Nanobody repertoire generated against the spike protein of ancestral SARS-CoV-2 remains efficacious against the rapidly evolving virus.

To date, all major modes of monoclonal antibody therapy targeting SARS-CoV-2 have lost significant efficacy against the latest circulating variants. As SARS-CoV-2 omicron sublineages account for over 90% of COVID-19 infections, evasion of immune responses generated by vaccination or exposure to previous variants poses a significant challenge. A compelling new therapeutic strategy against SARS-CoV-2 is that of single-domain antibodies, termed nanobodies, which address certain limitations of monoclonal antibodies. Here, we demonstrate that our high-affinity nanobody repertoire, generated against wild-type SARS-CoV-2 spike protein (Mast et al., 2021), remains effective against variants of concern, including omicron BA.4/BA.5; a subset is predicted to counter resistance in emerging XBB and BQ.1.1 sublineages. Furthermore, we reveal the synergistic potential of nanobody cocktails in neutralizing emerging variants. Our study highlights the power of nanobody technology as a versatile therapeutic and diagnostic tool to combat rapidly evolving infectious diseases such as SARS-CoV-2.

Automated, image-based quantification of peroxisome characteristics with perox-per-cell.

perox-per-cell automates cumbersome, image-based data collection tasks often encountered in peroxisome research. The software processes microscopy images to quantify peroxisome features in yeast cells. It uses off-the-shelf image processing tools to automatically segment cells and peroxisomes and then outputs quantitative metrics including peroxisome counts per cell and spatial areas. In validation tests, we found that perox-per-cell output agrees well with manually-quantified peroxisomal counts and cell instances, thereby enabling high-throughput quantification of peroxisomal characteristics. The software is available at https://github.com/AitchisonLab/perox-per-cell.

Multiple receptor tyrosine kinases regulate dengue infection of hepatocytes.

Introduction: Dengue is an arboviral disease causing severe illness in over 500,000 people each year. Currently, there is no way to constrain dengue in the clinic. Host kinase regulators of dengue virus (DENV) infection have the potential to be disrupted by existing therapeutics to prevent infection and/or disease progression. Methods: To evaluate kinase regulation of DENV infection, we performed kinase regression (KiR), a machine learning approach that predicts kinase regulators of infection using existing drug-target information and a small drug screen. We infected hepatocytes with DENV in vitro in the presence of a panel of 38 kinase inhibitors then quantified the effect of each inhibitor on infection rate. We employed elastic net regularization on these data to obtain predictions of which of 291 kinases are regulating DENV infection. Results: Thirty-six kinases were predicted to have a functional role. Intriguingly, seven of the predicted kinases - EPH receptor A4 (EPHA4), EPH receptor B3 (EPHB3), EPH receptor B4 (EPHB4), erb-b2 receptor tyrosine kinase 2 (ERBB2), fibroblast growth factor receptor 2 (FGFR2), Insulin like growth factor 1 receptor (IGF1R), and ret proto-oncogene (RET) - belong to the receptor tyrosine kinase (RTK) family, which are already therapeutic targets in the clinic. We demonstrate that predicted RTKs are expressed at higher levels in DENV infected cells. Knockdown of EPHB4, ERBB2, FGFR2, or IGF1R reduces DENV infection in hepatocytes. Finally, we observe differential temporal induction of ERBB2 and IGF1R following DENV infection, highlighting their unique roles in regulating DENV. Discussion: Collectively, our findings underscore the significance of multiple RTKs in DENV infection and advocate further exploration of RTK-oriented interventions against dengue.

CD4-mediated immunity shapes neutrophil-driven tuberculous pathology.

Pulmonary Mycobacterium tuberculosis (Mtb) infection results in highly heterogeneous lesions ranging from granulomas with central necrosis to those primarily comprised of alveolitis. While alveolitis has been associated with prior immunity in human post-mortem studies, the drivers of these distinct pathologic outcomes are poorly understood. Here, we show that these divergent lesion structures can be modeled in C3HeB/FeJ mice and are regulated by prior immunity. Using quantitative imaging, scRNAseq, and flow cytometry, we demonstrate that Mtb infection in the absence of prior immunity elicits dysregulated neutrophil recruitment and necrotic granulomas. In contrast, prior immunity induces rapid recruitment and activation of T cells, local macrophage activation, and diminished late neutrophil responses. Depletion studies at distinct infection stages demonstrated that neutrophils are required for early necrosis initiation and necrosis propagation at chronic stages, whereas early CD4 T cell responses prevent neutrophil feedforward circuits and necrosis. Together, these studies reveal fundamental determinants of tuberculosis lesion structure and pathogenesis, which have important implications for new strategies to prevent or treat tuberculosis.

