RAIN: machine learning-based identification for HIV-1 bNAbs.

Broadly neutralizing antibodies (bNAbs) are promising candidates for the treatment and prevention of HIV-1 infections. Despite their critical importance, automatic detection of HIV-1 bNAbs from immune repertoires is still lacking. Here, we develop a straightforward computational method for the Rapid Automatic Identification of bNAbs (RAIN) based on machine learning methods. In contrast to other approaches, which use one-hot encoding amino acid sequences or structural alignment for prediction, RAIN uses a combination of selected sequence-based features for the accurate prediction of HIV-1 bNAbs. We demonstrate the performance of our approach on non-biased, experimentally obtained and sequenced BCR repertoires from HIV-1 immune donors. RAIN processing leads to the successful identification of distinct HIV-1 bNAbs targeting the CD4-binding site of the envelope glycoprotein. In addition, we validate the identified bNAbs using an in vitro neutralization assay and we solve the structure of one of them in complex with the soluble native-like heterotrimeric envelope glycoprotein by single-particle cryo-electron microscopy (cryo-EM). Overall, we propose a method to facilitate and accelerate HIV-1 bNAbs discovery from non-selected immune repertoires.

Safety and protective efficacy of PfSPZ Vaccine administered to HIV-negative and -positive Tanzanian adults.

BACKGROUNDSanaria PfSPZ Vaccine, composed of attenuated Plasmodium falciparum (Pf) sporozoites (SPZ), protects against malaria. We conducted this clinical trial to assess the safety and efficacy of PfSPZ Vaccine in HIV-positive (HIV+) individuals, since the HIV-infection status of participants in mass vaccination programs may be unknown.METHODSThis randomized, double-blind, placebo-controlled trial enrolled 18- to 45-year-old HIV-negative (HIV-) and well-controlled HIV+ Tanzanians (HIV viral load <40 copies/mL, CD4 counts >500 cells/µL). Participants received 5 doses of PfSPZ Vaccine or normal saline (NS) over 28 days, followed by controlled human malaria infection (CHMI) 3 weeks later.RESULTSThere were no solicited adverse events in the 9 HIV- and 12 HIV+ participants. After CHMI, 6 of 6 NS controls, 1 of 5 HIV- vaccinees, and 4 of 4 HIV+ vaccinees were Pf positive by quantitative PCR (qPCR). After immunization, anti-Pf circumsporozoite protein (anti-PfCSP) (isotype and IgG subclass) and anti-PfSPZ antibodies, anti-PfSPZ CD4+ T cell responses, and Vδ2+ γδ CD3+ T cells were nonsignificantly higher in HIV- than in HIV+ vaccinees. Sera from HIV- vaccinees had significantly higher inhibition of PfSPZ invasion of hepatocytes in vitro and antibody-dependent complement deposition (ADCD) and Fcγ3B binding by anti-PfCSP and ADCD by anti-cell-traversal protein for ookinetes and SPZ (anti-PfCelTOS) antibodies.CONCLUSIONSPfSPZ Vaccine was safe and well tolerated in HIV+ vaccinees, but not protective. Vaccine efficacy was 80% in HIV- vaccinees (P = 0.012), whose sera had significantly higher inhibition of PfSPZ invasion of hepatocytes and enrichment of multifunctional PfCSP antibodies. A more potent PfSPZ vaccine or regimen is needed to protect those living with HIV against Pf infection in Africa.TRIAL REGISTRATIONClinicalTrials.gov NCT03420053.FUNDINGEquatorial Guinea Malaria Vaccine Initiative (EGMVI), made up of the Government of Equatorial Guinea Ministries of Mines and Hydrocarbons, and Health and Social Welfare, Marathon Equatorial Guinea Production Limited, Noble Energy, Atlantic Methanol Production Company, and EG LNG; Swiss government, through ESKAS scholarship grant no. 2016.0056; Intramural Research Program of the National Institute of Allergy and Infectious Diseases, NIH; NIH grant 1U01AI155354-01.

Safety and Immunogenicity of Radiation-Attenuated PfSPZ Vaccine in Equatoguinean Infants, Children, and Adults.

