Recombinant Full-length Plasmodium falciparum Circumsporozoite Protein-Based Vaccine Adjuvanted With Glucopyranosyl Lipid A-Liposome Quillaja saponaria 21: Results of Phase 1 Testing With Malaria Challenge.

BACKGROUND: Malaria is preventable yet causes >600 000 deaths annually. RTS,S, the first marketed malaria vaccine, has modest efficacy, but improvements are needed for eradication. METHODS: We conducted an open-label, dose escalation phase 1 study of a full-length recombinant circumsporozoite protein vaccine (rCSP) administered with adjuvant glucopyranosyl lipid A-liposome Quillaja saponaria 21 formulation (GLA-LSQ) on days 1, 29, and 85 or 1 and 490 to healthy, malaria-naive adults. The primary end points were safety and reactogenicity. The secondary end points were antibody responses and Plasmodium falciparum parasitemia after homologous controlled human malaria infection. RESULTS: Participants were enrolled into 4 groups receiving rCSP/GLA-LSQ: 10 µg × 3 (n = 20), 30 µg × 3 (n = 10), 60 µg × 3 (n = 10), or 60 µg × 2 (n = 9); 10 participants received 30 µg rCSP alone × 3, and there were 6 infectivity controls. Participants experienced no serious adverse events. Rates of solicited and unsolicited adverse events were similar among groups. All 26 participants who underwent controlled human malaria infection 28 days after final vaccinations developed malaria. Increasing vaccine doses induced higher immunoglobulin G titers but did not achieve previously established RTS,S benchmarks. CONCLUSIONS: rCSP/GLA-LSQ had favorable safety results. However, tested regimens did not induce protective immunity. Further investigation could assess whether adjuvant or schedule adjustments improve efficacy. CLINICAL TRIALS REGISTRATION: NCT03589794.

Coronavirus Disease 2019 Burden Among Unaccompanied Minors in US Custody.

BACKGROUND: Before the coronavirus disease 2019 (COVID-19) pandemic, crowded and unsanitary living conditions lacking medical expertise made US detention centers hotbeds for infectious disease outbreaks. There have been 30 000 COVID-19 cases, positivity rates exceeding 50%, and 9 deaths in Immigration and Customs Enforcement custody, but the extent of disease among children under the care of the Office of Refugee Resettlement (ORR) has not been well-documented. We sought to evaluate the burden of COVID-19 among unaccompanied minors under the ORR's responsibility. METHODS: We analyzed SARS-CoV-2 testing results of refugees and asylum seekers in facilities associated with the ORR from February 1, 2020, to November 18, 2020, courtesy of a Freedom of Information Act request. RESULTS: ORR facilities performed 7132 SARS-CoV-2 tests from March 13, 2020, to November 18, 2020. Overall, the SARS-CoV-2 positivity rate was 13.4%. Factors associated with higher positivity rates were age group (16-17 years old); identifying as male; undergoing testing in April, August, or September; staying in a for-profit versus a nonprofit facility; and detention in certain facilities. The mean detention time with a positive test was 14.8 ± 3.2 days. Greater than 10% of positive tests were in long-term detainees. CONCLUSIONS: The high SARS-CoV-2 test positivity rate raises concerns about an inability to limit the spread of SARS-CoV-2 within detention facilities housing unaccompanied migrant children, particularly those run by for-profit companies. Mandated measures for social distancing and vaccination among detainees and detention facility employees are needed to limit the spread of the virus.

STRIDE: a command-line HMM-based identifier and sub-classifier of Plasmodium falciparum RIFIN and STEVOR variant surface antigen families.

BACKGROUND: RIFINs and STEVORs are variant surface antigens expressed by P. falciparum that play roles in severe malaria pathogenesis and immune evasion. These two highly diverse multigene families feature multiple paralogs, making their classification challenging using traditional bioinformatic methods. RESULTS: STRIDE (STevor and RIfin iDEntifier) is an HMM-based, command-line program that automates the identification and classification of RIFIN and STEVOR protein sequences in the malaria parasite Plasmodium falciparum. STRIDE is more sensitive in detecting RIFINs and STEVORs than available PFAM and TIGRFAM tools and reports RIFIN subtypes and the number of sequences with a FHEYDER amino acid motif, which has been associated with severe malaria pathogenesis. CONCLUSIONS: STRIDE will be beneficial to malaria research groups analyzing genome sequences and transcripts of clinical field isolates, providing insight into parasite biology and virulence.

Epitope-Specific Antibody Responses to a Plasmodium falciparum Subunit Vaccine Target in a Malaria-Endemic Population.