SARS-CoV-2 Orf6 is positioned in the nuclear pore complex by Rae1 to inhibit nucleocytoplasmic transport.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) accessory protein Orf6 works as an interferon antagonist, in part, by inhibiting the nuclear import activated p-STAT1, an activator of interferon-stimulated genes, and the export of the poly(A) RNA. Insight into the transport regulatory function of Orf6 has come from the observation that Orf6 binds to the nuclear pore complex (NPC) components: Rae1 and Nup98. To gain further insight into the mechanism of Orf6-mediated transport inhibition, we examined the role of Rae1 and Nup98. We show that Rae1 alone is not necessary to support p-STAT1 import or nuclear export of poly(A) RNA. Moreover, the loss of Rae1 suppresses the transport inhibitory activity of Orf6. We propose that the Rae1/Nup98 complex strategically positions Orf6 within the NPC where it alters FG-Nup interactions and their ability to support nuclear transport. In addition, we show that Rae1 is required for normal viral protein production during SARS-CoV-2 infection presumably through its role in supporting Orf6 function.

Deleteome-Tools: Utilizing a compendium of yeast deletion strain transcriptomes to identify co-functional genes.

We developed an R codebase that uses a publicly-available compendium of transcriptomes from yeast single-gene deletion strains - the Deleteome - to predict gene function. Primarily, the codebase provides functions for identifying similarities between the transcriptomic signatures of deletion strains, thereby associating genes of interest with others that may be functionally related. We describe how our tool predicted a novel relationship between the yeast nucleoporin Nup170 and the Ctf18-RFC complex, which was confirmed experimentally, revealing a previously unknown link between nuclear pore complexes and the DNA replication machinery. We also discuss how our strategy for quantifying similarity between deletion strains differs from other approaches and why it has the potential to identify functional relationships that similar approaches may not. Deleteome-Tools is implemented in R and is freely available at https://github.com/AitchisonLab/Deleteome-Tools .

Nanobody repertoire generated against the spike protein of ancestral SARS-CoV-2 remains efficacious against the rapidly evolving virus.

To date, all major modes of monoclonal antibody therapy targeting SARS-CoV-2 have lost significant efficacy against the latest circulating variants. As SARS-CoV-2 omicron sublineages account for over 90% of COVID-19 infections, evasion of immune responses generated by vaccination or exposure to previous variants poses a significant challenge. A compelling new therapeutic strategy against SARS-CoV-2 is that of single domain antibodies, termed nanobodies, which address certain limitations of monoclonal antibodies. Here we demonstrate that our high-affinity nanobody repertoire, generated against wild-type SARS-CoV-2 spike protein (Mast, Fridy et al. 2021), remains effective against variants of concern, including omicron BA.4/BA.5; a subset is predicted to counter resistance in emerging XBB and BQ.1.1 sublineages. Furthermore, we reveal the synergistic potential of nanobody cocktails in neutralizing emerging variants. Our study highlights the power of nanobody technology as a versatile therapeutic and diagnostic tool to combat rapidly evolving infectious diseases such as SARS-CoV-2.

Predicting host-based, synthetic lethal antiviral targets from omics data.

Traditional antiviral therapies often have limited effectiveness due to toxicity and development of drug resistance. Host-based antivirals, while an alternative, may lead to non-specific effects. Recent evidence shows that virus-infected cells can be selectively eliminated by targeting synthetic lethal (SL) partners of proteins disrupted by viral infection. Thus, we hypothesized that genes depleted in CRISPR KO screens of virus-infected cells may be enriched in SL partners of proteins altered by infection. To investigate this, we established a computational pipeline predicting SL drug targets of viral infections. First, we identified SARS-CoV-2-induced changes in gene products via a large compendium of omics data. Second, we identified SL partners for each altered gene product. Last, we screened CRISPR KO data for SL partners required for cell viability in infected cells. Despite differences in virus-induced alterations detected by various omics data, they share many predicted SL targets, with significant enrichment in CRISPR KO-depleted datasets. Comparing data from SARS-CoV-2 and influenza infections, we found possible broad-spectrum, host-based antiviral SL targets. This suggests that CRISPR KO data are replete with common antiviral targets due to their SL relationship with virus-altered states and that such targets can be revealed from analysis of omics datasets and SL predictions.