The radiation-attenuated Plasmodium falciparum sporozoites (PfSPZ) Vaccine has demonstrated safety and immunogenicity in 5-month-old to 50-year-old Africans in multiple trials. Except for one, each trial has restricted enrollment to either infants and children or adults < 50 years old. This trial was conducted in Equatorial Guinea and assessed the safety, tolerability, and immunogenicity of three direct venous inoculations of 1.8 × 106 or 2.7 × 106 PfSPZ, of PfSPZ Vaccine, or normal saline administered at 8-week intervals in a randomized, double-blind, placebo-controlled trial stratified by age (6-11 months and 1-5, 6-10, 11-17, 18-35, and 36-61 years). All doses were successfully administered. In all, 192/207 injections (93%) in those aged 6-61 years were rated as causing no or mild pain. There were no significant differences in solicited adverse events (AEs) between vaccinees and controls in any age group (P ≥ 0.17). There were no significant differences between vaccinees and controls with respect to the rates or severity of unsolicited AEs or laboratory abnormalities. Development of antibodies to P. falciparum circumsporozoite protein occurred in 67/69 vaccinees (97%) and 0/15 controls. Median antibody levels were highest in infants and 1-5-year-olds and declined progressively with age. Antibody responses in children were greater than in adults protected against controlled human malaria infection. Robust immunogenicity, combined with a benign AE profile, indicates children are an ideal target for immunization with PfSPZ Vaccine.

Effects of Age, Gender and Soil-Transmitted Helminth Infection on Prevalence of Plasmodium Infection among Population Living in Bata District, Equatorial Guinea.

INTRODUCTION: Malaria and soil-transmitted helminth (STH) co-infection is an important parasitic infection affecting populations in co-endemic countries including Equatorial Guinea. To date, the health impact of STH and malaria co-infection is inconclusive. The current study aimed to report the malaria and STH infection epidemiology in the continental region of Equatorial Guinea. METHODS: We performed a cross-sectional study between October 2020 and January 2021 in the Bata district of Equatorial Guinea. Participants aged 1-9 years, 10-17 years and above 18 were recruited. Fresh venous blood was collected for malaria testing via mRDTs and light microscopy. Stool specimens were collected, and the Kato-Katz technique was used to detect the presence of Ascaris lumbricoides, Trichuris trichiura, hookworm spp. and intestinal Schistosoma eggs. RESULTS: A total of 402 participants were included in this study. An amount of 44.3% of them lived in urban areas, and only 51.9% of them reported having bed nets. Malaria infections were detected in 34.8% of the participants, while 50% of malaria infections were reported in children aged 10-17 years. Females had a lower prevalence of malaria (28.8%) compared with males (41.7%). Children of 1-9 years carried more gametocytes compared with other age groups. An amount of 49.3% of the participants infected with T. trichiura had malaria parasites compared with those infected with A. lumbricoides (39.6%) or both (46.8%). CONCLUSIONS: The overlapping problem of STH and malaria is neglected in Bata. The current study forces the government and other stakeholders involved in the fight against malaria and STH to consider a combined control program strategy for both parasitic infections in Equatorial Guinea.

Field evaluation of the novel One Step Malaria Pf and Pf/Pv rapid diagnostic tests and the proportion of HRP-2 gene deletion identified on samples collected in the Pwani region, Tanzania.

Background: Malaria rapid diagnostic tests (mRDTs) have played an important role in the early detection of clinical malaria in an endemic area. While several mRDTs are currently on the market, the availability of mRDTs with high sensitivity and specificity will merit the fight against malaria. We evaluated the field performance of a novel One Step Malaria (P.f/P.v) Tri-line and One Step Malaria (P.f) rapid test kits in Pwani, Tanzania. Methods: In a cross-sectional study conducted in Bagamoyo and Kibiti districts in Tanzania, symptomatic patients were tested using the SD BIOLINE, One Step Malaria (P.f/P.v) Tri-line and One Step Malaria (P.f) rapid test kits, microscope, and quantitative Polymerase Chain Reaction (qPCR). An additional qPCR assay was carried out to detect Histidine-Rich Protein 2 (HRP-2) gene deletion on mRDT negative but microscope and qPCR positive samples. Microscope results confirmed by qPCR were used for analysis, where qPCR was used as a reference method. Results: The sensitivity and specificity of One Step P.f/P.v Tri-line mRDTs were 96.0% (CI 93.5-97.7%) and 98.3% (CI 96.8-99.2%), respectively. One Step P.f mRDT had sensitivity and specificity of 95.2% (CI 92.5-97.1%) and 97.9% (CI 96.3-99.0%) respectively. Positive predictive value (PPV) was 97.6% (CI 95.4-98.7%) and negative predictive value (NPV) was 96.2% (CI 95.5-98.3%) for the One Step P.f/P.v Tri-line mRDTs respectively, while One Step P.f mRDT had positive predictive value (PPV) and negative predictive value (NPV) of 97.0% (CI 94.8-98.3%) and 96.7 (CI 94.9-97.9%) respectively. 9.8% (CI 7.84-11.76) of all samples tested and reported to be malaria-negative by mRDT had HRP-2 gene deletion. Conclusion: One Step Malaria P.f/P.v Tri-line and One Step Malaria P.f rapid test kits have similar sensitivity and specificity as the standard mRDT that is currently in the market, demonstrating the potential to contribute in the fight against malaria in endemic settings. However, the identified malaria parasites population with HRP-2 gene deletion pose a threat to the current mRDT usability in the field and warrants further investigations.

