Non-small cell lung cancer (NSCLC) is known for high relapse rates despite resection in early stages. present the results of a phase I clinical trial in which a dendritic cell (DC) vaccine targeting patient neoantigens is evaluated in patients with resected NSCLC. Vaccine manufacturing is feasible enrolled patients. Toxicity is limited to grade 1-2 adverse events. Systemic T cell responses in five out of six vaccinated patients, with T cell responses remaining detectable up to 19 months nation. Single-cell analysis indicates that the responsive T cell population is polyclonal and exhibits entire spectrum of T cell differentiation states, including a naive-like state, but excluding exhausted states. Three of six vaccinated patients experience disease recurrence during the follow-up period Collectively, these data support the feasibility, safety, and immunogenicity of this treatment NSCLC.

Throughout their life cycle, viruses interact with cellular host factors, thereby influencing propagation, host range, cell tropism and pathogenesis. The hepatitis E virus (HEV) is an underestimated RNA virus in which knowledge of the virus–host interaction network to date is limited. Here, two related high-throughput mammalian two-hybrid approaches (MAPPIT and KISS) were used to screen for HEV-interacting host proteins. Promising hits were examined on protein function, involved pathway(s), and their relation to other viruses. We identified 37 ORF2 hits, 187 for ORF3 and 91 for ORF4. Several hits had functions in the life cycle of distinct viruses. We focused on SHARPIN and RNF5 as candidate hits for ORF3, as they are involved in the RLR-MAVS pathway and interferon (IFN) induction during viral infections. Knocking out (KO) SHARPIN and RNF5 resulted in a different IFN response upon ORF3 transfection, compared to wild-type cells. Moreover, infection was increased in SHARPIN KO cells and decreased in RNF5 KO cells. In conclusion, MAPPIT and KISS are valuable tools to study virus–host interactions, providing insights into the poorly understood HEV life cycle. We further provide evidence for two identified hits as new host factors in the HEV life cycle.

In the human thymus, a CD10+ PD-1+ TCRαβ+ differentiation pathway diverges from the conventional single positive T cell lineages at the early double-positive stage. Here, we identify the progeny of this unconventional lineage in antigen-inexperienced blood. These unconventional T cells (UTCs) in thymus and blood share a transcriptomic profile, characterized by hallmark transcription factors (i.e., ZNF683 and IKZF2), and a polyclonal TCR repertoire with autoreactive features, exhibiting a bias toward early TCRα chain rearrangements. Single-cell RNA sequencing confirms a common developmental trajectory between the thymic and blood UTCs and clearly delineates this unconventional lineage in blood. Besides MME+ recent thymic emigrants, effector-like clusters are identified in this heterogeneous lineage. Expression of Helios and KIR and a decreased CD8β expression are characteristics of this lineage. This UTC lineage could be identified in adult blood and intestinal tissues. In summary, our data provide a comprehensive characterization of the polyclonal unconventional lineage in antigen-inexperienced blood and identify the adult progeny.

The hepatitis E virus (HEV) is an underestimated RNA virus of which the viral life cycle and pathogenicity remain partially understood and for which specific antivirals are lacking. The virus exists in two forms: nonenveloped HEV that is shed in feces and transmits between hosts; and membrane-associated, quasi-enveloped HEV that circulates in the blood. It is suggested that both forms employ different mechanisms for cellular entry and internalization but little is known about the exact mechanisms. Interestingly, the membrane of enveloped HEV is enriched with phosphatidylserine, a natural ligand for the T-cell immunoglobulin and mucin domain-containing protein 1 (TIM1) during apoptosis and involved in ‘apoptotic mimicry’, a process by which viruses hijack the apoptosis pathway to promote infection. We here investigated the role of TIM1 in the entry process of HEV. We determined that HEV infection with particles derived from culture supernatant, which are cloaked by host-derived membranes (eHEV), was significantly impaired after knockout of TIM1, whereas infection with intracellular HEV particles (iHEV) was unaffected. eHEV infection was restored upon TIM1 expression; and enhanced after ectopic TIM1 expression. The significance of TIM1 during entry was further confirmed by viral binding assay, and point mutations of the PS-binding pocket diminished eHEV infection. In addition, Annexin V, a PS-binding molecule also significantly reduced infection. Taken together, our findings support a role for TIM1 in eHEV-mediated cell entry, facilitated by the PS present on the viral membrane, a strategy HEV may use to promote viral spread throughout the infected body.