SUMOylation at the inner nuclear membrane facilitates nuclear envelope biogenesis during mitosis.

As eukaryotic cells progress through cell division, the nuclear envelope (NE) membrane must expand to accommodate the formation of progeny nuclei. In Saccharomyces cerevisiae, closed mitosis allows visualization of NE biogenesis during mitosis. During this period, the SUMO E3 ligase Siz2 binds the inner nuclear membrane (INM) and initiates a wave of INM protein SUMOylation. Here, we show these events increase INM levels of phosphatidic acid (PA), an intermediate of phospholipid biogenesis, and are necessary for normal mitotic NE membrane expansion. The increase in INM PA is driven by the Siz2-mediated inhibition of the PA phosphatase Pah1. During mitosis, this results from the binding of Siz2 to the INM and dissociation of Spo7 and Nem1, a complex required for the activation of Pah1. As cells enter interphase, the process is then reversed by the deSUMOylase Ulp1. This work further establishes a central role for temporally controlled INM SUMOylation in coordinating processes, including membrane expansion, that regulate NE biogenesis during mitosis.

Nuclear pore complexes mediate subtelomeric gene silencing by regulating PCNA levels on chromatin.

The nuclear pore complex (NPC) physically interacts with chromatin and regulates gene expression. The Saccharomyces cerevisiae inner ring nucleoporin Nup170 has been implicated in chromatin organization and the maintenance of gene silencing in subtelomeric regions. To gain insight into how Nup170 regulates this process, we used protein-protein interactions, genetic interactions, and transcriptome correlation analyses to identify the Ctf18-RFC complex, an alternative proliferating cell nuclear antigen (PCNA) loader, as a facilitator of the gene regulatory functions of Nup170. The Ctf18-RFC complex is recruited to a subpopulation of NPCs that lack the nuclear basket proteins Mlp1 and Mlp2. In the absence of Nup170, PCNA levels on DNA are reduced, resulting in the loss of silencing of subtelomeric genes. Increasing PCNA levels on DNA by removing Elg1, which is required for PCNA unloading, rescues subtelomeric silencing defects in nup170Δ. The NPC, therefore, mediates subtelomeric gene silencing by regulating PCNA levels on DNA.

Distinct immune responses associated with vaccination status and protection outcomes after malaria challenge.

Understanding immune mechanisms that mediate malaria protection is critical for improving vaccine development. Vaccination with radiation-attenuated Plasmodium falciparum sporozoites (PfRAS) induces high level of sterilizing immunity against malaria and serves as a valuable tool for the study of protective mechanisms. To identify vaccine-induced and protection-associated responses during malarial infection, we performed transcriptome profiling of whole blood and in-depth cellular profiling of PBMCs from volunteers who received either PfRAS or noninfectious mosquito bites, followed by controlled human malaria infection (CHMI) challenge. In-depth single-cell profiling of cell subsets that respond to CHMI in mock-vaccinated individuals showed a predominantly inflammatory transcriptome response. Whole blood transcriptome analysis revealed that gene sets associated with type I and II interferon and NK cell responses were increased in prior to CHMI while T and B cell signatures were decreased as early as one day following CHMI in protected vaccinees. In contrast, non-protected vaccinees and mock-vaccinated individuals exhibited shared transcriptome changes after CHMI characterized by decreased innate cell signatures and inflammatory responses. Additionally, immunophenotyping data showed different induction profiles of vδ2+ γδ T cells, CD56+ CD8+ T effector memory (Tem) cells, and non-classical monocytes between protected vaccinees and individuals developing blood-stage parasitemia, following treatment and resolution of infection. Our data provide key insights in understanding immune mechanistic pathways of PfRAS-induced protection and infective CHMI. We demonstrate that vaccine-induced immune response is heterogenous between protected and non-protected vaccinees and that inducted-malaria protection by PfRAS is associated with early and rapid changes in interferon, NK cell and adaptive immune responses. Trial Registration: ClinicalTrials.gov NCT01994525.