Understanding the role of serological and clinical data on assessing the dynamic of malaria transmission: a case study of Bagamoyo district, Tanzania.

Introduction: naturally acquired blood-stage malaria antibodies and malaria clinical data have been reported to be useful in monitoring malaria change over time and as a marker of malaria exposure. This study assessed the total immunoglobulin G (IgG) levels to Plasmodium falciparum schizont among infants (5-17 months), estimated malaria incidence using routine health facility-based surveillance data and predicted trend relation between anti-schizont antibodies and malaria incidence in Bagamoyo. Methods: 252 serum samples were used for assessment of total IgG by enzyme-linked immunosorbent assay and results were expressed in arbitrary units (AU). 147/252 samples were collected in 2021 during a blood-stage malaria vaccine trial [ClinicalTrials.gov NCT04318002], and 105/252 were archived samples of malaria vaccine trial conducted in 2012 [ClinicalTrials.gov NCT00866619]. Malaria incidence was calculated from outpatient clinic data of malaria rapid test or blood smear positive results retrieved from District-Health-Information-Software-2 (DHIS2) between 2013 and 2020. Cross-sectional data from both studies were analysed using STATA version 14. Results: this study demonstrated a decline in total anti-schizont IgG levels from 490.21AU in 2012 to 97.07AU in 2021 which was related to a fall in incidence from 58.25 cases/1000 person-year in 2013 to 14.28 cases/1000 person-year in 2020. We also observed a significant difference in incidence when comparing high and low malaria transmission areas and by gender. However, we did not observe differences when comparing total anti-schizont antibodies by gender and study year. Conclusion: total anti-schizont antibody levels appear to be an important serological marker of exposure for assessing the dynamic of malaria transmission in infants living in malaria-endemic regions.

Increased levels of anti-PfCSP antibodies in post-pubertal females versus males immunized with PfSPZ Vaccine does not translate into increased protective efficacy.

Background: While prior research has shown differences in the risk of malaria infection and sickness between males and females, little is known about sex differences in vaccine-induced immunity to malaria. Identifying such differences could elucidate important aspects of malaria biology and facilitate development of improved approaches to malaria vaccination. Methods: Using a standardized enzyme-linked immunosorbent assay, IgG antibodies to the major surface protein on Plasmodium falciparum (Pf) sporozoites (SPZ), the Pf circumsporozoite protein (PfCSP), were measured before and two weeks after administration of a PfSPZ-based malaria vaccine (PfSPZ Vaccine) to 5-month to 61-year-olds in 11 clinical trials in Germany, the US and five countries in Africa, to determine if there were differences in vaccine elicited antibody response between males and females and if these differences were associated with differential protection against naturally transmitted Pf malaria (Africa) or controlled human malaria infection (Germany, the US and Africa). Results: Females ≥ 11 years of age made significantly higher levels of antibodies to PfCSP than did males in most trials, while there was no indication of such differences in infants or children. Although adult females had higher levels of antibodies, there was no evidence of improved protection compared to males. In 2 of the 7 trials with sufficient data, protected males had significantly higher levels of antibodies than unprotected males, and in 3 other trials protected females had higher levels of antibodies than did unprotected females. Conclusion: Immunization with PfSPZ Vaccine induced higher levels of antibodies in post-pubertal females but showed equivalent protection in males and females. We conclude that the increased antibody levels in post-pubertal females did not contribute substantially to improved protection. We hypothesize that while antibodies to PfCSP (and PfSPZ) may potentially contribute directly to protection, they primarily correlate with other, potentially protective immune mechanisms, such as antibody dependent and antibody independent cellular responses in the liver.

Diagnostic performance and comparison of ultrasensitive and conventional rapid diagnostic test, thick blood smear and quantitative PCR for detection of low-density Plasmodium falciparum infections during a controlled human malaria infection study in Equatorial Guinea.