The Wiskott–Aldrich syndrome (WAS) is an X-linked primary immune deficiency caused by a mutation in the WAS gene. This leads to altered or absent WAS protein (WASp) expression and function resulting in thrombocytopenia, eczema, recurrent infections, and autoimmunity. In T cells, WASp is required for immune synapse formation. Patients with WAS show reduced numbers of peripheral blood T lymphocytes and an altered T-cell receptor repertoire. In vitro, their peripheral T cells show decreased proliferation and cytokine production upon aCD3/aCD28 stimulation. It is unclear whether these T-cell defects are acquired during peripheral activation or are, in part, generated during thymic development. Here, we assessed the role of WASp during T-cell differentiation using artificial thymic organoid cultures and in the thymus of humanized mice. Although CRISPR/Cas9 WAS knockout hematopoietic stem and progenitor cells (HSPCs) rearranged the T-cell receptor and differentiated to T-cell receptor (TCR)+ CD4+ CD8+ double-positive (DP) cells similar to wild-type HSPCs, a partial defect in the generation of CD8 single-positive (SP) cells was observed, suggesting that WASp is involved in their positive selection. TCR repertoire analysis of the DP and CD8+ SP population, however, showed a polyclonal repertoire with no bias toward autoreactivity. To our knowledge, this is the first study of the role of WASp in human T-cell differentiation and on TCR repertoire generation.

Messenger RNA (mRNA) has become a promising tool in therapeutic cancer vaccine strategies. Owing to its flexible design and rapid production, mRNA is an attractive antigen delivery format for cancer vaccines targeting mutated peptides expressed in a tumor-the so-called neoantigens. These neoantigens are rarely shared between patients, and inclusion of these antigens in a vaccine requires the production of individual batches of patient-tailored mRNA. The authors have developed MIDRIXNEO, a personalized mRNA-loaded dendritic cell vaccine targeting tumor neoantigens, which is currently being evaluated in a phase 1 clinical study in lung cancer patients. To facilitate this study, the authors set up a Good Manufacturing Practice (GMP)-compliant production process for the manufacture of small batches of personalized neoantigen-encoding mRNA. In this article, the authors describe the complete mRNA production process and the extensive quality assessment to which the mRNA is subjected. Validation runs have shown that the process delivers mRNA of reproducible, high quality. This process is now successfully applied for the production of neoantigen-encoding mRNA for the clinical evaluation of MIDRIXNEO. To the authors' knowledge, this is the first time that a GMP-based production process of patient-tailored neoantigen mRNA has been described.

T cell receptor (TCR)-redirected T cells target intracellular antigens such as Wilms’ tumor 1 (WT1), a tumor-associated antigen overexpressed in several malignancies, including acute myeloid leukemia (AML). For both chimeric antigen receptor (CAR)- and TCR-redirected T cells, several clinical studies indicate that T cell subsets with a less-differentiated phenotype (e.g. stem cell memory T cells, TSCM) survive longer and mediate superior anti-tumor effects in vivo as opposed to more terminally differentiated T cells. Cytokines added during in vitro and ex vivo culture of T cells play an important role in driving the phenotype of T cells for adoptive transfer. Using the OP9-DL1 co-culture system, we have shown previously that we are able to generate in vitro, starting from clinically relevant stem cell sources, T cells with a single tumor antigen (TA)-specific TCR. This method circumvents possible TCR chain mispairing and unwanted toxicities that might occur when introducing a TA-specific TCR in peripheral blood lymphocytes. We now show that we are able to optimize our in vitro culture protocol, by adding IL-21 during maturation, resulting in generation of TA-specific T cells with a less-differentiated phenotype and enhanced in vitro anti-tumor effects. We believe the favorable TSCM-like phenotype of these in vitro generated T cells preludes superior in vivo persistence and anti-tumor efficacy. Therefore, these TA-specific T cells could be of use as a valuable new form of patient-tailored T cell immunotherapy for malignancies for which finding a suitable CAR-T target antigen is challenging, such as AML.

