Malaria merozoite surface proteins (MSPs) are thought to have important roles in red blood cell (RBC) invasion and their exposure on the parasite surface makes them attractive vaccine candidates. However, their role in invasion has not been directly demonstrated and their biological functions remain unknown. One of the most abundant merozoite surface proteins is PfMSP2, a likely ancestral protein that has been maintained in the Plasmodium falciparum lineage and is a focus of vaccine development. Using CRISPR-Cas9 gene editing, we removed PfMSP2 from two different P. falciparum lines with no impact on parasite replication or phenotype in vitro, demonstrating that it is not essential for RBC invasion. Interestingly, loss of PfMSP2 led to increased inhibitory potency of antibodies targeting other merozoite proteins involved in invasion, particularly PfAMA1. In a solid-phase model, increasing concentrations of PfMSP2 protein reduced binding of different antibodies against PfAMA1 in a dose-dependent manner. These data suggest that PfMSP2 can modulate the susceptibility of merozoites to protective inhibitory antibodies. The results of this study change our understanding of the potential functions of PfMSP2 and establish a new concept in malaria where a surface protein can reduce the protective efficacy of antibodies targeting a different antigen. These findings have important implications for understanding malaria immunity and informing vaccine development.

Abstract Introduction Next-generation malaria vaccines are urgently needed to provide greater efficacy and longevity. Antibodies targeting blood-stage merozoites can confer protection against clinical malaria through multiple Fc-mediated functions. In particular, merozoite surface protein 2 ( Pf MSP2), is a known target of protective antibodies that can clear merozoites via multiple antibody Fc-mediated functions, making is a highly promising vaccine candidate. Methods We developed Pf MSP2 as a self-amplifying RNA (saRNA) vaccine, which was successfully validated for in vitro expression in mammalian cells. Subsequently, the Pf MSP2-saRNA was formulated as lipid nanoparticles (LNP) and evaluated for immunogenicity in mice in a 3-dose regimen comparing 1 μg and 10 μg doses. We evaluated the induction of antibodies with functional activities relevant to protective immunity. Results Our Pf MSP2-saRNA vaccine induced antigen-specific IgG responses that recognised the surface of whole merozoites. Both 1 μg and 10 μg dosing induced comparable antibodies to Pf MSP2, and responses were predominantly murine cytophilic IgG subclasses. These vaccine-induced antibodies demonstrated potent Fc-mediated functions, including complement fixation and binding of human Fcγ-receptor I (FcγRI), after only two doses, which remained consistent after the third dose. Conclusions Pf MSP2 is highly immunogenic using the saRNA vaccine platform in a dose-sparing regimen, and induces antibodies with multiple Fc-mediated functions associated with protective immunity in humans. This saRNA platform is a promising strategy to develop highly efficacious vaccines requiring lower and fewer doses.

Prioritising malaria vaccine targets requires understanding immunity to genetically and structurally diverse parasite antigens, influencing antibody measurements and durability. We measured total IgG levels to 25 Plasmodium falciparum antigens and assessed their association with protection and antigen features. Antibodies were quantified in two longitudinal cohorts of Papua New Guinean children (5-14 years; n=647) experiencing high or moderate transmission. Associations between antibody levels and time to first clinical malaria episode were evaluated using Cox regression and Bayesian antibody-kinetics models, incorporating antigen genetic diversity and structural properties. In high-transmission settings, antibody levels were elevated and stable, with the strongest protection observed for conserved, low-diversity antigens dominated by the 3D7-reference-matching haplotypes and enriched for intrinsically disordered and alpha-helical regions. In moderate transmission, antibody levels were variable, decayed over time, and reflected recent exposure. These findings identify antigen diversity as a key modifier of malaria immunity and underscore the importance of antigen features.

