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Abstract Background Despite treatment with highly effective antimalarial drugs, malaria annually claims the lives of over half a million children under 5-years of age in sub-Saharan Africa. Cerebral malaria (CM), defined as Plasmodium falciparum infection with coma, is the severe malaria syndrome with the highest mortality. Studies in the CM mouse model suggest that a T cell-mediated response underlies CM pathology, opening a new target for therapy in humans. This trial aims to establish the preliminary safety of one such novel therapy, the glutamine antagonist 6-diazo-5-oxo-L-norleucine (DON). Methods In this phase I/IIa dose-escalation clinical trial, a single dose of intravenous (IV) DON is administered to three participants groups—healthy adults and adults with uncomplicated malaria, then pediatric participants with CM—to primarily assess safety. The secondary objective of this trial is to assess pharmacokinetics of DON over a range of doses. The open-label adult portion of the trial enrolls 40 healthy adults concurrently with 40 adults with uncomplicated malaria. Cohorts of 10 participants receive a single IV dose of DON with doses escalating between cohorts from 0.1 mg/kg, 1.0 mg/kg, 5.0 mg/kg, to 10 mg/kg. Following subsequent safety review, a randomized, double-blind, and placebo-controlled pediatric study enrolls 72 participants aged 6 months to 14 years with CM. The pediatric portion of the study minimally spans three malaria seasons including a planned interim analysis after 50% of pediatric enrollments. The first half of pediatric participants receive DON 0.1 mg/kg, 1.0 mg/kg, or placebo. Dosing for the second half of pediatric participants is informed by the safety and preliminary efficacy results of those previously enrolled. The pediatric portion of the study has an exploratory outcome evaluating the preliminary efficacy of DON. Efficacy is assessed by diagnostics predictive of CM outcome: electroencephalography (EEG), magnetic resonance imaging (MRI), and transcranial doppler (TCD), measured before and after DON administration. All participants with malaria receive standard of care antimalarials in accordance with local guidelines, regardless of study drug dose group. Discussion This preliminary safety and efficacy study evaluates DON, a candidate adjunctive therapy for pediatric CM. If results support DON preliminary safety and efficacy, follow-up phase II and III clinical trials will be indicated. Trial registration This trial was registered on ClinicalTrials.gov on 28 July 2022 (NCT05478720).

Abstract There are nearly 65 million people with chronic heart failure (CHF) globally, with no treatment directed at the pathologic cause of the disease, the loss of functioning cardiomyocytes. We have an allogeneic cardiac patch comprised of cardiomyocytes and human fibroblasts on a bioresorbable matrix. This patch increases blood flow to the damaged heart and improves left ventricular (LV) function in an immune competent rat model of ischemic CHF. After 6 months of treatment in an immune competent Yucatan mini swine ischemic CHF model, this patch restores LV contractility without constrictive physiology, partially reversing maladaptive LV and right ventricular remodeling, increases exercise tolerance, without inducing any cardiac arrhythmias or a change in myocardial oxygen consumption. Digital spatial profiling in mice with patch placement 3 weeks after a myocardial infarction shows that the patch induces a CD45pos immune cell response that results in an infiltration of dendritic cells and macrophages with high expression of macrophages polarization to the anti-inflammatory reparative M2 phenotype. Leveraging the host native immune system allows for the potential use of immunomodulatory therapies for treatment of chronic inflammatory diseases not limited to ischemic CHF.