γδ and αβ T cells have unique roles in immunity and both originate in the thymus from T-lineage committed precursors through distinct but unclear mechanisms. Here, we show that Notch1 activation is more stringently required for human γδ development compared to αβ-lineage differentiation and performed paired mRNA and miRNA profiling across 11 discrete developmental stages of human T cell development in an effort to identify the potential Notch1 downstream mechanism. Our data suggest that the miR-17-92 cluster is a Notch1 target in immature thymocytes and that miR-17 can restrict BCL11B expression in these Notch-dependent T cell precursors. We show that enforced miR-17 expression promotes human γδ T cell development and, consistently, that BCL11B is absolutely required for αβ but less for γδ T cell development. This study suggests that human γδ T cell development is mediated by a stage-specific Notch-driven negative feedback loop through which miR-17 temporally restricts BCL11B expression and provides functional insights into the developmental role of the disease-associated genes BCL11B and the miR-17-92 cluster in a human context.

In the mouse thymus, invariant γδ T cells are generated at well-defined times during development and acquire effector functions before exiting the thymus. However, whether such thymic programming and age-dependent generation of invariant γδ T cells occur in humans is not known. Here we found that, unlike postnatal γδ thymocytes, human fetal γδ thymocytes were functionally programmed (e.g., IFNγ, granzymes) and expressed low levels of terminal deoxynucleotidyl transferase (TdT). This low level of TdT resulted in a low number of N nucleotide insertions in the complementarity-determining region-3 (CDR3) of their TCR repertoire, allowing the usage of short homology repeats within the germline-encoded VDJ segments to generate invariant/public cytomegalovirus-reactive CDR3 sequences (TRGV8-TRJP1-CATWDTTGWFKIF, TRDV2-TRDD3-CACDTGGY, and TRDV1-TRDD3-CALGELGD). Furthermore, both the generation of invariant TCRs and the intrathymic acquisition of effector functions were due to an intrinsic property of fetal hematopoietic stem and precursor cells (HSPCs) caused by high expression of the RNA-binding protein Lin28b. In conclusion, our data indicate that the human fetal thymus generates, in an HSPC/Lin28b-dependent manner, invariant γδ T cells with programmed effector functions.

Recent approval of chimeric antigen receptor (CAR) T cell therapy by the European Medicines Agency (EMA)/Federal and Drug Administration (FDA) and the remarkable results of CAR T clinical trials illustrate the curative potential of this therapy. While CARs against a multitude of different antigens are being developed and tested (pre)clinically, there is still a need for optimization. The use of single-chain variable fragments (scFvs) as targeting moieties hampers the quick generation of functional CARs and could potentially limit the efficacy. Instead, nanobodies may largely circumvent these difficulties. We used an available nanobody library generated after immunization of llamas against Cluster of Differentiation (CD) 20 through DNA vaccination or against the ectodomain of CD33 using soluble protein. The nanobody specific sequences were amplified by PCR and cloned by Gibson Assembly into a retroviral vector containing two different second-generation CAR constructs. After transduction in T cells, we observed high cell membrane nanoCAR expression in all cases. Following stimulation of nanoCAR-expressing T cells with antigen-positive cell lines, robust T cell activation, cytokine production and tumor cell lysis both in vitro and in vivo was observed. The use of nanobody technology in combination with PCR and Gibson Assembly allows for the rapid and effective generation of compact CARs.

Human thymic CD8αα+ CD10+ PD-1+ αβ T cells selected through early agonist selection have been proposed as the putative thymic precursors of the human CD8αα+ intestinal intraepithelial lymphocytes (IELs). However, the progeny of these thymic precursor cells in human blood or tissues has not yet been characterized. Here, we studied the phenotypical and transcriptional differentiation of the thymic IEL precursor (IELp) lineage upon in vitro exposure to cytokines prominent in the peripheral tissues such as interleukin-15 (IL-15) and the inflammatory cytokines interleukin-12 (IL-12) and interleukin-18 (IL-18). We showed that only the CD1a− fraction of the CD10+ PD-1+ IELp population was able to proliferate with IL-15, suggesting that this subset had acquired functionality. These cells downregulated PD-1 expression and completely lost CD10 expression, whereas other surface markers such as CD95 and CXCR3 remained highly expressed. RNA-seq analysis of the IL-15-cultured cells clearly showed induction of innate-like and effector genes. Induction of the cytotoxic machinery by the CD10+ PD-1+ population was acquired in the presence of IL-15 and was further augmented by inflammatory cytokines. Our data suggest that only the CD1a− CD10+ PD-1+ population exits the thymus and survives in the periphery. Furthermore, PD-1 and CD10 expression is not an intrinsic property of this lineage, but rather characterizes a transient stage in differentiation. CD95 and CXCR3 expression combined with the absence of CD28, CCR7, and CD6 expression might be more powerful markers to define this lineage in the periphery.

