essential to standardize preclinical assays to measure the potency of such interventions in animal models. METHODS: Two assay configurations were studied using transgenic Plasmodium berghei expressing Plasmodium falciparum full-length circumsporozoite protein. The assays measured (1) reduction in parasite infection of the liver (liver burden) following an intravenous (i.v) administration of sporozoites and (2) protection from parasitaemia following mosquito bite challenge. Two human CSP mAbs, AB311 and AB317, were compared for their ability to inhibit infection. Multiple independent experiments were conducted to define assay variability and resultant impact on the ability to discriminate differences in mAb functional activity. RESULTS: Overall, the assays produced highly consistent results in that all individual experiments showed greater functional activity for AB317 compared to AB311 as calculated by the dose required for 50% inhibition (ID50) as well as the serum concentration required for 50% inhibition (IC50). The data were then used to model experimental designs with adequate statistical power to rigorously screen, compare, and rank order novel anti-CSP mAbs. CONCLUSION: The results indicate that in vivo assays described here can provide reliable information for comparing the functional activity of mAbs. The results also provide guidance regarding selection of the appropriate experimental design, dose selection, and group sizes.
Assuntos
Anticorpos Monoclonais/imunologia , Parasitemia/prevenção & controle , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Antiprotozoários/sangue , Modelos Animais de Doenças , Feminino , Concentração Inibidora 50 , Fígado/parasitologia , Malária Falciparum/imunologia , Malária Falciparum/terapia , Camundongos , Camundongos Endogâmicos C57BL , Organismos Geneticamente Modificados , Carga Parasitária , Plasmodium berghei/genética , Plasmodium falciparum/genética , Proteínas de Protozoários/genéticaRESUMO
Acquired resistance against antimalarial drugs has further increased the need for an effective malaria vaccine. The current leading candidate, RTS,S, is a recombinant circumsporozoite protein (CSP)-based vaccine against Plasmodium falciparum that contains 19 NANP repeats followed by a thrombospondin repeat domain. Although RTS,S has undergone extensive clinical testing and has progressed through phase III clinical trials, continued efforts are underway to enhance its efficacy and duration of protection. Here, we determined that two monoclonal antibodies (mAbs 311 and 317), isolated from a recent controlled human malaria infection trial exploring a delayed fractional dose, inhibit parasite development in vivo by at least 97%. Crystal structures of antibody fragments (Fabs) 311 and 317 with an (NPNA)3 peptide illustrate their different binding modes. Notwithstanding, one and three of the three NPNA repeats adopt similar well-defined type I ß-turns with Fab311 and Fab317, respectively. Furthermore, to explore antibody binding in the context of P. falciparum CSP, we used negative-stain electron microscopy on a recombinant shortened CSP (rsCSP) construct saturated with Fabs. Both complexes display a compact rsCSP with multiple Fabs bound, with the rsCSP-Fab311 complex forming a highly organized helical structure. Together, these structural insights may aid in the design of a next-generation malaria vaccine.