Summary: Background: While vaccines have established utility against COVID-19, phase 3 efficacy studies have generally not comprehensively evaluated protection provided by previous infection or hybrid immunity (previous infection plus vaccination). Individual patient data from US government-supported harmonized vaccine trials provide an unprecedented sample population to address this issue. We characterized the protective efficacy of previous SARS-CoV-2 infection and hybrid immunity against COVID-19 early in the pandemic over three-to six-month follow-up and compared with vaccine-associated protection. Methods: In this post-hoc cross-protocol analysis of the Moderna, AstraZeneca, Janssen, and Novavax COVID-19 vaccine clinical trials, we allocated participants into four groups based on previous-infection status at enrolment and treatment: no previous infection/placebo; previous infection/placebo; no previous infection/vaccine; and previous infection/vaccine. The main outcome was RT-PCR-confirmed COVID-19 >7–15 days (per original protocols) after final study injection. We calculated crude and adjusted efficacy measures. Findings: Previous infection/placebo participants had a 92% decreased risk of future COVID-19 compared to no previous infection/placebo participants (overall hazard ratio [HR] ratio: 0.08; 95% CI: 0.05–0.13). Among single-dose Janssen participants, hybrid immunity conferred greater protection than vaccine alone (HR: 0.03; 95% CI: 0.01–0.10). Too few infections were observed to draw statistical inferences comparing hybrid immunity to vaccine alone for other trials. Vaccination, previous infection, and hybrid immunity all provided near-complete protection against severe disease. Interpretation: Previous infection, any hybrid immunity, and two-dose vaccination all provided substantial protection against symptomatic and severe COVID-19 through the early Delta period. Thus, as a surrogate for natural infection, vaccination remains the safest approach to protection. Funding: National Institutes of Health.

Abstract Estuaries provide many ecosystem services and host a majority of the world’s population. Here, the response of microbial communities after a record-breaking flood event in a highly urbanized estuary was followed. Hurricane Harvey (hereafter Harvey) was a category 4 hurricane that made landfall on the Texas coast in 2017 and lashed the Houston area with 1.4–1.7 × 1010 m3 of rainfall, disrupting the natural gradients of nutrients and salinity. Here, we utilized metagenomics to analyze how Harvey altered the microbial community of Galveston Bay over five weeks following the storm. We hypothesized that the community would shift from a marine dominated community to that of a terrestrial and freshwater origin. We found that following the storm there were changes in the distribution of species with specific metabolic capacities, such as Cyanobacteria, enriched in oxygenic photosynthesis and nitrogen fixation genes, as well as Verrucomicrobia and Betaproteobacteria, with high prevalence of the SOX complex and anoxygenic photosynthesis genes. On the other hand, dominant members of the community with more diverse metabolic capabilities showed less fluctuations in their distribution. Our results highlight how massive precipitation disturbances can alter microbial communities and how the coalescence of diverse microorganisms creates a resilient community able to maintain ecosystem services even when the system is in an altered state.

We formulate the bedload sediment flux probability distribution from the Lagrangian dynamics of individual grains. Individual particles obey Langevin equations wherein the stochastic forces driving particle motions are switched on and off by particle entrainment and deposition. The flux is calculated as the rate of many such particles crossing a control surface within a specified observation time. Flux distributions inherit observation time dependence from the on–off motions of particles. At the longest observation times, distributions converge to sharp peaks around classically expected values, but at short times, fluctuations are erratic. We relate this scale dependence of bedload transport rates to the movement characteristics of individual sediment grains. This work provides a statistical mechanics description for the fluctuations and observation-scale dependence of sediment transport rates.

Abstract Background Despite elevated prevalence rates of prenatal (antenatal) anxiety across studies (13–21%), and prenatal people’s use of the Internet to search for pregnancy-related information and support, research investigating prenatal people’s experiences with online mental health communication, such as blogs, is lacking. This study examined blog entries focused on anxiety in pregnancy to better understand prenatal people’s Internet discourse concerning their experiences with anxiety. Methods A Google search using the keywords “anxiety,” “pregnant,” and “blog” resulted in N = 18 blogs that met inclusion criteria (public blog written in English describing a personal experience with prenatal anxiety in 250 words or more). Blog content was analyzed using a thematic analytic approach based on grounded theory principles. Results Three main themes capturing prenatal people’s experiences with anxiety as written in public blog content were developed from qualitative analyses: 1) etiology (subthemes: before pregnancy, during the current pregnancy, related to a previous pregnancy), 2) triggers (subthemes: uncertainty, perceived lack of control, and guilt and shame for not having a normal pregnancy), and 3) symptoms (subthemes: intertwined emotional, cognitive and physical symptoms, in addition to behavioural symptoms). Conclusions Our findings demonstrate a need for perinatal professionals to address anxiety symptoms and triggers in pregnancy. One way to address this may be by providing credible information regarding prenatal mental and physical health to pregnant people through online mediums, such as blogs. Bloggers often discussed experiencing a combination of emotional, cognitive, physical, and behavioural symptoms, which suggests that medical and mental health professionals should work collaboratively to provide care for prenatal people experiencing anxiety. Furthermore, Cognitive Behavioural Therapy (CBT) addresses these types of symptoms, which suggests that interventions developed or adapted to meet this populations’ needs could employ this therapeutic approach. Future research should explore the reasons why prenatal people experiencing anxiety engage with blogs, the characteristics of bloggers and readers, the impact of the blogging experience on both the blogger and their audience, and the information quality of blog content.