Circumsporozoite protein (CSP) coats the Plasmodium falciparum sporozoite surface and is a major malaria subunit vaccine target. We measured epitope-specific reactivity to field-derived CSP haplotypes in serum samples from Malian adults and children on a custom peptide microarray. Compared to children, adults showed greater antibody responses and responses to more variants in regions proximal to and within the central repeat region. Children acquired short-lived immunity to an epitope proximal to the central repeat region but not to the central repeat region itself. This approach has the potential to differentiate immunodominant from protective epitope-specific responses when combined with longitudinal infection data.

COVID-19 Outbreaks in US Immigrant Detention Centers: The Urgent Need to Adopt CDC Guidelines for Prevention and Evaluation.

There have been several significant outbreaks of COVID-19 in federal immigrant detention centers, which lack clear and consistent guidelines across Department of Homeland Security (DHS) agencies to limit the spread of COVID-19. The Centers for Disease Control and Prevention (CDC) has issued detailed guidelines for the control, prevention, and evaluation of COVID-19 in detention facilities. Although the DHS's Immigration and Customs Enforcement agency has stated that it complies with CDC recommendations, its policies significantly differ from these CDC guidelines, placing detainees at risk for contracting COVID-19. This submission urges the adoption of CDC guidelines across DHS-associated facilities. Such a policy change has the potential to protect and save the lives of the most vulnerable populations under the auspices of the federal government.

A "Natural Death": The Political Battlefield of Infections and Migrant Children's Bodies.

Before September 2018, no child had died in United States Border Patrol custody in a decade. Since then, 7 detained children have died in the past 10 months. Migrant children's bodies have become the latest political battlefield, and these children have been caught in the crossfire. This piece focuses on the recent deaths of several migrant children from overwhelming infections in United States detainment centers. The circumstances surrounding these illnesses bring to the fore concerns about the care of these children, suggesting infectious disease outbreaks in these detainment centers, delays in bringing children to medical attention, and inadequate medical expertise in their care. There is an urgent need for advocacy by clinicians and professional societies to resolve this crisis.

Dose-Dependent Infectivity of Aseptic, Purified, Cryopreserved Plasmodium falciparum 7G8 Sporozoites in Malaria-Naive Adults.

Direct venous inoculation of 3.2 × 103 aseptic, purified, cryopreserved, vialed Plasmodium falciparum (Pf) strain NF54 sporozoites, PfSPZ Challenge (NF54), has been used for controlled human malaria infection (CHMI) in the United States, 4 European countries, and 6 African countries. In nonimmune adults, this results in 100% infection rates. We conducted a double-blind, randomized, dose-escalation study to assess the infectivity of the 7G8 clone of Pf (PfSPZ Challenge [7G8]). Results showed dose-dependent infectivity from 43% for 8 × 102 PfSPZ to 100% for 4.8 × 103 PfSPZ. PfSPZ Challenge (7G8) will allow for more complete assessment by CHMI of antimalarial vaccines and drugs.

Serologic responses to the PfEMP1 DBL-CIDR head structure may be a better indicator of malaria exposure than those to the DBL-α tag.

BACKGROUND: Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) antigens play a critical role in host immune evasion. Serologic responses to these antigens have been associated with protection from clinical malaria, suggesting that antibodies to PfEMP1 antigens may contribute to natural immunity. The first N-terminal constitutive domain in a PfEMP1 is the Duffy binding-like alpha (DBL-α) domain, which contains a 300 to 400 base pair region unique to each particular protein (the DBL-α "tag"). This DBL-α tag has been used as a marker of PfEMP1 diversity and serologic responses in malaria-exposed populations. In this study, using sera from a malaria-endemic region, responses to DBL-α tags were compared to responses to the corresponding entire DBL-α domain (or "parent" domain) coupled with the succeeding cysteine-rich interdomain region (CIDR). METHODS: A protein microarray populated with DBL-α tags, the parent DBL-CIDR head structures, and downstream PfEMP1 protein fragments was probed with sera from Malian children (aged 1 to 6 years) and adults from the control arms of apical membrane antigen 1 (AMA1) vaccine clinical trials before and during a malaria transmission season. Serological responses to the DBL-α tag and the DBL-CIDR head structure were measured and compared in children and adults, and throughout the season. RESULTS: Malian serologic responses to a PfEMP1's DBL-α tag region did not correlate with seasonal malaria exposure, or with responses to the parent DBL-CIDR head structure in either children or adults. Parent DBL-CIDR head structures were better indicators of malaria exposure. CONCLUSIONS: Larger PfEMP1 domains may be better indicators of malaria exposure than short, variable PfEMP1 fragments such as DBL-α tags. PfEMP1 head structures that include conserved sequences appear particularly well suited for study as serologic predictors of malaria exposure.