BACKGROUND: Progress towards malaria elimination has stagnated, partly because infections persisting at low parasite densities comprise a large reservoir contributing to ongoing malaria transmission and are difficult to detect. This study compared the performance of an ultrasensitive rapid diagnostic test (uRDT) designed to detect low density infections to a conventional RDT (cRDT), expert microscopy using Giemsa-stained thick blood smears (TBS), and quantitative polymerase chain reaction (qPCR) during a controlled human malaria infection (CHMI) study conducted in malaria exposed adults (NCT03590340). METHODS: Blood samples were collected from healthy Equatoguineans aged 18-35 years beginning on day 8 after CHMI with 3.2 × 103 cryopreserved, infectious Plasmodium falciparum sporozoites (PfSPZ Challenge, strain NF54) administered by direct venous inoculation. qPCR (18s ribosomal DNA), uRDT (Alere™ Malaria Ag P.f.), cRDT [Carestart Malaria Pf/PAN (PfHRP2/pLDH)], and TBS were performed daily until the volunteer became TBS positive and treatment was administered. qPCR was the reference for the presence of Plasmodium falciparum parasites. RESULTS: 279 samples were collected from 24 participants; 123 were positive by qPCR. TBS detected 24/123 (19.5% sensitivity [95% CI 13.1-27.8%]), uRDT 21/123 (17.1% sensitivity [95% CI 11.1-25.1%]), cRDT 10/123 (8.1% sensitivity [95% CI 4.2-14.8%]); all were 100% specific and did not detect any positive samples not detected by qPCR. TBS and uRDT were more sensitive than cRDT (TBS vs. cRDT p = 0.015; uRDT vs. cRDT p = 0.053), detecting parasitaemias as low as 3.7 parasites/µL (p/µL) (TBS and uRDT) compared to 5.6 p/µL (cRDT) based on TBS density measurements. TBS, uRDT and cRDT did not detect any of the 70/123 samples positive by qPCR below 5.86 p/µL, the qPCR density corresponding to 3.7 p/µL by TBS. The median prepatent periods in days (ranges) were 14.5 (10-20), 18.0 (15-28), 18.0 (15-20) and 18.0 (16-24) for qPCR, TBS, uRDT and cRDT, respectively; qPCR detected parasitaemia significantly earlier (3.5 days) than the other tests. CONCLUSIONS: TBS and uRDT had similar sensitivities, both were more sensitive than cRDT, and neither matched qPCR for detecting low density parasitaemia. uRDT could be considered an alternative to TBS in selected applications, such as CHMI or field diagnosis, where qualitative, dichotomous results for malaria infection might be sufficient.

Multi-Dose Priming Regimens of PfSPZ Vaccine: Safety and Efficacy against Controlled Human Malaria Infection in Equatoguinean Adults.

Plasmodium falciparum sporozoites (PfSPZ) Vaccine is composed of radiation-attenuated, aseptic, purified cryopreserved PfSPZ. Multiple clinical trials empirically assessing two to six doses have shown multi-dose priming (-two to four doses the first week) to be optimal for protection in both 4- and 16-week regimens. In this randomized, double-blind, normal saline (NS), placebo-controlled trial, four groups (G) of 18- to 32-year-old Equatoguineans received multi-dose priming regimens with or without a delayed final dose at 4 or 16 weeks (9 × 105 PfSPZ/dose). The regimens were G1: days 1, 3, 5, 7, and 113; G2: days 1, 3, 5, and 7; G3: days 1, 3, 5, 7, and 29; and G4: days 1, 8, and 29). All doses were 9 × 105 PfSPZ. Tolerability, safety, immunogenicity, and vaccine efficacy (VE) against homologous-controlled human malaria infection (CHMI) 6-7 weeks after vaccination were assessed to down-select the best regimen. All four regimens were safe and well tolerated, with no significant differences in adverse events (AEs) between vaccinees (N = 84) and NS controls (N = 20) or between regimens. Out of 19 controls, 13 developed Pf parasitemia by quantitative polymerase chain reaction (qPCR) after CHMI. Only the vaccine regimen administered on study days 1, 8, and 29 gave significant protection (7/21 vaccinees versus 13/19 controls infected, VE 51.3%, P = 0.03, Barnard's test, two-tailed). There were no significant differences in antibodies against Pf circumporozoite protein (PfCSP), a major SPZ antigen, between protected and nonprotected vaccinees or controls pre-CHMI. The six controls not developing Pf parasitemia had significantly higher antibodies to blood stage antigens Pf exported protein 1 (PfEXP1) and Pf merozoite surface protein 1 (PfMSP1) than the controls who developed parasitemia, suggesting naturally acquired immunity against Pf-limited infections in controls. This study identified a safe, protective, 4-week, multi-dose prime vaccination regimen for assessment in future trials of PfSPZ Vaccine.

Analysis of nucleic acids extracted from rapid diagnostic tests reveals a significant proportion of false positive test results associated with recent malaria treatment.