ABSTRACT Cerebral malaria (CM), the deadliest complication of Plasmodium infection, is a complex and unpredictable disease. However, our understanding of the host and parasite factors that cause CM is limited. Using a mouse model of CM, experimental CM (ECM), we performed a three-way comparison between ECM-susceptible C57BL/6 mice infected with ECM-causing Plasmodium ANKA parasites [ANKA(C57BL/6)], ECM-resistant BALB/c mice infected with Plasmodium ANKA [ANKA(BALB/c)], and C57BL/6 mice infected with Plasmodium NK65 that does not cause ECM [NK65(C57BL/6)]. All ANKA(C57BL/6) mice developed CM. In contrast, in ANKA(BALB/c) and NK65(C57BL/6), infections do not result in CM and proceed similarly in terms of parasite growth, disease course, and host immune response. However, parasite gene expression in ANKA(BALB/c) was remarkably different than that in ANKA(C57BL/6) but similar to the gene expression in NK65(C57BL/6). Thus, Plasmodium ANKA has an ECM-specific gene expression profile that is activated only in susceptible hosts, providing evidence that the host has a critical influence on the outcome of infection. IMPORTANCE Hundreds of thousands of lives are lost each year due to the brain damage caused by malaria disease. The overwhelming majority of these deaths occur in young children living in sub-Saharan Africa. Thus far, there are no vaccines against this deadly disease, and we still do not know why fatal brain damage occurs in some children while others have milder, self-limiting disease progression. Our research provides an important clue to this problem. Here, we showed that the genetic background of the host has an important role in determining the course and the outcome of the disease. Our research also identified parasite molecules that can potentially be targeted in vaccination and therapy approaches.

Abstract Background During the perinatal period (including pregnancy and up to 12 months after childbirth), expectant and new mothers are at an elevated risk of developing depression. Inadequate knowledge about perinatal depression and treatment options may contribute to the low help-seeking rates exhibited by perinatal people. The Internet can be an accessible source of information about perinatal depression; however, the quality of this information remains to be evaluated. The purpose of this study was to assess the quality of perinatal depression information websites. Methods After review, 37 websites were included in our sample. To assess overall website quality, we rated websites based on their reading level (Simple Measure of Gobbledegook; SMOG), information quality (DISCERN), usability (Patient Education Materials Assessment Tool; PEMAT), and visual design (Visual Aesthetics of Website Inventory; VisAWI). Results Websites often exceeded the National Institute of Health's recommended reading level of grades 6–8, with scores ranging from 6.8 to 13.5. Website information quality ratings ranged from 1.8 to 4.3 out of 5, with websites often containing insufficient information about treatment choices. Website usability ratings were negatively impacted by the lack of information summaries, visual aids, and tangible tools. Visual design ratings ranged from 3.2 to 6.6 out of 7, with a need for more creative design elements to enhance user engagement. Conclusions This study outlines the characteristics of high-quality perinatal depression information websites. Our findings illustrate that perinatal depression websites are not meeting the needs of users in terms of reading level, information quality, usability, and visual design. Our results may be helpful in guiding healthcare providers to reliable, evidence-based online resources for their perinatal patients.