Expanding and improving nanobody repertoires using a yeast display method: Targeting SARS-CoV-2.

COVID-19, caused by the coronavirus SARS-CoV-2, represents a serious worldwide health issue, with continually emerging new variants challenging current therapeutics. One promising alternate therapeutic avenue is represented by nanobodies, small single-chain antibodies derived from camelids with numerous advantageous properties and the potential to neutralize the virus. For identification and characterization of a broad spectrum of anti-SARS-CoV-2 Spike nanobodies, we further optimized a yeast display method, leveraging a previously published mass spectrometry-based method, using B-cell complementary DNA from the same immunized animals as a source of VHH sequences. Yeast display captured many of the sequences identified by the previous approach, as well as many additional sequences that proved to encode a large new repertoire of nanobodies with high affinities and neutralization activities against different SARS-CoV-2 variants. We evaluated DNA shuffling applied to the three complementarity-determining regions of antiviral nanobodies. The results suggested a surprising degree of modularity to complementarity-determining region function. Importantly, the yeast display approach applied to nanobody libraries from immunized animals allows parallel interrogation of a vast number of nanobodies. For example, we employed a modified yeast display to carry out massively parallel epitope binning. The current yeast display approach proved comparable in efficiency and specificity to the mass spectrometry-based approach, while requiring none of the infrastructure and expertise required for that approach, making these highly complementary approaches that together appear to comprehensively explore the paratope space. The larger repertoires produced maximize the likelihood of discovering broadly specific reagents and those that powerfully synergize in mixtures.

Viral protein engagement of GBF1 induces host cell vulnerability through synthetic lethality.

Viruses co-opt host proteins to carry out their lifecycle. Repurposed host proteins may thus become functionally compromised; a situation analogous to a loss-of-function mutation. We term such host proteins as viral-induced hypomorphs. Cells bearing cancer driver loss-of-function mutations have successfully been targeted with drugs perturbing proteins encoded by the synthetic lethal (SL) partners of cancer-specific mutations. Similarly, SL interactions of viral-induced hypomorphs can potentially be targeted as host-based antiviral therapeutics. Here, we use GBF1, which supports the infection of many RNA viruses, as a proof-of-concept. GBF1 becomes a hypomorph upon interaction with the poliovirus protein 3A. Screening for SL partners of GBF1 revealed ARF1 as the top hit, disruption of which selectively killed cells that synthesize 3A alone or in the context of a poliovirus replicon. Thus, viral protein interactions can induce hypomorphs that render host cells selectively vulnerable to perturbations that leave uninfected cells otherwise unscathed. Exploiting viral-induced vulnerabilities could lead to broad-spectrum antivirals for many viruses, including SARS-CoV-2.

Persistent Nonviral Plasmid Vector in Nasal Tissues Causes False-Positive SARS-CoV-2 Diagnostic Nucleic Acid Tests.

Biomedical personnel can become contaminated with nonhazardous reagents used in the laboratory. We describe molecular studies performed on nasal secretions collected longitudinally from asymptomatic laboratory coworkers to determine if they were infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) circulating in the community or with SARS-CoV-2 DNA from a plasmid vector. Participants enrolled in a prospective study of incident SARS-CoV-2 infection had nasal swabs collected aseptically by study staff at enrollment, followed by weekly self-collection of anterior nasal swabs. SARS-CoV-2 diagnosis was performed by a real-time PCR test targeting the nucleocapsid gene. PCR tests targeting SARS-CoV-2 nonstructural protein 10 (nsp10), nsp14, and envelope and three regions of the plasmid vector were performed to differentiate amplification of SARS-CoV-2 RNA from the plasmid vector's DNA. Nasal swabs from four asymptomatic coworkers with positive real-time PCR results for the SARS-CoV-2 nucleocapsid targets were negative when tested for SARS-CoV-2 nsp10, nsp14, and envelope protein. However, nucleic acids extracted from these nasal swabs amplified DNA regions of the plasmid vector used by the coworkers, including the ampicillin and neomycin/kanamycin resistance genes, the promoter-nucleocapsid junction, and unique codon-optimized regions. Nasal swabs from these individuals tested positive repeatedly, including during isolation. Longitudinal detection of plasmid DNA with SARS-CoV-2 nucleocapsid in nasal swabs suggests persistence in nasal tissues or colonizing bacteria. Nonviral plasmid vectors, while regarded as safe laboratory reagents, can interfere with molecular diagnostic tests. These reagents should be handled using proper personal protective equipment to prevent contamination of samples or laboratory personnel. IMPORTANCE Asymptomatic laboratory workers who tested positive for SARS-CoV-2 for days to months were found to harbor a laboratory plasmid vector containing SARS-CoV-2 DNA, which they had worked with in the past, in their nasal secretions. While prior studies have documented contamination of research personnel with PCR amplicons, our observation is novel, as these individuals shed the laboratory plasmid over days to months, including during isolation in their homes. This suggests that the plasmid was in their nasal tissues or that bacteria containing the plasmid had colonized their noses. While plasmids are generally safe, our detection of plasmid DNA in the nasal secretions of laboratory workers for weeks after they had stopped working with the plasmid shows the potential for these reagents to interfere with clinical tests and emphasizes that occupational exposures in the preceding months should be considered when interpreting diagnostic clinical tests.