BACKGROUND: Surveillance programmes often use malaria rapid diagnostic tests (RDTs) to determine the proportion of the population carrying parasites in their peripheral blood to assess the malaria transmission intensity. Despite an increasing number of reports on false-negative and false-positive RDT results, there is a lack of systematic quality control activities for RDTs deployed in malaria surveillance programmes. METHODS: The diagnostic performance of field-deployed RDTs used for malaria surveys was assessed by retrospective molecular analysis of the blood retained on the tests. RESULTS: Of the 2865 RDTs that were collected in 2018 on Bioko Island and analysed in this study, 4.7% had a false-negative result. These false-negative RDTs were associated with low parasite density infections. In 16.6% of analysed samples, masked pfhrp2 and pfhrp3 gene deletions were identified, in which at least one Plasmodium falciparum strain carried a gene deletion. Among all positive RDTs analysed, 28.4% were tested negative by qPCR and therefore considered to be false-positive. Analysing the questionnaire data collected from the participants, this high proportion of false-positive RDTs could be explained by P. falciparum histidine rich protein 2 (PfHRP2) antigen persistence after recent malaria treatment. CONCLUSION: Malaria surveillance depending solely on RDTs needs well-integrated quality control procedures to assess the extent and impact of reduced sensitivity and specificity of RDTs on malaria control programmes.

Transcriptional correlates of malaria in RTS,S/AS01-vaccinated African children: a matched case-control study.

Background: In a phase 3 trial in African infants and children, the RTS,S/AS01 vaccine (GSK) showed moderate efficacy against clinical malaria. We sought to further understand RTS,S/AS01-induced immune responses associated with vaccine protection. Methods: Applying the blood transcriptional module (BTM) framework, we characterized the transcriptomic response to RTS,S/AS01 vaccination in antigen-stimulated (and vehicle control) peripheral blood mononuclear cells sampled from a subset of trial participants at baseline and month 3 (1-month post-third dose). Using a matched case-control study design, we evaluated which of these 'RTS,S/AS01 signature BTMs' associated with malaria case status in RTS,S/AS01 vaccinees. Antigen-specific T-cell responses were analyzed by flow cytometry. We also performed a cross-study correlates analysis where we assessed the generalizability of our findings across three controlled human malaria infection studies of healthy, malaria-naive adult RTS,S/AS01 recipients. Results: RTS,S/AS01 vaccination was associated with downregulation of B-cell and monocyte-related BTMs and upregulation of T-cell-related BTMs, as well as higher month 3 (vs. baseline) circumsporozoite protein-specific CD4+ T-cell responses. There were few RTS,S/AS01-associated BTMs whose month 3 levels correlated with malaria risk. In contrast, baseline levels of BTMs associated with dendritic cells and with monocytes (among others) correlated with malaria risk. The baseline dendritic cell- and monocyte-related BTM correlations with malaria risk appeared to generalize to healthy, malaria-naive adults. Conclusions: A prevaccination transcriptomic signature associates with malaria in RTS,S/AS01-vaccinated African children, and elements of this signature may be broadly generalizable. The consistent presence of monocyte-related modules suggests that certain monocyte subsets may inhibit protective RTS,S/AS01-induced responses. Funding: Funding was obtained from the NIH-NIAID (R01AI095789), NIH-NIAID (U19AI128914), PATH Malaria Vaccine Initiative (MVI), and Ministerio de Economía y Competitividad (Instituto de Salud Carlos III, PI11/00423 and PI14/01422). The RNA-seq project has been funded in whole or in part with Federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under grant number U19AI110818 to the Broad Institute. This study was also supported by the Vaccine Statistical Support (Bill and Melinda Gates Foundation award INV-008576/OPP1154739 to R.G.). C.D. was the recipient of a Ramon y Cajal Contract from the Ministerio de Economía y Competitividad (RYC-2008-02631). G.M. was the recipient of a Sara Borrell-ISCIII fellowship (CD010/00156) and work was performed with the support of Department of Health, Catalan Government grant (SLT006/17/00109). This research is part of the ISGlobal's Program on the Molecular Mechanisms of Malaria which is partially supported by the Fundación Ramón Areces and we acknowledge support from the Spanish Ministry of Science and Innovation through the 'Centro de Excelencia Severo Ochoa 2019-2023' Program (CEX2018-000806-S), and support from the Generalitat de Catalunya through the CERCA Program.

Rapid Identification of SARS-CoV-2 Variants of Concern Using a Portable peakPCR Platform.