Juvenile white sharks (JWS) of the Northeastern Pacific population are present in nearshore southern California waters and form mixed size class (~1.5-3 m) aggregations for weeks to months, often within 500 m of shore. These nearshore beach habitats are heavily used for human recreation (e.g., surfing, swimming, body boarding, wading, and standup paddleboarding) and the amount of spatio-temporal overlap between JWS and humans is currently unknown. Increases in human population and the Northeastern Pacific population of white sharks have raised concern over human beach safety. To determine spatio-temporal JWS-human overlap at various spatial scales (e.g., across the entire southern California coastline, across different distances from shore, and within specific beach locations), 26 beach locations across southern California were surveyed monthly resulting in 1644 aerial drone surveys between January 2019 to March 2021. Thirteen environmental variables were assessed to predict when spatio-temporal overlap between JWS and water users was highest. Coast-wide distribution of JWS was clumped, limiting human-shark co-occurrence to specific locations, with 1096 of 1204 JWS observations occurring at Carpinteria and Del Mar Beach locations. Nearshore distribution indicated JWS are often close enough to the wave break to interact with some water users (median = 101 m, range = 2-702 m), although JWS had the most spatial overlap with stand-up paddlers. Daily human-shark co-occurrence was 97% at beaches where JWS aggregations had formed, and human activity showed high spatial overlap at shark aggregation sites. Although there is higher seasonal human-shark spatio-temporal overlap where aggregations form in southern California, the number of unprovoked shark bites across southern California is extremely low. This study provides evidence that high human-shark spatio-temporal overlap does not lead to an increased bite frequency in southern California, and there are a number of possible explanations as to why JWS are not biting water users despite daily encounters.

Parental mental health services in neonatal follow-up programs (NFUPs) are lacking though needed. This study aimed to determine (1) the unmet mental health needs of parents and (2) the parent and provider perspectives on barriers and opportunities to increase mental health service access. Study 1: Parents in a central Canadian NFUP (N = 49) completed a mixed-method online survey (analyzed descriptively and by content analysis) to elucidate their mental health, related service use, barriers to service use, and service preferences. Study 2: Virtual focus groups with NFUP service providers (N = 5) were run to inform service improvements (analyzed by reflexive thematic analysis). The results show that parents endorsed a 2–4 times higher prevalence of clinically significant depression (59.2%), anxiety (51.0%), and PTSD (26.5%) than the general postpartum population. Most parents were not using mental health services (55.1%) due to resource insecurity among parents (e.g., time, cost) and the organization (e.g., staffing, training, referrals). Consolidating parents’ and service providers’ perspectives revealed four opportunities for service improvements: bridging services, mental health screening, online psychoeducation, and peer support. Findings clarify how a central Canadian NFUP can address parental mental health in ways that are desired by parents and feasible for service providers.

Dengue virus causes a range of inflammatory pathology but understanding critical phases of the infection during human infection has been challenging. Here the author’s present immunotranscriptomic changes during the acute and clearance phases of a dengue virus serotype 2 human challenge model.

Multivalent vaccines that confer protection to multiple serotypes of Dengue virus have been established. Here the authors examine the presence of vaccine induced multivalent antibodies and how these link to protection in a human challenge model of Dengue virus.

Whether and how migratory organisms exhibit interindividual behavioral and/or physiological variation across movement strategies remains an open question. The Dark-eyed Junco (Junco hyemalis) is a migratory songbird known for its intraspecies variation displayed in relation to morphology, song repertoires, and migration. Thus, studies focusing on juncos can reveal how migratory strategy may covary with other individual-scale factors and, therefore, identify the selective forces driving intraspecies variations throughout its distribution. We used Dark-eyed Junco hydrogen stable isotope feather values (δ²H) and implemented a Bayesian framework to infer the breeding and molting origin of migratory juncos captured on their winter grounds in Oklahoma, USA. We modeled the distribution of feather hydrogen stable isotope values as a function of five morphological variables including body mass and fat deposition measured in Oklahoma during the winter. We then investigated the trade-off between longer and more energetically costly migration strategies, in relation to diet preferences through carbon (δ13C) and nitrogen (δ15N) stable isotope analysis, from feather values. Dark-eyed Juncos wintering in south central U.S. likely originate from multiple breeding populations in northern U.S. and Canada. Body condition at the wintering ground (e.g., mass) had no effect on feather hydrogen stable isotope abundance. However, we found a positive correlation between nitrogen and hydrogen stable isotopes, suggesting that a trophic level shift toward insect consumption might occur in individuals migrating from southern latitudes. Increased insect-derived protein consumption might be explained by reduced fatty-acid reserves necessary to complete a shorter migratory journey.