Low rate of SARS-CoV-2 incident infection identified by weekly screening PCR in a prospective year-long cohort study.

BACKGROUND: Asymptomatic and pre-symptomatic SARS-CoV-2 infections may contribute to ongoing community transmission, however, the benefit of routine screening of asymptomatic individuals in low-risk populations is unclear. METHODS: To identify SARS-CoV-2 infections 553 seronegative individuals were prospectively followed for 52 weeks. From 4/2020-7/2021, participants submitted weekly self-collected nasal swabs for rtPCR and completed symptom and exposure surveys. RESULTS: Incident SARS2-CoV-2 infections were identified in 9/553 (1.6%) participants. Comparisons of SARS2-CoV-2(+) to SARS2-CoV-2(-) participants revealed significantly more close contacts outside the household (median: 5 versus 3; p = 0.005). The incidence of infection was higher among unvaccinated/partially vaccinated than among fully vaccinated participants (9/7,679 versus 0/6,845 person-weeks; p = 0.004). At notification of positive test result, eight cases were symptomatic and one pre-symptomatic. CONCLUSIONS: These data suggest that weekly SARS2-CoV2 surveillance by rtPCR did not efficiently detect pre-symptomatic infections in unvaccinated participants.

Dengue activates mTORC2 signaling to counteract apoptosis and maximize viral replication.

The mechanistic target of rapamycin (mTOR) functions in two distinct complexes: mTORC1, and mTORC2. mTORC1 has been implicated in the pathogenesis of flaviviruses including dengue, where it contributes to the establishment of a pro-viral autophagic state. Activation of mTORC2 occurs upon infection with some viruses, but its functional role in viral pathogenesis remains poorly understood. In this study, we explore the consequences of a physical protein-protein interaction between dengue non-structural protein 5 (NS5) and host cell mTOR proteins during infection. Using shRNA to differentially target mTORC1 and mTORC2 complexes, we show that mTORC2 is required for optimal dengue replication. Furthermore, we show that mTORC2 is activated during viral replication, and that mTORC2 counteracts virus-induced apoptosis, promoting the survival of infected cells. This work reveals a novel mechanism by which the dengue flavivirus can promote cell survival to maximize viral replication.

A genome-wide CRISPR-Cas9 screen identifies CENPJ as a host regulator of altered microtubule organization during Plasmodium liver infection.

Prior to initiating symptomatic malaria, a single Plasmodium sporozoite infects a hepatocyte and develops into thousands of merozoites, in part by scavenging host resources, likely delivered by vesicles. Here, we demonstrate that host microtubules (MTs) dynamically reorganize around the developing liver stage (LS) parasite to facilitate vesicular transport to the parasite. Using a genome-wide CRISPR-Cas9 screen, we identified host regulators of cytoskeleton organization, vesicle trafficking, and ER/Golgi stress that regulate LS development. Foci of γ-tubulin localized to the parasite periphery; depletion of centromere protein J (CENPJ), a novel regulator identified in the screen, exacerbated this re-localization and increased infection. We demonstrate that the Golgi acts as a non-centrosomal MT organizing center (ncMTOC) by positioning γ-tubulin and stimulating MT nucleation at parasite periphery. Together, these data support a model where the Plasmodium LS recruits host Golgi to form MT-mediated conduits along which host organelles are recruited to PVM and support parasite development.