The need for tools that facilitate rapid detection and continuous monitoring of SARS-CoV-2 variants of concern (VOCs) is greater than ever, as these variants are more transmissible and therefore increase the pressure of COVID-19 on healthcare systems. To address this demand, we aimed at developing and evaluating a robust and fast diagnostic approach for the identification of SARS-CoV-2 VOC-associated spike gene mutations. Our diagnostic assays detect the E484K and N501Y single-nucleotide polymorphisms (SNPs) as well as a spike gene deletion (HV69/70) and can be run on standard laboratory equipment or on the portable rapid diagnostic technology platform peakPCR. The assays achieved excellent diagnostic performance when tested with RNA extracted from culture-derived SARS-CoV-2 VOC lineages and clinical samples collected in Equatorial Guinea, Central-West Africa. Simplicity of usage and the relatively low cost are advantages that make our approach well suitable for decentralized and rapid testing, especially in resource-limited settings.

Safety, immunogenicity and efficacy of PfSPZ Vaccine against malaria in infants in western Kenya: a double-blind, randomized, placebo-controlled phase 2 trial.

The radiation-attenuated Plasmodium falciparum sporozoite (PfSPZ) vaccine provides protection against P. falciparum infection in malaria-naïve adults. Preclinical studies show that T cell-mediated immunity is required for protection and is readily induced in humans after vaccination. However, previous malaria exposure can limit immune responses and vaccine efficacy (VE) in adults. We hypothesized that infants with less previous exposure to malaria would have improved immunity and protection. We conducted a multi-arm, randomized, double-blind, placebo-controlled trial in 336 infants aged 5-12 months to determine the safety, tolerability, immunogenicity and efficacy of the PfSPZ Vaccine in infants in a high-transmission malaria setting in western Kenya ( NCT02687373 ). Groups of 84 infants each received 4.5 × 105, 9.0 × 105 or 1.8 × 106 PfSPZ Vaccine or saline three times at 8-week intervals. The vaccine was well tolerated; 52 (20.6%) children in the vaccine groups and 20 (23.8%) in the placebo group experienced related solicited adverse events (AEs) within 28 d postvaccination and most were mild. There was 1 grade 3-related solicited AE in the vaccine group (0.4%) and 2 in the placebo group (2.4%). Seizures were more common in the highest-dose group (14.3%) compared to 6.0% of controls, with most being attributed to malaria. There was no significant protection against P. falciparum infection in any dose group at 6 months (VE in the 9.0 × 105 dose group = -6.5%, P = 0.598, the primary statistical end point of the study). VE against clinical malaria 3 months after the last dose in the highest-dose group was 45.8% (P = 0.027), an exploratory end point. There was a dose-dependent increase in antibody responses that correlated with VE at 6 months in the lowest- and highest-dose groups. T cell responses were undetectable across all dose groups. Detection of Vδ2+Vγ9+ T cells, which have been correlated with induction of PfSPZ Vaccine T cell immunity and protection in adults, were infrequent. These data suggest that PfSPZ Vaccine-induced T cell immunity is age-dependent and may be influenced by Vδ2+Vγ9+ T cell frequency. Since there was no significant VE at 6 months in these infants, these vaccine regimens will likely not be pursued further in this age group.

Incidence of Plasmodium falciparum malaria infection in 6-month to 45-year-olds on selected areas of Bioko Island, Equatorial Guinea.

BACKGROUND: Extensive malaria control measures have been implemented on Bioko Island, Equatorial Guinea over the past 16 years, reducing parasite prevalence and malaria-related morbidity and mortality, but without achieving elimination. Malaria vaccines offer hope for reducing the burden to zero. Three phase 1/2 studies have been conducted successfully on Bioko Island to evaluate the safety and efficacy of whole Plasmodium falciparum (Pf) sporozoite (SPZ) malaria vaccines. A large, pivotal trial of the safety and efficacy of the radiation-attenuated Sanaria® PfSPZ Vaccine against P. falciparum is planned for 2022. This study assessed the incidence of malaria at the phase 3 study site and characterized the influence of socio-demographic factors on the burden of malaria to guide trial design. METHODS: A cohort of 240 randomly selected individuals aged 6 months to 45 years from selected areas of North Bioko Province, Bioko Island, was followed for 24 weeks after clearance of parasitaemia. Assessment of clinical presentation consistent with malaria and thick blood smears were performed every 2 weeks. Incidence of first and multiple malaria infections per person-time of follow-up was estimated, compared between age groups, and examined for associated socio-demographic risk factors. RESULTS: There were 58 malaria infection episodes observed during the follow up period, including 47 first and 11 repeat infections. The incidence of malaria was 0.25 [95% CI (0.19, 0.32)] and of first malaria was 0.23 [95% CI (0.17, 0.30)] per person per 24 weeks (0.22 in 6-59-month-olds, 0.26 in 5-17-year-olds, 0.20 in 18-45-year-olds). Incidence of first malaria with symptoms was 0.13 [95% CI (0.09, 0.19)] per person per 24 weeks (0.16 in 6-59-month-olds, 0.10 in 5-17-year-olds, 0.11 in 18-45-year-olds). Multivariate assessment showed that study area, gender, malaria positivity at screening, and household socioeconomic status independently predicted the observed incidence of malaria. CONCLUSION: Despite intensive malaria control efforts on Bioko Island, local transmission remains and is spread evenly throughout age groups. These incidence rates indicate moderate malaria transmission which may be sufficient to support future larger trials of PfSPZ Vaccine. The long-term goal is to conduct mass vaccination programmes to halt transmission and eliminate P. falciparum malaria.