Background: Acute viral infections induce a rapid and transient increase in antibody-secreting plasmablasts. At convalescence, memory B cells (MBC) and long-lived plasma cells (LLPC) are responsible for long-term humoral immunity. Following an acute viral infection, the specific properties and relationships between antibodies produced by these B cell compartments are poorly understood. Methods: We utilized a controlled human challenge model of primary dengue virus serotype 2 (DENV2) infection to study acute and convalescent B-cell responses. Findings: The level of DENV2 replication was correlated with the magnitude of the plasmablast response. Functional analysis of plasmablast-derived monoclonal antibodies showed that the DENV2-specific response was dominated by cells producing DENV2 serotype-specific antibodies. DENV2-neutralizing antibodies targeted quaternary structure epitopes centered on domain III of the viral envelope protein (EDIII). Functional analysis of MBC and serum antibodies from the same subjects six months post-challenge revealed maintenance of the serotype-specific response in both compartments. The serum response mainly targeted DENV2 serotype-specific epitopes on EDIII. Interpretation: Our data suggest overall functional alignment of DENV2-specific responses from the plasmablast, through the MBC and LLPC compartments following primary DENV2 inflection. These results provide enhanced resolution of the temporal and specificity of the B cell compartment in viral infection and serve as framework for evaluation of B cell responses in challenge models. Funding: This study was supported by the Bill and Melinda Gates Foundation and the National Institutes of Health. Keywords: Dengue, Viral infection, Humoral immunity, Antibody

Dengue virus (DENV) is a mosquito-borne flavivirus that causes serious human disease. The current lack of an effective vaccine to simultaneously protect against the four serotypes of DENV in seronegative individuals is a major unmet medical need. Further, the immunological basis for protective immunity in the setting of DENV infection or vaccination is not fully understood. Our team has developed a live attenuated tetravalent dengue virus vaccine that provides complete protection in a human model of dengue virus challenge. The goal of this study was to define, in the context of protective human vaccination, the quality of vaccine-induced DENV-specific CD8+ and CD4+ T cells and the temporal dynamics associated with their formation and maintenance. Multifunctional, DENV-specific CD8+ and CD4+ T cells developed 8–14 days after vaccination and were maintained for at least 6 months. Virus-specific CD8 T+ cells were a mixture of effector memory T cells (TEM) and effector memory T cells re-expressing CD45RA (TEMRA), with TEM cells predominating until day 21 post-vaccination and TEMRA cells thereafter. The majority of virus-specific CD4+ T cells were TEM with a small fraction being TEMRA. The frequency of virus-specific CD8+ and CD4+ T cells were further skewed to the TEMRA phenotype following either a second dose of the tetravalent vaccine or challenge with a single serotype of DENV. Collectively, our study has defined the phenotypic profile of antiviral CD8+ and CD4+ T cells associated with protective immunity to DENV infection and the kinetics of their formation and maintenance.

Abstract Objectives There is an incomplete understanding of the host humoral immune response to severe acute respiratory syndrome (SARS)‐coronavirus (CoV)‐2, which underlies COVID‐19, during acute infection. Host factors such as age and sex as well as the kinetics and functionality of antibody responses are important factors to consider as vaccine development proceeds. The receptor‐binding domain of the CoV spike (RBD‐S) protein mediates host cell binding and infection and is a major target for vaccine design to elicit neutralising antibodies. Methods We assessed serum anti‐SARS‐CoV‐2 RBD‐S IgG, IgM and IgA antibodies by a two‐step ELISA and neutralising antibodies in a cross‐sectional study of hospitalised COVID‐19 patients of varying disease severities. Anti‐RBD‐S IgG levels were also determined in asymptomatic seropositives. Results We found equivalent levels of anti‐RBD‐S antibodies in male and female patients and no age‐related deficiencies even out to 93 years of age. The anti‐RBD‐S response was evident as little as 6 days after onset of symptoms and for at least 5 weeks after symptom onset. Anti‐RBD‐S IgG, IgM and IgA responses were simultaneously induced within 10 days after onset, with anti‐RBD‐S IgG sustained over a 5‐week period. Anti‐RBD‐S antibodies strongly correlated with neutralising activity. Lastly, anti‐RBD‐S IgG responses were higher in symptomatic COVID‐19 patients during acute infection compared with asymptomatic seropositive donors. Conclusion Our results suggest that anti‐RBD‐S IgG reflect functional immune responses to SARS‐CoV‐2, but do not completely explain age‐ and sex‐related disparities in COVID‐19 fatalities.