Assuntos
Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Vacinas Antimaláricas/imunologia , Malária Falciparum/terapia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Animais , Anticorpos Antiprotozoários/química , Antígenos de Protozoários/química , Antígenos de Protozoários/isolamento & purificação , Antígenos de Protozoários/uso terapêutico , Ensaios Clínicos Fase II como Assunto , Cristalografia por Raios X , Mapeamento de Epitopos , Epitopos/química , Epitopos/imunologia , Humanos , Vacinas Antimaláricas/química , Vacinas Antimaláricas/uso terapêutico , Malária Falciparum/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/isolamento & purificação , Proteínas de Protozoários/uso terapêutico , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/uso terapêutico , Sequências Repetitivas de Aminoácidos/imunologia , Relação Estrutura-AtividadeRESUMO
BACKGROUND: The circumsporozoite protein (CSP) of Plasmodium is a key surface antigen that induces antibodies and T-cells, conferring immune protection in animal models and humans. However, much of the work on CSP and immunity has been developed based on studies using rodent or non-human primate CSP antigens, which may not be entirely translatable to CSP expressed by human malaria parasites, especially considering the host specificity of the different species. METHODS: Using a genetically engineered strain of Plasmodium berghei that expresses luciferase, GFP and the Plasmodium falciparum orthologue of CSP, the effect of laboratory preparation, mosquito treatment and mouse factors on sporozoite infectivity was assessed using an in vivo bioluminescence assay on mice. This assay was compared with a PCR-based protection assay using an already described monoclonal antibody that can provide sterile protection against sporozoite challenge. RESULTS: Bioluminescence assay demonstrated similar detection levels of the quantity and kinetics of liver-stage infection, compared to PCR-based detection. This assay was used to evaluate treatment of sporozoite and delivery method on mouse infectivity, as well as the effects of age, sex and strain of mice. Finally, this assay was used to test the protective capacity of monoclonal antibody AB317; results strongly recapitulate the findings of previous work on this antibody. CONCLUSIONS: The PbGFP-Luc line and in vivo bioluminescence imaging provide highly sensitive read-outs of liver-stage infection in mice, and this method can be useful to reliably evaluate potency of pre-erythrocytic interventions.
Assuntos
Malária/imunologia , Plasmodium berghei/fisiologia , Animais , Anopheles/parasitologia , Feminino , Ensaios de Triagem em Larga Escala , Fígado/parasitologia , Luciferases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/fisiologia , Plasmodium berghei/genética , Plasmodium falciparum/genética , Proteínas de Protozoários/metabolismo , Esporozoítos/crescimento & desenvolvimentoRESUMO
Recent studies have shown that immune responses against the cell-traversal protein for Plasmodium ookinetes and sporozoites (CelTOS) can inhibit parasite infection. While these studies provide important evidence toward the development of vaccines targeting this protein, it remains unknown whether these responses could engage the Plasmodium falciparum CelTOS in vivo Using a newly developed rodent malaria chimeric parasite expressing the P. falciparum CelTOS (PfCelTOS), we evaluated the protective effect of in vivo immune responses elicited by vaccination and assessed the neutralizing capacity of monoclonal antibodies specific against PfCelTOS. Mice immunized with recombinant P. falciparum CelTOS in combination with the glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE) or glucopyranosyl lipid adjuvant-liposome-QS21 (GLA-LSQ) adjuvant system significantly inhibited sporozoite hepatocyte infection. Notably, monoclonal antibodies against PfCelTOS strongly inhibited oocyst development of P. falciparum and Plasmodium berghei expressing PfCelTOS in Anopheles gambiae mosquitoes. Taken together, our results demonstrate that anti-CelTOS responses elicited by vaccination or passive immunization can inhibit sporozoite and ookinete infection and impair vector transmission.
Assuntos
Antígenos de Protozoários/imunologia , Vacinas Antimaláricas/imunologia , Malária Falciparum/imunologia , Malária Falciparum/parasitologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Esporozoítos/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/genética , Modelos Animais de Doenças , Hepatócitos/efeitos dos fármacos , Hepatócitos/parasitologia , Imunização , Imunização Passiva , Estágios do Ciclo de Vida , Malária Falciparum/prevenção & controle , Malária Falciparum/transmissão , Camundongos , Plasmodium falciparum/crescimento & desenvolvimento , Proteínas de Protozoários/genética , Proteínas RecombinantesRESUMO
Hemozoin is a crystal synthesized by Plasmodium parasites during hemoglobin digestion in the erythrocytic stage. The hemozoin released when the parasites egress from the red blood cell, which is complexed with parasite DNA, is cleared from the circulation by circulating and tissue-resident monocytes and macrophages, respectively. Recently, we reported that intravenous administration of purified hemozoin complexed with Plasmodium berghei DNA (HzPbDNA) resulted in an innate immune response that blocked liver stage development of sporozoites that was dose-dependent and time-limited. Here, we further characterize the organismal, cellular, and molecular events associated with this protective innate response in the liver and report that a large proportion of the IV administered HzPbDNA localized to F4/80+ cells in the liver and that the rapid and strong protection against liver-stage development waned quickly such that by 1 week post-HzPbDNA treatment animals were fully susceptible to infection. RNAseq of the liver after IV administration of HzPbDNA demonstrated that the rapid and robust induction of genes associated with the acute phase response, innate immune activation, cellular recruitment, and IFN-γ signaling observed at day 1 was largely absent at day 7. RNAseq analysis implicated NK cells as the major cellular source of IFN-γ. In vivo cell depletion and IFN-γ neutralization experiments supported the hypothesis that tissue-resident macrophages and NK cells are major contributors to the protective response and the NK cell-derived IFN-γ is key to induction of the mechanisms that block sporozoite development in the liver. These findings advance our understanding of the innate immune responses that prevent liver stage malaria infection.