Systems analysis of immune responses to attenuated P. falciparum malaria sporozoite vaccination reveals excessive inflammatory signatures correlating with impaired immunity.

Immunization with radiation-attenuated sporozoites (RAS) can confer sterilizing protection against malaria, although the mechanisms behind this protection are incompletely understood. We performed a systems biology analysis of samples from the Immunization by Mosquito with Radiation Attenuated Sporozoites (IMRAS) trial, which comprised P. falciparum RAS-immunized (PfRAS), malaria-naive participants whose protection from malaria infection was subsequently assessed by controlled human malaria infection (CHMI). Blood samples collected after initial PfRAS immunization were analyzed to compare immune responses between protected and non-protected volunteers leveraging integrative analysis of whole blood RNA-seq, high parameter flow cytometry, and single cell CITEseq of PBMCs. This analysis revealed differences in early innate immune responses indicating divergent paths associated with protection. In particular, elevated levels of inflammatory responses early after the initial immunization were detrimental for the development of protective adaptive immunity. Specifically, non-classical monocytes and early type I interferon responses induced within 1 day of PfRAS vaccination correlated with impaired immunity. Non-protected individuals also showed an increase in Th2 polarized T cell responses whereas we observed a trend towards increased Th1 and T-bet+ CD8 T cell responses in protected individuals. Temporal differences in genes associated with natural killer cells suggest an important role in immune regulation by these cells. These findings give insight into the immune responses that confer protection against malaria and may guide further malaria vaccine development. Trial registration: ClinicalTrials.gov NCT01994525.

Preimmunization correlates of protection shared across malaria vaccine trials in adults.

Identifying preimmunization biological characteristics that promote an effective vaccine response offers opportunities for illuminating the critical immunological mechanisms that confer vaccine-induced protection, for developing adjuvant strategies, and for tailoring vaccination regimens to individuals or groups. In the context of malaria vaccine research, studying preimmunization correlates of protection can help address the need for a widely effective malaria vaccine, which remains elusive. In this study, common preimmunization correlates of protection were identified using transcriptomic data from four independent, heterogeneous malaria vaccine trials in adults. Systems-based analyses showed that a moderately elevated inflammatory state prior to immunization was associated with protection against malaria challenge. Functional profiling of protection-associated genes revealed the importance of several inflammatory pathways, including TLR signaling. These findings, which echo previous studies that associated enhanced preimmunization inflammation with protection, illuminate common baseline characteristics that set the stage for an effective vaccine response across diverse malaria vaccine strategies in adults.

Longitudinal immune profiling after radiation-attenuated sporozoite vaccination reveals coordinated immune processes correlated with malaria protection.

Background: Identifying immune processes required for liver-stage sterilizing immunity to malaria remains an open problem. The IMRAS trial comprised 5x immunizations with radiation-attenuated sporozoites resulting in 55% protection from subsequent challenge. Methods: To identify correlates of vaccination and protection, we performed detailed systems immunology longitudinal profiling of the entire trial time course including whole blood transcriptomics, detailed PBMC cell phenotyping and serum antigen array profiling of 11 IMRAS radiation-attenuated sporozoite (RAS) vaccinees at up to 21 timepoints each. Results: RAS vaccination induced serum antibody responses to CSP, TRAP, and AMA1 in all vaccinees. We observed large numbers of differentially expressed genes associated with vaccination response and protection, with distinctly differing transcriptome responses elicited after each immunization. These included inflammatory and proliferative responses, as well as increased abundance of monocyte and DC subsets after each immunization. Increases in Vδ2 γδ; T cells and MAIT cells were observed in response to immunization over the course of study, and CD1c+ CD40+ DC abundance was significantly associated with protection. Interferon responses strongly differed between protected and non-protected individuals with high interferon responses after the 1st immunization, but not the 2nd-5th. Blood transcriptional interferon responses were correlated with abundances of different circulating classical and non-classical monocyte populations. Conclusions: This study has revealed multiple coordinated immunological processes induced by vaccination and associated with protection. Our work represents the most detailed immunological profiling of a RAS vaccine trial performed to date and will guide the design and interpretation of future malaria vaccine trials.