Somatic hypermutation of antibodies can occur in infants but are difficult to track. Here the authors present a new method called MIDCIRS for deep quantitative repertoire sequencing with few cells, and show infants as young as 3 months can expand antibody lineage complexity in response to malaria infection.

Malaria: Inhibiting parasite invasion of red blood cells A vaccine targeting a protein complex that allows malaria-causing parasite to enter red blood cells has been produced. Malaria caused by the parasite Plasmodium falciparum is an oft-deadly infectious disease without an effective vaccine. A team of researchers at the National Institutes of Health led by Prakash Srinivasan, currently at the Johns Hopkins Malaria Research Institute, United States, demonstrated the efficacy of a vaccine candidate that works by priming a host’s immune system to a parasitic protein complex required to form a junction with red blood cells, allowing entry and proliferation of the pathogen. The group’s vaccine conferred more effective protection in monkeys than prior candidates that targeted only one component of the parasitic protein complex. This research warrants a closer look into how this candidate, and others targeting the protein complex, can be used to prevent malaria in humans.

Malaria is a deadly infectious disease associated with fundamental changes in the composition of the memory B cell (MBC) compartment, most notably a large expansion of T-bet+ MBCs, termed atypical MBCs. However, we know little about the precursors of atypical MBCs and the conditions that drive their differentiation. We compared the responses of human tonsil naïve B cells, MBCs, and germinal center B cells to a variety of stimulatory conditions. We determined that prolonged antigen presentation in the presence of CpG and IFN-γ induced maximal expression of T-bet and other phenotypic markers of malaria-associated atypical MBCs primarily in naïve B cells in vitro. Importantly T-bet+ naïve-derived B cells resembled atypical MBCs in their hypo-responsiveness to signaling through their B cell receptors. Thus, naïve B cells can be induced to differentiate into phenotypically and functionally atypical-like MBCs in vitro under conditions that may prevail in chronic infectious diseases in vivo.

Cerebral malaria is the deadliest complication of malaria, a febrile infectious disease caused by Plasmodium parasite. Any of the five human Plasmodium species can cause disease, but, for unknown reasons, in approximately 2 million cases each year P. falciparum progresses to severe disease, ultimately resulting in half a million deaths. The majority of these deaths are in children under the age of five. Currently, there is no way to predict which child will progress to severe disease and there are no adjunctive therapies to halt the symptoms after onset. Herein, we discuss what is known about the disease mechanism of one form of severe malaria, cerebral malaria, and how we might exploit this understanding to rescue children in the throes of cerebral disease.

Antibodies acquired naturally through repeated exposure to Plasmodium falciparum are essential in the control of blood-stage malaria. Antibody-dependent functions may include neutralization of parasite–host interactions, complement activation, and activation of Fc receptor functions. A role of antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells in protection from malaria has not been established. Here we show that IgG isolated from adults living in a malaria-endemic region activated ADCC by primary human NK cells, which lysed infected red blood cells (RBCs) and inhibited parasite growth in an in vitro assay for ADCC-dependent growth inhibition. RBC lysis by NK cells was highly selective for infected RBCs in a mixed culture with uninfected RBCs. Human antibodies to P. falciparum antigens PfEMP1 and RIFIN were sufficient to promote NK-dependent growth inhibition. As these results implicate acquired immunity through NK-mediated ADCC, antibody-based vaccines that target bloodstream parasites should consider this new mechanism of action.

Many chronic infections, including malaria and HIV, are associated with a large expansion of CD21-CD27- 'atypical' memory B cells (MBCs) that exhibit reduced B cell receptor (BCR) signaling and effector functions. Little is known about the conditions or transcriptional regulators driving atypical MBC differentiation. Here we show that atypical MBCs in malaria-exposed individuals highly express the transcription factor T-bet, and that T-bet expression correlates inversely with BCR signaling and skews toward IgG3 class switching. Moreover, a longitudinal analysis of a subset of children suggested a correlation between the incidence of febrile malaria and the expansion of T-bethi B cells. The Th1-cytokine containing supernatants of malaria-stimulated PBMCs plus BCR cross linking induced T-bet expression in naïve B cells that was abrogated by neutralizing IFN-γ or blocking the IFN-γ receptor on B cells. Accordingly, recombinant IFN-γ plus BCR cross-linking drove T-bet expression in peripheral and tonsillar B cells. Consistent with this, Th1-polarized Tfh (Tfh-1) cells more efficiently induced T-bet expression in naïve B cells. These data provide new insight into the mechanisms underlying atypical MBC differentiation.