Assuntos
Hemeproteínas , Imunidade Inata , Interferon gama , Fígado , Malária , Plasmodium berghei , Esporozoítos , Animais , Plasmodium berghei/imunologia , Esporozoítos/imunologia , Malária/imunologia , Malária/prevenção & controle , Malária/parasitologia , Hemeproteínas/imunologia , Camundongos , Fígado/parasitologia , Fígado/imunologia , Interferon gama/imunologia , Interferon gama/metabolismo , Camundongos Endogâmicos C57BL , Macrófagos/imunologia , Macrófagos/parasitologia , DNA de Protozoário/genética , FemininoRESUMO
Resurgence in malaria has been noted in 2022 with 249 million clinical cases resulting in 608,000 deaths, mostly in children under five. Two vaccines, RTS, S, and more recently R21, targeting the circumsporozoite protein (CSP) are recommended by the WHO but are not yet widely available. Strong humoral responses to neutralize sporozoites before they can infect the hepatocytes are important for vaccine-mediated protection. Suboptimal protection conferred by these first-generation vaccines highlight the need for approaches to improve vaccine-induced immune responses. With the recent success of mRNA-LNP vaccines against COVID-19, there is growing interest in leveraging this approach to enhance malaria vaccines. Here, we present the development of a novel chemokine fusion mRNA vaccine aimed at boosting immune responses to PfCSP by targeting the immunogen to immature dendritic cells (iDC). Vaccination of mice with mRNA encoding full-length CSP fused to macrophage inflammatory protein 3 alpha (MIP3α) encapsulated within lipid nanoparticles (LNP) elicited robust CD4+ T cell responses and enhanced antibody titers against NANP repeat epitopes compared to a conventional CSP mRNA-LNP vaccine. Importantly, the CSP-MIP3α fusion vaccine provided significantly greater protection against liver infection upon challenge with P. berghei PfCSP transgenic sporozoites. This enhanced protection was associated with multifunctional CD4+ T cells levels and anti-NANP repeat titers. This study highlights the potential to augment immune responses to PfCSP through iDC targeting and bolster protection against malaria liver infection.
RESUMO
New strategies are needed to reduce the incidence of malaria, and promising approaches include vaccines targeting the circumsporozoite protein (CSP). To improve upon the malaria vaccine, RTS,S/AS01, it is essential to standardize preclinical assays to measure the potency of next-generation vaccines against this benchmark. We focus on RTS,S/AS01-induced antibody responses and functional activity in conjunction with robust statistical analyses. Transgenic Plasmodium berghei sporozoites containing full-length P. falciparum CSP (tgPb-PfCSP) allow two assessments of efficacy: quantitative reduction in liver infection following intravenous challenge, and sterile protection from mosquito bite challenge. Two or three doses of RTS,S/AS01 were given intramuscularly at 3-week intervals, with challenge 2-weeks after the last vaccination. Minimal inter- and intra-assay variability indicates the reproducibility of the methods. Importantly, the range of this model is suitable for screening more potent vaccines. Levels of induced anti-CSP antibody 2A10 equivalency were also associated with activity: 105 µg/mL (95% CI: 68.8, 141) reduced liver infection by 50%, whereas 285 µg/mL (95% CI: 166, 404) is required for 50% sterile protection from mosquito bite challenge. Additionally, the liver burden model was able to differentiate between protected and non-protected human plasma samples from a controlled human malaria infection study, supporting these models' relevance and predictive capability. Comparison in animal models of CSP-based vaccine candidates to RTS,S/AS01 is now possible under well controlled conditions. Assessment of the quality of induced antibodies, likely a determinant of durability of protection in humans, should be possible using these methods.