A new method for sequencing the hypervariable Plasmodium falciparum gene var2csa from clinical samples.

BACKGROUND: VAR2CSA, a member of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family, mediates the binding of P. falciparum-infected erythrocytes to chondroitin sulfate A, a surface-associated molecule expressed in placental cells, and plays a central role in the pathogenesis of placental malaria. VAR2CSA is a target of naturally acquired immunity and, as such, is a leading vaccine candidate against placental malaria. This protein is very polymorphic and technically challenging to sequence. Published var2csa sequences, mostly limited to specific domains, have been generated through the sequencing of cloned PCR amplicons using capillary electrophoresis, a method that is both time consuming and costly, and that performs poorly when applied to clinical samples that are commonly polyclonal. A next-generation sequencing platform, Pacific Biosciences (PacBio), offers an alternative approach to overcome these issues. METHODS: PCR primers were designed that target a 5 kb segment in the 5' end of var2csa and the resulting amplicons were sequenced using PacBio sequencing. The primers were optimized using two laboratory strains and were validated on DNA from 43 clinical samples, extracted from dried blood spots on filter paper or from cryopreserved P. falciparum-infected erythrocytes. Sequence reads were assembled using the SMRT-analysis ConsensusTools module. RESULTS: Here, a PacBio sequencing-based approach for recovering a segment encoding the majority of VAR2CSA's extracellular region is described; this segment includes the totality of the first four domains in the 5' end of var2csa (~5 kb), from clinical malaria samples. The feasibility of the method is demonstrated, showing a high success rate from cryopreserved samples and more limited success from dried blood spots stored at room temperature, and characterized the genetic variation of the var2csa locus. CONCLUSIONS: This method will facilitate a detailed analysis of var2csa genetic variation and can be adapted to sequence other hypervariable P. falciparum genes.

Hemoglobin C Trait Provides Protection From Clinical Falciparum Malaria in Malian Children.

BACKGROUND: Hemoglobin C trait, like hemoglobin S trait, protects against severe malaria in children, but it is unclear whether hemoglobin C trait also protects against uncomplicated malaria. We hypothesized that Malian children with hemoglobin C trait would have a lower risk of clinical malaria than children with hemoglobin AA. METHODS: Three hundred children aged 0-6 years were enrolled in a cohort study of malaria incidence in Bandiagara, Mali, with continuous passive and monthly active follow-up from June 2009 to June 2010. RESULTS: Compared to hemoglobin AA children (n = 242), hemoglobin AC children (n = 39) had a longer time to first clinical malaria episode (hazard ratio [HR], 0.19; P = .001; 364 median malaria-free days vs 181 days), fewer episodes of clinical malaria, and a lower cumulative parasite burden. Similarly, hemoglobin AS children (n = 14) had a longer time to first clinical malaria episode than hemoglobin AA children (HR, 0.15; P = .015; 364 median malaria-free days vs 181 days), but experienced the most asymptomatic malaria infections of any group. CONCLUSIONS: Both hemoglobin C and S traits exerted a protective effect against clinical malaria episodes, but appeared to do so by mechanisms that differentially affect the response to infecting malaria parasites.

Stable malaria incidence despite scaling up control strategies in a malaria vaccine-testing site in Mali.

BACKGROUND: The recent decline in malaria incidence in many African countries has been attributed to the provision of prompt and effective anti-malarial treatment using artemisinin-based combination therapy (ACT) and to the widespread distribution of long-lasting, insecticide-treated bed nets (LLINs). At a malaria vaccine-testing site in Bandiagara, Mali, ACT was introduced in 2004, and LLINs have been distributed free of charge since 2007 to infants after they complete the Expanded Programme of Immunization (EPI) schedule and to pregnant women receiving antenatal care. These strategies may have an impact on malaria incidence. METHODS: To document malaria incidence, a cohort of 400 children aged 0 to 14 years was followed for three to four years up to July 2013. Monthly cross-sectional surveys were done to measure the prevalence of malaria infection and anaemia. Clinical disease was measured both actively and passively through continuous availability of primary medical care. Measured outcomes included asymptomatic Plasmodium infection, anaemia and clinical malaria episodes. RESULTS: The incidence rate of clinical malaria varied significantly from June 2009 to July 2013 without a clear downward trend. A sharp seasonality in malaria illness incidence was observed with higher clinical malaria incidence rates during the rainy season. Parasite and anaemia point prevalence also showed seasonal variation with much higher prevalence rates during rainy seasons compared to dry seasons. CONCLUSIONS: Despite the scaling up of malaria prevention and treatment, including the widespread use of bed nets, better diagnosis and wider availability of ACT, malaria incidence did not decrease in Bandiagara during the study period.