Early whole blood transcriptional responses to radiation-attenuated Plasmodium falciparum sporozoite vaccination in malaria naïve and malaria pre-exposed adult volunteers.

BACKGROUND: Vaccination with radiation-attenuated Plasmodium falciparum sporozoites is known to induce protective immunity. However, the mechanisms underlying this protection remain unclear. In this work, two recent radiation-attenuated sporozoite vaccination studies were used to identify potential transcriptional correlates of vaccination-induced protection. METHODS: Longitudinal whole blood RNAseq transcriptome responses to immunization with radiation-attenuated P. falciparum sporozoites were analysed and compared across malaria-naïve adult participants (IMRAS) and malaria-experienced adult participants (BSPZV1). Parasite dose and method of delivery differed between trials, and immunization regimens were designed to achieve incomplete protective efficacy. Observed protective efficacy was 55% in IMRAS and 20% in BSPZV1. Study vaccine dosings were chosen to elicit both protected and non-protected subjects, so that protection-associated responses could be identified. RESULTS: Analysis of comparable time points up to 1 week after the first vaccination revealed a shared cross-study transcriptional response programme, despite large differences in number and magnitude of differentially expressed genes between trials. A time-dependent regulatory programme of coherent blood transcriptional modular responses was observed, involving induction of inflammatory responses 1-3 days post-vaccination, with cell cycle responses apparent by day 7 in protected individuals from both trials. Additionally, strongly increased induction of inflammation and interferon-associated responses was seen in non-protected IMRAS participants. All individuals, except for non-protected BSPZV1 participants, showed robust upregulation of cell-cycle associated transcriptional responses post vaccination. CONCLUSIONS: In summary, despite stark differences between the two studies, including route of vaccination and status of malaria exposure, responses were identified that were associated with protection after PfRAS vaccination. These comprised a moderate early interferon response peaking 2 days post vaccination, followed by a later proliferative cell cycle response steadily increasing over the first 7 days post vaccination. Non-protection is associated with deviations from this model, observed in this study with over-induction of early interferon responses in IMRAS and failure to mount a cell cycle response in BSPZV1.

Genomic Surveillance Enables the Identification of Co-infections With Multiple SARS-CoV-2 Lineages in Equatorial Guinea.

COVID-19 disease caused by SARS-CoV-2 represents an ongoing global public health emergency. Rapid identification of emergence, evolution, and spread of SARS-CoV-2 variants of concern (VOC) would enable timely and tailored responses by public health decision-making bodies. Yet, global disparities in current SARS-CoV-2 genomic surveillance activities reveal serious geographical gaps. Here, we discuss the experiences and lessons learned from the SARS-CoV-2 monitoring and surveillance program at the Public Health Laboratory on Bioko Island, Equatorial Guinea that was implemented as part of the national COVID-19 response and monitoring activities. We report how three distinct SARS-CoV-2 variants have dominated the epidemiological situation in Equatorial Guinea since March 2020. In addition, a case of co-infection of two SARS-CoV-2 VOC, Beta and Delta, in a clinically asymptomatic and fully COVID-19 vaccinated man living in Equatorial Guinea is presented. To our knowledge, this is the first report of a person co-infected with Beta and Delta VOC globally. Rapid identification of co-infections is relevant since these might provide an opportunity for genetic recombination resulting in emergence of novel SARS-CoV-2 lineages with enhanced transmission or immune evasion potential.

Molecular malaria surveillance using a novel protocol for extraction and analysis of nucleic acids retained on used rapid diagnostic tests.