RESUMO
Over 75% of malaria-attributable deaths occur in children under the age of 5 years. However, the first malaria vaccine recommended by the World Health Organization (WHO) for pediatric use, RTS,S/AS01 (Mosquirix), has modest efficacy. Complementary strategies, including monoclonal antibodies, will be important in efforts to eradicate malaria. Here we characterize the circulating B cell repertoires of 45 RTS,S/AS01 vaccinees and discover monoclonal antibodies for development as potential therapeutics. We generated >28,000 antibody sequences and tested 481 antibodies for binding activity and 125 antibodies for antimalaria activity in vivo. Through these analyses we identified correlations suggesting that sequences in Plasmodium falciparum circumsporozoite protein, the target antigen in RTS,S/AS01, may induce immunodominant antibody responses that limit more protective, but subdominant, responses. Using binding studies, mouse malaria models, biomanufacturing assessments and protein stability assays, we selected AB-000224 and AB-007088 for advancement as a clinical lead and backup. We engineered the variable domains (Fv) of both antibodies to enable low-cost manufacturing at scale for distribution to pediatric populations, in alignment with WHO's preferred product guidelines. The engineered clone with the optimal manufacturing and drug property profile, MAM01, was advanced into clinical development.
Assuntos
Anticorpos Monoclonais , Malária , Animais , Pré-Escolar , Humanos , Lactente , Camundongos , Anticorpos Monoclonais/uso terapêutico , Linfócitos B , Malária/prevenção & controle , Vacinas AntimaláricasRESUMO
Mosquito salivary proteins play a crucial role in regulating hemostatic responses at the bite site during blood feeding. In this study, we investigate the function of Anopheles gambiae salivary apyrase (AgApyrase) in Plasmodium transmission. Our results demonstrate that salivary apyrase interacts with and activates tissue plasminogen activator, facilitating the conversion of plasminogen to plasmin, a human protein previously shown to be required for Plasmodium transmission. Microscopy imaging shows that mosquitoes ingest a substantial amount of apyrase during blood feeding which reduces coagulation in the blood meal by enhancing fibrin degradation and inhibiting platelet aggregation. Supplementation of Plasmodium infected blood with apyrase significantly enhanced Plasmodium infection in the mosquito midgut. In contrast, AgApyrase immunization inhibited Plasmodium mosquito infection and sporozoite transmission. This study highlights a pivotal role for mosquito salivary apyrase for regulation of hemostasis in the mosquito blood meal and for Plasmodium transmission to mosquitoes and to the mammal host, underscoring the potential for new strategies to prevent malaria transmission.
RESUMO
The generation of high-quality antibody responses to Plasmodium falciparum (Pf) circumsporozoite protein (PfCSP), the primary surface antigen of Pf sporozoites, is paramount to the development of an effective malaria vaccine. Here we present an in-depth structural and functional analysis of a panel of potent antibodies encoded by the immunoglobulin heavy chain variable (IGHV) gene IGHV3-33, which is among the most prevalent and potent antibody families induced in the anti-PfCSP immune response and targets the Asn-Ala-Asn-Pro (NANP) repeat region. Cryo-electron microscopy (cryo-EM) reveals a remarkable spectrum of helical antibody-PfCSP structures stabilized by homotypic interactions between tightly packed fragments antigen binding (Fabs), many of which correlate with somatic hypermutation. We demonstrate a key role of these mutated homotypic contacts for high avidity binding to PfCSP and in protection from Pf malaria infection. Together, these data emphasize the importance of anti-homotypic affinity maturation in the frequent selection of IGHV3-33 antibodies and highlight key features underlying the potent protection of this antibody family.