Abstract Introduction Malaria is a leading health problem with high disease burden and mortality rates worldwide. Currently approved vaccines target the sporozoite form of Plasmodium falciparum that initially infects the liver, but only provide modest protection against malaria in young children. There is an urgent need to develop next-generation malaria vaccines that target multiple parasite developmental stages for greater efficacy. Antibodies to merozoites, which are involved in blood-stage replication, and are associated with clinical illness, have multiple functional activities and can protect against malaria. A promising merozoite vaccine candidate is Merozoite Surface Protein 2 ( Pf MSP2). Antibodies to Pf MSP2 can promote multiple antibody Fc-mediated functional activities to clear merozoites. Methods We developed and evaluated monovalent and bivalent (3D7 and FC27 variants) Pf MSP2-based mRNA vaccines. We designed and codon-optimised mRNA, which was validated for in vitro expression in mammalian cells, and subsequently formulated as lipid nanoparticles for vaccination of mice in a 3-dose regimen. Vaccination with recombinant Pf MSP2 protein with adjuvant was performed for comparison. We evaluated the induction of antibodies and functional activities relevant to protective immunity. Results mRNA vaccines induced prominent IgG responses using monovalent (3D7 allele) and bivalent (3D7 and FC27 alleles) vaccines encoding near full-length Pf MSP2, and antibodies recognised the surface of whole merozoites. Vaccine responses were equivalent to, or superior than, a recombinant protein-based Pf MSP2 vaccine. The bivalent vaccine induced equivalent antibodies to the two Pf MSP2 alleles. Vaccination induced cytophilic IgG subclasses with multiple functional activities, including complement fixation, binding of human Fcγ-receptors I and IIa, and opsonic phagocytosis. Conclusions Pf MSP2 is highly immunogenic using the mRNA vaccine platform and induces antibodies with multiple functional activities associated with protective immunity in humans. Combining Pf MSP2 with other merozoite and sporozoite antigens is a promising strategy to develop highly efficacious vaccines to achieve malaria control and elimination goals.

Defining antibody correlates of protection against clinical malaria in areas of low and unstable transmission is challenging because of limited malaria cases in these areas. Additionally, clinical malaria affects both adults and children in areas of low and unstable transmission, but it is unclear whether antibody correlates of protection against malaria differ with age. Blood samples were obtained from 5753 individuals in Kenyan highland area with low and seasonal malaria transmission in 2007 and recorded episodes of clinical malaria in this population from 2007 to 2017. Using a nested case–control study design, participants who developed clinical malaria (cases) were matched by age and village to those who did not (controls). Immunoglobulin (Ig)G, IgG1, IgG3, IgA and IgM responses to 16 Plasmodium falciparum antigens were compared in individuals < 5 years old (80 cases vs. 240 controls), 5–14 years old (103 cases vs. 309 controls) and ≥ 15 years old (118 cases vs. 354 controls). Antibody level was correlated with risk of clinical malaria, adjusted for malaria exposure markers. In all age groups, most antibodies were not associated with risk of clinical malaria. In children < 5 years, higher levels of IgG to GLURP-R2 and MSP-2, IgG1 to GLURP-R2, and IgG3 to MSP-2 were associated with reduced risk of clinical malaria, while higher IgG3 levels to CSP were associated with increased risk of clinical malaria. In children 5–14 years and individuals ≥ 15 years, higher antibody levels to multiple P. falciparum antigens were associated with an increased risk of clinical malaria, and none were associated with decreased risk of clinical malaria. Antibody correlates of protection against clinical malaria were observed only in children < 5 years old in this area of low and unstable malaria transmission. In older children and adults in this area, some antibody responses correlated with increased risk of clinical malaria. Future studies in low malaria transmission areas should evaluate the comparative contributions of cellular and humoral immunity to protection from clinical malaria in young children versus older children and adults.