Advancements in high-throughput sequencing and molecular identifier-based error correction have opened the door to antibody repertoire sequencing with single mutation precision, increasing both the breadth and depth of immune response characterization. However, improvements in sequencing technology cannot resolve one key aspect of antibody repertoire sequencing accuracy: the possibility of undocumented novel germline alleles. Somatic hypermutation (SHM) calling requires a reference germline sequence, and the antibody variable region gene alleles collected by the IMGT database, although large in number, are not comprehensive. Mismatches, resulted from single nucleotide polymorphisms or other genetic variation, between the true germline sequence and the closest IMGT allele can inflate SHM counts, leading to inaccurate antibody repertoire analysis. Here, we developed a streamlined approach to novel allele prediction and validation using bulk PBMC antibody repertoire sequencing data and targeted genomic DNA amplification and sequencing using PBMCs from only 4 ml of blood to quickly and effectively improve the fidelity of antibody repertoire analysis. This approach establishes a framework for comprehensively annotating novel alleles using a small amount of blood sample, which is extremely useful in studying young children’s immune systems.

Infection caused by the four serotypes of dengue virus (DENV-1-4) is a leading cause of mosquito-borne disease. Clinically-severe dengue disease is more common when secondary dengue infection occurs following prior infection with a heterologous dengue serotype. Other flaviviruses such as yellow fever virus, Japanese encephalitis virus, and Zika virus, can also elicit antibodies which are cross-reactive to DENV. As candidate dengue vaccines become available in endemic settings and for individuals who have received other flavivirus vaccines, it is important to examine vaccine safety and immunogenicity in these flavivirus-experienced populations. We performed a randomized, controlled trial of the National Institutes of Health live attenuated tetravalent dengue vaccine candidate (TV003) in fifty-eight individuals with prior exposure to flavivirus infection or vaccine. As in prior studies of this vaccine in flavivirus-naive volunteers, flavivirus-experienced subjects received two doses of vaccine six months apart and were followed closely for clinical events, laboratory changes, viremia, and neutralizing antibody titers. TV003 was well tolerated with few adverse events other than rash, which was predominately mild. Following one dose, 87% of vaccinees had an antibody response to all four serotypes (tetravalent response), suggesting a robust immune response. In addition, 76% of vaccinees were viremic; mean peak titers ranged from 0.68–1.1 log10 PFU/mL and did not differ by serotype. The second dose of TV003 was not associated with viremia, rash, or a sustained boost in antibody titers indicating that a single dose of the vaccine is likely sufficient to prevent viral replication and thus protect against disease. In comparison to the viremia and neutralizing antibody response elicited by TV003 in flavivirus-naïve subjects from prior studies, we found that subjects who were flavivirus-exposed prior to vaccination exhibited slightly higher DENV-3 viremia, higher neutralizing antibody titers to DENV-2, -3, and -4, and a higher tetravalent response frequency after TV003 administration. In summary, we demonstrate that the NIH tetravalent dengue vaccine TV003 is well-tolerated in flavivirus-experienced individuals and elicits robust post-vaccination neutralizing antibody titers.ClinicalTrials.gov NCT01506570.