Immunotherapeutic strategies targeting the rare leukemic stem cell compartment might provide salvage to the high relapse rates currently observed in acute myeloid leukemia (AML). We applied gene expression profiling for comparison of leukemic blasts and leukemic stem cells with their normal counterparts. Here, we show that the T-cell receptor γ chain alternate reading frame protein (TARP) is over-expressed in de novo pediatric (n=13) and adult (n=17) AML sorted leukemic stem cells and blasts compared to hematopoietic stem cells and normal myeloblasts (15 healthy controls). Moreover, TARP expression was significantly associated with a fms-like tyrosine kinase receptor-3 internal tandem duplication in pediatric AML. TARP overexpression was confirmed in AML cell lines (n=9), and was found to be absent in B-cell acute lymphocytic leukemia (n=5) and chronic myeloid leukemia (n=1). Sequencing revealed that both a classical TARP transcript, as described in breast and prostate adenocarcinoma, and an AML-specific alternative TARP transcript, were present. Protein expression levels mostly matched transcript levels. TARP was shown to reside in the cytoplasmic compartment and showed sporadic endoplasmic reticulum co-localization. TARP-T-cell receptor engineered cytotoxic T-cells in vitro killed AML cell lines and patient leukemic cells co-expressing TARP and HLA-A*0201. In conclusion, TARP qualifies as a relevant target for immunotherapeutic T-cell therapy in AML.

Recent clinical trials have shown that adoptive chimeric antigen receptor (CAR) T cell therapy is a very potent and possibly curative option in the treatment of B cell leukemias and lymphomas. However, targeting a single antigen may not be sufficient, and relapse due to the emergence of antigen negative leukemic cells may occur. A potential strategy to counter the outgrowth of antigen escape variants is to broaden the specificity of the CAR by incorporation of multiple antigen recognition domains in tandem. As a proof of concept, we here describe a bispecific CAR in which the single chain variable fragment (scFv) is replaced by a tandem of two single-antibody domains or nanobodies (nanoCAR). High membrane nanoCAR expression levels are observed in retrovirally transduced T cells. NanoCARs specific for CD20 and HER2 induce T cell activation, cytokine production and tumor lysis upon incubation with transgenic Jurkat cells expressing either antigen or both antigens simultaneously. The use of nanobody technology allows for the production of compact CARs with dual specificity and predefined affinity.

Infections with hepatitis C virus (HCV) represent a worldwide health burden and a prophylactic vaccine is still not available. Liver transplantation (LT) is often the only option for patients with HCV-induced end-stage liver disease. However, immediately after transplantation, the liver graft becomes infected by circulating virus, resulting in accelerated progression of liver disease. Although the efficacy of HCV treatment using direct-acting antivirals has improved significantly, immune compromised LT-patients and patients with advanced liver disease remain difficult to treat. As an alternative approach, interfering with viral entry could prevent infection of the donor liver. We generated a human monoclonal antibody (mAb), designated 2A5, which targets the HCV envelope. The neutralizing activity of mAb 2A5 was assessed using multiple prototype and patient-derived HCV pseudoparticles (HCVpp), cell culture produced HCV (HCVcc), and a human-liver chimeric mouse model. Neutralization levels observed for mAb 2A5 were generally high and mostly superior to those obtained with AP33, a well-characterized HCV-neutralizing monoclonal antibody. Using humanized mice, complete protection was observed after genotype 1a and 4a HCV challenge, while only partial protection was achieved using gt1b and 6a isolates. Epitope mapping revealed that mAb 2A5 binding is conformation-dependent and identified the E2-region spanning amino acids 434 to 446 (epitope II) as the predominant contact domain.CONCLUSION:mAb 2A5 shows potent anti-HCV neutralizing activity both in vitro and in vivo and could hence represent a valuable candidate to prevent HCV recurrence in LT-patients. In addition, the detailed identification of the neutralizing epitope can be applied for the design of prophylactic HCV vaccines.