Seroreactivity to Plasmodium falciparum erythrocyte membrane protein 1 intracellular domain in malaria-exposed children and adults.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) antigens mediate parasite sequestration and host immune evasion. Reactivity to 21 PfEMP1 fragments on a protein microarray was measured in serum samples from Malian children aged 1-6 years and adults. Seroreactivity to PfEMP1 fragments was higher in adults than in children; intracellular conserved fragments were more widely recognized than were extracellular hypervariable fragments. Over a malaria season, children maintained this differential seroreactivity and recognized additional intracellular PfEMP1 fragments. This approach has the potential to identify conserved, seroreactive extracellular PfEMP1 domains critical for protective immunity to malaria.

Gene expression analyses reveal differences in children's response to malaria according to their age.

In Bandiagara, Mali, children experience on average two clinical malaria episodes per year. However, even in the same transmission area, the number of uncomplicated symptomatic infections, and their parasitemia, can vary dramatically among children. We simultaneously characterize host and parasite gene expression profiles from 136 Malian children with symptomatic falciparum malaria and examine differences in the relative proportion of immune cells and parasite stages, as well as in gene expression, associated with infection and or patient characteristics. Parasitemia explains much of the variation in host and parasite gene expression, and infections with higher parasitemia display proportionally more neutrophils and fewer T cells, suggesting parasitemia-dependent neutrophil recruitment and/or T cell extravasation to secondary lymphoid organs. The child's age also strongly correlates with variations in gene expression: Plasmodium falciparum genes associated with age suggest that older children carry more male gametocytes, while variations in host gene expression indicate a stronger innate response in younger children and stronger adaptive response in older children. These analyses highlight the variability in host responses and parasite regulation during P. falciparum symptomatic infections and emphasize the importance of considering the children's age when studying and treating malaria infections.

Immune gene expression changes more during a malaria transmission season than between consecutive seasons.

Plasmodium parasites, the causative organism of malaria, caused over 600,000 deaths in 2022. In Mali, Plasmodium falciparum causes the majority of malaria cases and deaths and is transmitted seasonally. Anti-malarial immunity develops slowly over repeated exposures to P. falciparum and some aspects of this immunity (e.g., antibody titers) wane during the non-transmission, dry season. Here, we sequenced RNA from 33 pediatric blood samples collected during P. falciparum infections at the beginning or end of a transmission season, and characterized the host and parasite gene expression profiles for paired, consecutive infections. We found that human gene expression changes more over the course of one transmission season than between seasons, with signatures of partial development of an adaptive immune response during one transmission season and stability in gene expression during the dry season. Additionally, we found that P. falciparum gene expression did not vary with timing during the season and remained stable both across and between seasons, despite varying human immune pressures. Our results provide insights into the dynamics of anti-malarial immune response development over short time frames that could be exploited by future vaccine and prevention efforts. IMPORTANCE: Our work seeks to understand how the immune response to Plasmodium falciparum malaria changes between infections that occur during low and high malaria transmission seasons, and highlights that immune gene expression changes more during the high transmission season. This provides important insight into the dynamics of the anti-malarial immune response that are important to characterize over these short time frames to better understand how to exploit this immune response with future vaccine efforts.

Children with hemoglobin C or S trait have low serologic responses to a subset of malaria variant surface antigens.

Children with hemoglobin AC or AS have decreased susceptibility to clinical malaria. Parasite variant surface antigen (VSA) presentation on the surface of infected erythrocytes is altered in erythrocytes with hemoglobin C (Hb AC) or sickle trait (Hb AS) mutations in vitro. The protective role of incomplete or altered VSA presentation against clinical malaria in individuals with Hb AC or AS is unclear. Using a high-throughput protein microarray, we sought to use serological responses to VSAs as a measure of host exposure to VSAs among Malian children with Hb AC, Hb AS, or wildtype hemoglobin (Hb AA). In uncomplicated malaria, when compared to Hb AA children, Hb AC children had significantly lower serological responses to extracellular Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) domains but did not differ in responses to intracellular PfEMP1 domains and other VSAs, including members of the repetitive interspersed family (RIFIN) and subtelomeric variable open reading frame (STEVOR) family. Healthy children with Hb AC and Hb AS genotypes recognized fewer extracellular PfEMP1s compared to children with Hb AA, especially CD36-binding PfEMP1s. These reduced serologic responses may reflect reduced VSA presentation or lower parasite exposure in children with Hb AC or AS and provide insights into mechanisms of protection.