Cerebral malaria (CM) is a severe complication of Plasmodium falciparum infection that results in thousands of deaths each year, mostly in African children. The in vivo mechanisms underlying this fatal condition are not entirely understood. Using the animal model of experimental cerebral malaria (ECM), we sought mechanistic insights into the pathogenesis of CM. Fatal disease was associated with alterations in tight junction proteins, vascular breakdown in the meninges / parenchyma, edema, and ultimately neuronal cell death in the brainstem, which is consistent with cerebral herniation as a cause of death. At the peak of ECM, we revealed using intravital two-photon microscopy that myelomonocytic cells and parasite-specific CD8+ T cells associated primarily with the luminal surface of CNS blood vessels. Myelomonocytic cells participated in the removal of parasitized red blood cells (pRBCs) from cerebral blood vessels, but were not required for the disease. Interestingly, the majority of disease-inducing parasite-specific CD8+ T cells interacted with the lumen of brain vascular endothelial cells (ECs), where they were observed surveying, dividing, and arresting in a cognate peptide-MHC I dependent manner. These activities were critically dependent on IFN-γ, which was responsible for activating cerebrovascular ECs to upregulate adhesion and antigen-presenting molecules. Importantly, parasite-specific CD8+ T cell interactions with cerebral vessels were impaired in chimeric mice rendered unable to present EC antigens on MHC I, and these mice were in turn resistant to fatal brainstem pathology. Moreover, anti-adhesion molecule (LFA-1 / VLA-4) therapy prevented fatal disease by rapidly displacing luminal CD8+ T cells from cerebrovascular ECs without affecting extravascular T cells. These in vivo data demonstrate that parasite-specific CD8+ T cell-induced fatal vascular breakdown and subsequent neuronal death during ECM is associated with luminal, antigen-dependent interactions with cerebrovasculature.

ABSTRACT Malaria is an infectious disease caused by parasites of several Plasmodium spp. Cerebral malaria (CM) is a common form of severe malaria resulting in nearly 700,000 deaths each year in Africa alone. At present, there is no adjunctive therapy for CM. Although the mechanisms underlying the pathogenesis of CM are incompletely understood, it is likely that both intrinsic features of the parasite and the human host's immune response contribute to disease. The kinase mammalian target of rapamycin (mTOR) is a central regulator of immune responses, and drugs that inhibit the mTOR pathway have been shown to be antiparasitic. In a mouse model of CM, experimental CM (ECM), we show that the mTOR inhibitor rapamycin protects against ECM when administered within the first 4 days of infection. Treatment with rapamycin increased survival, blocked breakdown of the blood-brain barrier and brain hemorrhaging, decreased the influx of both CD4+ and CD8+ T cells into the brain and the accumulation of parasitized red blood cells in the brain. Rapamycin induced marked transcriptional changes in the brains of infected mice, and analysis of transcription profiles predicted that rapamycin blocked leukocyte trafficking to and proliferation in the brain. Remarkably, animals were protected against ECM even though rapamycin treatment significantly increased the inflammatory response induced by infection in both the brain and spleen. These results open a new avenue for the development of highly selective adjunctive therapies for CM by targeting pathways that regulate host and parasite metabolism. IMPORTANCE Malaria is a highly prevalent infectious disease caused by parasites of several Plasmodium spp. Malaria is usually uncomplicated and resolves with time; however, in about 1% of cases, almost exclusively among young children, malaria becomes severe and life threatening, resulting in nearly 700,000 deaths each year in Africa alone. Among the most severe complications of Plasmodium falciparum infection is cerebral malaria with a fatality rate of 15 to 20%, despite treatment with antimalarial drugs. Cerebral malaria takes a second toll on African children, leaving survivors at high risk of debilitating neurological defects. At present, we have no effective adjunctive therapies for cerebral malaria, and developing such therapies would have a large impact on saving young lives in Africa. Here we report results that open a new avenue for the development of highly selective adjunctive therapies for cerebral malaria by targeting pathways that regulate host and parasite metabolism.

Protective antibodies in Plasmodium falciparum malaria are only acquired after years of repeated infections. Chronic malaria exposure is associated with a large increase in atypical memory B cells (MBCs) that resemble B cells expanded in a variety of persistent viral infections. Understanding the function of atypical MBCs and their relationship to classical MBCs will be critical to developing effective vaccines for malaria and other chronic infections. We show that VH gene repertoires and somatic hypermutation rates of atypical and classical MBCs are indistinguishable indicating a common developmental history. Atypical MBCs express an array of inhibitory receptors and B cell receptor (BCR) signaling is stunted in atypical MBCs resulting in impaired B cell responses including proliferation, cytokine production and antibody secretion. Thus, in response to chronic malaria exposure, atypical MBCs appear to differentiate from classical MBCs becoming refractory to BCR-mediated activation and potentially interfering with the acquisition of malaria immunity.

Modified herpes simplex viruses that are unable to produce glycoprotein D may make effective vaccines.