Infections with hepatitis C virus (HCV) represent a worldwide health burden and a prophylactic vaccine is still not available. Liver transplantation (LT) is often the only option for patients with HCV-induced end-stage liver disease. However, the liver graft becomes infected by circulating virus, resulting in accelerated progression of liver disease. Although the efficacy of HCV treatment has improved significantly, immune compromised LT-patients and patients with advanced liver disease remain difficult to treat. As an alternative approach, interfering with viral entry could prevent infection of the donor liver. In the present study, we describe the full characterization of the human monoclonal antibody (mAb) 2A5 raised against the HCV envelope. Results of GNA binding ELISA showed high affinity of mAb2A5 towards HCV envelope glycoproteins E1E2 of different strains. mAb2A5 efficiently cross-neutralized HCVpp and HCVcc from all genotypes and completely protected humanized mice from genotypes 1a and 4a challenge. Partial protection from genotypes 1b and 6a was achieved however sequence analysis of E1E2 from non-protected mice did not reveal resistance. Epitope mapping using a synthetic peptide library and alanine mutants of full length E1E2 identified the E2-region spanning amino acids 434 to 446 (epitope II) as the predominant contact domain. In addition, the binding activity was completely lost against denatured E1E2 indicating a conformation-dependent epitope. Interestingly, competition ELISA showed no interference with the binding activity of the murine mAb AP33 that target epitope I in the E2 protein. Finally, sequence alignment revealed highly conserved residues critical for mAb binding among different HCV genotypes. In conclusion, mAb2A5 shows potent anti-HCV neutralizing activity and could hence provide an effective strategy to prevent HCV recurrence in chronically infected HCV LT-patients. In addition, the detailed identification of the neutralizing epitope can be applied for the design of prophylactic HCV vaccines with cross-protection activity.

Recent clinical studies indicate that adoptive T-cell therapy and especially chimeric antigen receptor (CAR) T-cell therapy is a very potent and potentially curative treatment for B-lineage hematologic malignancies. Currently, autologous peripheral blood T cells are used for adoptive T-cell therapy. Adoptive T cells derived from healthy allogeneic donors may have several advantages; however, the expected occurrence of graft versus host disease (GvHD) as a consequence of the diverse allogeneic T-cell receptor (TCR) repertoire expressed by these cells compromises this approach. Here, we generated T cells from cord blood hematopoietic progenitor cells (HPCs) that were transduced to express an antigen receptor (AR): either a CAR or a TCR with or without built-in CD28 co-stimulatory domains. These AR-transgenic HPCs were culture-expanded on an OP9-DL1 feeder layer and subsequently differentiated to CD5+CD7+ T-lineage precursors, to CD4+ CD8+ double positive cells and finally to mature AR+ T cells. The AR+ T cells were largely naive CD45RA+CD62L+ T cells. These T cells had mostly germline TCRα and TCRβ loci and therefore lacked surface-expressed CD3/TCRαβ complexes. The CD3− AR-transgenic cells were mono-specific, functional T cells as they displayed specific cytotoxic activity. Cytokine production, including IL-2, was prominent in those cells bearing ARs with built-in CD28 domains. Data sustain the concept that cord blood HPC derived, in vitro generated allogeneic CD3− AR+ T cells can be used to more effectively eliminate malignant cells, while at the same time limiting the occurrence of GvHD.

To facilitate studies on Vpr function in replicating HIV-1, we aimed to tag the protein in an infectious virus. First we showed that N-, but not C-terminal HA/FLAG tagging of Vpr protein preserves Vpr cytopathicity. Cloning the tags into proviral DNA however ablated viral production and replication. By construction of additional viral variants we could show this defect was not protein-but RNA-dependent and sequence specific, and characterized by oversplicing of the genomic RNA. Simulation of genomic RNA folding suggested that introduction of the tag sequence induced an alternative folding structure in a region enriched in splice sites and splicing regulatory sequences. In silico predictions identified the HA/His(6)-Vpr tagging in HIV-1 to affect mRNA folding less than HA/FLAG-Vpr tagging. In vitro infectivity and mRNA splice pattern improved but did not reach wild-type values. Thus, sequence-specific insertions may interfere with mRNA splicing, possibly due to altered RNA folding. Our results point to the complexity of viral RNA genome sequence interactions. This should be taken into consideration when designing viral manipulation strategies, for both research as for biological interventions.