The use of malaria rapid diagnostic tests (RDTs) as a source for nucleic acids that can be analyzed via nucleic acid amplification techniques has several advantages, including minimal amounts of blood, sample collection, simplified storage and shipping conditions at room temperature. We have systematically developed and extensively evaluated a procedure to extract total nucleic acids from used malaria RDTs. The co-extraction of DNA and RNA molecules from small volumes of dried blood retained on the RDTs allows detection and quantification of P. falciparum parasites from asymptomatic patients with parasite densities as low as 1 Pf/µL blood using reverse transcription quantitative PCR. Based on the extraction protocol we have developed the ENAR (Extraction of Nucleic Acids from RDTs) approach; a complete workflow for large-scale molecular malaria surveillance. Using RDTs collected during a malaria indicator survey we demonstrated that ENAR provides a powerful tool to analyze nucleic acids from thousands of RDTs in a standardized and high-throughput manner. We found several, known and new, non-synonymous single nucleotide polymorphisms in the propeller region of the kelch 13 gene among isolates circulating on Bioko Island, Equatorial Guinea.

Immune system development varies according to age, location, and anemia in African children.

Children from low- and middle-income countries, where there is a high incidence of infectious disease, have the greatest need for the protection afforded by vaccination, but vaccines often show reduced efficacy in these populations. An improved understanding of how age, infection, nutrition, and genetics influence immune ontogeny and function is key to informing vaccine design for this at-risk population. We sought to identify factors that shape immune development in children under 5 years of age from Tanzania and Mozambique by detailed immunophenotyping of longitudinal blood samples collected during the RTS,S malaria vaccine phase 3 trial. In these cohorts, the composition of the immune system is dynamically transformed during the first years of life, and this was further influenced by geographical location, with some immune cell types showing an altered rate of development in Tanzanian children compared to Dutch children enrolled in the Generation R population-based cohort study. High-titer antibody responses to the RTS,S/AS01E vaccine were associated with an activated immune profile at the time of vaccination, including an increased frequency of antibody-secreting plasmablasts and follicular helper T cells. Anemic children had lower frequencies of recent thymic emigrant T cells, isotype-switched memory B cells, and plasmablasts; modulating iron bioavailability in vitro could recapitulate the B cell defects observed in anemic children. Our findings demonstrate that the composition of the immune system in children varies according to age, geographical location, and anemia status.

Whole blood transcriptome changes following controlled human malaria infection in malaria pre-exposed volunteers correlate with parasite prepatent period.

Malaria continues to be one of mankind's most devastating diseases despite the many and varied efforts to combat it. Indispensable for malaria elimination and eventual eradication is the development of effective vaccines. Controlled human malaria infection (CHMI) is an invaluable tool for vaccine efficacy assessment and investigation of early immunological and molecular responses against Plasmodium falciparum infection. Here, we investigated gene expression changes following CHMI using RNA-Seq. Peripheral blood samples were collected in Bagamoyo, Tanzania, from ten adults who were injected intradermally (ID) with 2.5x104 aseptic, purified, cryopreserved P. falciparum sporozoites (Sanaria® PfSPZ Challenge). A total of 2,758 genes were identified as differentially expressed following CHMI. Transcriptional changes were most pronounced on day 5 after inoculation, during the clinically silent liver phase. A secondary analysis, grouping the volunteers according to their prepatent period duration, identified 265 genes whose expression levels were linked to time of blood stage parasitemia detection. Gene modules associated with these 265 genes were linked to regulation of transcription, cell cycle, phosphatidylinositol signaling and erythrocyte development. Our study showed that in malaria pre-exposed volunteers, parasite prepatent period in each individual is linked to magnitude and timing of early gene expression changes after ID CHMI.

drLumi: An open-source package to manage data, calibrate, and conduct quality control of multiplex bead-based immunoassays data analysis.

Multiplex bead-based immunoassays are used to measure concentrations of several analytes simultaneously. These assays include control standard curves (SC) to reduce between-plate variability and normalize quantitation of analytes of biological samples. Suboptimal calibration might result in large random error and decreased number of samples with analyte concentrations within the limits of quantification. Suboptimal calibration may be a consequence of poor fitness of the functions used for the SC, the treatment of the background noise and the method used to estimate the limits of quantification. Currently assessment of fitness of curves is largely dependent on operator and that may add additional error. Moreover, there is no software to automate data managing and quality control. In this article we present a R package, drLumi, with functions for managing data, calibrating assays and performing quality control. To optimize the assay the package implements: i) three dose-response functions, ii) four approaches for treating background noise and iii) three methods for estimating limits of quantifications. Other implemented functions are focused on the quality control of the fitted standard curve: detection of outliers, estimation of the confidence or prediction interval, and estimation of summary statistics. With demonstration purpose, we apply the software to 30 cytokines, chemokines and growth factors measured in a multiplex bead-based immunoassay in a study aiming to measure correlates of risk or protection from malaria of the RTS,S malaria vaccine nested in the Phase 3 randomized controlled trial of this vaccine.