Assuntos
Vacinas Antimaláricas , Malária Falciparum , Malária , Humanos , Microscopia Crioeletrônica , Plasmodium falciparum/genética , Malária/prevenção & controle , Malária Falciparum/prevenção & controle , Proteínas de Protozoários/química , Anticorpos , Anticorpos AntiprotozoáriosRESUMO
Human monoclonal antibodies (hmAbs) targeting the Plasmodium falciparum circumsporozoite protein (PfCSP) on the sporozoite surface are a promising tool for preventing malaria infection. However, their mechanisms of protection remain unclear. Here, using 13 distinctive PfCSP hmAbs, we provide a comprehensive view of how PfCSP hmAbs neutralize sporozoites in host tissues. Sporozoites are most vulnerable to hmAb-mediated neutralization in the skin. However, rare but potent hmAbs additionally neutralize sporozoites in the blood and liver. Efficient protection in tissues mainly associates with high-affinity and high-cytotoxicity hmAbs inducing rapid parasite loss-of-fitness in the absence of complement and host cells in vitro. A 3D-substrate assay greatly enhances hmAb cytotoxicity and mimics the skin-dependent protection, indicating that the physical stress imposed on motile sporozoites by the skin is crucial for unfolding the protective potential of hmAbs. This functional 3D cytotoxicity assay can thus be useful for downselecting potent anti-PfCSP hmAbs and vaccines.
Assuntos
Vacinas Antimaláricas , Malária Falciparum , Malária , Animais , Humanos , Plasmodium falciparum , Proteínas de Protozoários , Imunoglobulinas , EsporozoítosRESUMO
IGHV3-33-encoded antibodies are prevalent in the human humoral response against the Plasmodium falciparum circumsporozoite protein (PfCSP). Among VH3-33 antibodies, cross-reactivity between PfCSP major repeat (NANP), minor (NVDP), and junctional (NPDP) motifs is associated with high affinity and potent parasite inhibition. However, the molecular basis of antibody cross-reactivity and the relationship with efficacy remain unresolved. Here, we perform an extensive structure-function characterization of 12 VH3-33 anti-PfCSP monoclonal antibodies (mAbs) with varying degrees of cross-reactivity induced by immunization of mice expressing a human immunoglobulin gene repertoire. We identify residues in the antibody paratope that mediate cross-reactive binding and delineate four distinct epitope conformations induced by antibody binding, with one consistently associated with high protective efficacy and another that confers comparably potent inhibition of parasite liver invasion. Our data show a link between molecular features of cross-reactive VH3-33 mAb binding to PfCSP and mAb potency, relevant for the development of antibody-based interventions against malaria.
Assuntos
Malária Falciparum , Malária , Camundongos , Humanos , Animais , Plasmodium falciparum/genética , Anticorpos Antiprotozoários , Proteínas de Protozoários/genética , Epitopos , Anticorpos Monoclonais , Malária Falciparum/parasitologiaRESUMO
Pre-erythrocytic malaria vaccines that induce high-titer, durable antibody responses can potentially provide protection from infection. Here, we engineered a virus-like particle (VLP)-based vaccine targeting a recently described vulnerable epitope at the N-terminus of the central repeat region of the Plasmodium falciparum circumsporozoite protein that is recognized by the potently inhibitory monoclonal antibody L9 and show that immunization with L9 VLPs induces strong antibody responses that provide protection from blood-stage malaria in a mouse infection model.