Autologous T cells transduced to express chimeric antigen receptors (CAR) directed against CD19, a B cell antigen, are reported to induce complete remission in patients with leukemia or lymphoma of the B cell lineage. Although potentially very effective, this treatment strategy has major drawbacks. First, CAR therapy is based on autologous T cells and therefore dependent on the nature and quality of T cells present in the peripheral blood of these patients at the time of treatment. Poor quality of the T cells may cause treatment failure in some patients. In addition, therapy based on autologous cells is tailor-made i.e. CAR+ T cells have to be generated de novo for every patient. Finally, autologous cell therapy requires different, more complicated logistics than conventional therapy. We therefore investigate whether a general purpose, allogeneic CAR therapy based on HLA-matched cord blood obtained from cord blood banks can be devised. Here, we investigated whether functional CAR+ T cells can be generated in vitro that do not express an endogenous T cell receptor to avoid alloreactivity causing graft versus host reactions. We compared carcino-embryonic antigen (CEA)- specific CARs of the first generation (intracellular CD3ζ signaling chain), of the 2nd generation (intracellular CD3ζ and CD28 signaling chain) and of the 3rd generation (intracellular CD3ζ, CD28 and OX40 signaling chain). CD34+ progenitor cells were isolated from human cord blood or postnatal thymus and subsequently transduced with one of the three green fluorescent protein (GFP)-encoding CAR constructs. Transduced cells were subsequently co-cultured on OP9DL1 in the presence of stem cell factor, Flt3-ligand and interleukin-7. Unlike T cell receptor transduced precursors (1), expansion was not enhanced by transduction of the chimeric receptor. Expansion was highest with first generation CARs whereas second and third generation CARs displayed only restricted expansion. Similar to T cell receptor transduced progenitors, CAR transduced cells show an accelerated differentiation during co-culture compared to the non-transduced cells: first committed CD5+ CD7+ T precursors appear, then CD4+ CD8+ double positive cells (DP) and finally CD1- CD27- single positive or double negative (DN) mature T cells. In cultures transduced with 2nd and 3rd generation CARs, few transduced cells passed through the proliferative DP pathway but rather differentiated to mature CD1- CD27- non-proliferative DN cells without passing through the DP stage. This phenomenon is responsible for the limited expansion seen with precursors transgenic for 2nd or 3rd generation CARs. However, in all cultures CAR+ DP cells were generated and, as shown for TCR transgenic cells (1), we were able to induce CEA specific maturation after co-culturing these DP cells with a cell line expressing CEA or by antibody-induced cross-linking of the CAR, giving rise to CD1- CD27+ matured cells. The observations described above are compatible with data obtained in mice showing that strong T cell receptor (TCR) activation during thymocyte differentiation inhibits the generation of DP cells and induces maturation to DN cells. Both the spontaneously and induced mature CAR+ cells were TCR and CD3 negative, suggesting that the expression of a CAR in early T cell precursors shuts down rearrangements of the endogenous TCR chains. Moreover, these cells lack NK marker expression (CD56, NKG2D) and show expression of T cell markers (CD5, CD7, CD2), confirming their T cell nature. In conclusion, the CAR+ CD3/TCR negative cells are T cells as these are derived from T cell precursors (CD5+, DP cells) and express various membrane and nuclear T cell markers. Mature CD1- CD27- CAR+ cells can be expanded to large cell numbers using T cell expansion protocols. They displayed cytokine production specific for CEA+ tumor lines as well as specific cytotoxicity. Moreover, the 2nd and 3rd generation CAR expressing cells showed increased specific cytokine production when compared to the first generation CAR expressing cells. These results show that the cord blood-derived CAR+ cells have potent functional activity similar to peripheral blood derived CAR+ T cells. We believe that these in vitro generated CAR+ cells developed from HLA-matched cord blood progenitors may be ideal as an adjunct to cord blood transplantation. (1) Snauwaert et al, Leukemia, 2014 Disclosures No relevant conflicts of interest to declare.

Malaria, which is the result of Plasmodium falciparum infection, is a global health threat that resulted in 655,000 deaths and 216 million clinical cases in 2010 alone. Recent phase 3 trials with malaria vaccine candidate RTS,S/AS01 (RTS,S) in children has demonstrated modest efficacy against clinical and severe malaria. RTS,S targets the pre-erythrocytic phase of the disease and induces high antibody titers against the P. falciparum circumsporozoite protein (CSP) and a moderate CD4(+) T cell response. The individual contribution of these adaptive immune responses to protection from infection remains unknown. Here, we found that prophylactic administration of anti-CSP mAbs derived from an RTS,S-vaccinated recipient fully protected mice with humanized livers from i.v.- and mosquito bite–delivered P. falciparum sporozoite challenge. Titers of anti-CSP that conveyed full protection were within the range observed in human RTS,S vaccine recipients. Increasing anti-CSP titers resulted in a dose-dependent reduction of the liver parasite burden. These data indicate that RTS,S-induced antibodies are protective and provide sterilizing immunity against P. falciparum infection when reaching or exceeding a critical plasma concentration.