Introduction Antibodies targeting the blood-stage of Plasmodium falciparum play a critical role in naturally acquired immunity to malaria by limiting blood-stage parasitemia. One mode of action of antibodies is the direct inhibition of merozoite invasion of erythrocytes through targeting invasion ligands. However, evasion of inhibitory antibodies may be mediated in P. falciparum by switching between various ligand-mediated merozoite invasion pathways. Here, we investigated the potential roles of invasion ligands PfRH1, PfRH2a and PfRH2b in immune evasion through phenotypic variation, and their importance as targets of human invasion-inhibitory antibodies. Methods Serum samples from malaria-exposed children and adults in Kenya were examined for their ability to inhibit P. falciparum invasion, using parasites with disrupted pfrh1, pfrh2a or pfrh2b genes. Results and Discussion The loss of PfRH1 and PfRH2b substantially impacted on susceptibility to inhibitory antibodies, suggesting that variation in the use of these ligands contributes to immune evasion. The effect was less prominent with loss of PfRH2a. Differential inhibition of the knockout and parental lines points to PfRH1 and PfRH2b as targets of acquired growth inhibitory antibodies whereas PfRH2a appeared to be a minor target. There was limited relatedness of the inhibitory responses between different isolates or compared to parasites with deletions of erythrocyte-binding antigens. This further suggests that there is a substantial amount of antigenic diversity in invasion pathways to facilitate immune evasion. These findings provide evidence that PfRH1 and PfRH2b are significant targets of inhibitory antibodies and variation in their expression may facilitate immune evasion. Targeting of multiple invasion ligands in vaccine design is likely to be required to achieve potent inhibitory antibodies and protective efficacy against malaria.

IntroductionInfection remains one of the most common causes of death in neonates. However, early detection of neonatal infections to inform treatment decisions remains clinically and technically challenging due to the non-specific nature of symptoms, and the lack of a sufficiently accurate diagnostic test. Neonatal infections and sepsis in adults have been associated with increased CD64 expression on neutrophils. We investigated whole blood CD64 (wbCD64) and neutrophil elastase (NE) in neonates who were evaluated and treated for potential infection and evaluated the potential for these biomarkers as diagnostic tools.MethodsNeonates were prospectively recruited from two neonatal units. Whole blood samples were collected at the time of clinical evaluation for potential infection, if antimicrobials were also initiated. Whole blood CD64 and NE, as a marker of the neutrophil count, were measured by enzyme-linked immunosorbent assays (ELISA). Correlations between wbCD64, NE, and standard hematologic indices were evaluated and diagnostic performance of wbCD64 in relation to infections analyzed using logistic regression and receiver operating characteristic (ROC) curves.ResultsSamples were analyzed from a total of 178 episodes of infection evaluation from 163 neonates. Whole blood CD64 and NE had a positive, non-linear correlation. Infection was diagnosed in 45% (80/178) of episodes, and 31% (55/178) had infection that was microbiologically confirmed. There was no association identified between wbCD64 and infections, and wbCD64 had poor diagnostic performance for infection detection. Evaluation of wbCD64 relative to levels of NE did not improve diagnostic performance. WbCD64 levels were significantly higher among a subgroup of neonates aged >48 hours who had microbiologically-confirmed bacterial bloodstream infections (BSI), with optimal sensitivity and specificity for BSI detection 53% and 87% respectively.ConclusionWbCD64 is generally not significantly associated with infection in neonates, but shows some association with bacterial bloodstream infections. The diagnostic performance of wbCD64, with or without NE, does not afford sufficient diagnostic accuracy to aid antimicrobial therapeutic decisions for neonatal infections.

T-follicular CD4 T (Tfh) cells play essential roles in antibody induction during infection and following vaccination. In humans, peripheral Tfh (pTfh) cells are commonly analysed based on expression of CXCR3 and CCR6, with different subsets of pTfh (pTfh1, pTfh2, pTfh17) associated with antibody induction in a context-dependent manner. In malaria, the specific roles of pTfh subsets in antibody development is not clear. Several studies in human malaria infection and vaccination have identified an important role of pTfh2 cells, which associate with antibody development while pTfh1 cells do not. However, in vitro studies and animal models highlight that pTfh1 cells are key drivers of cytophilic antibody development, which are protective. To dissect these contradictions, we mapped the heterogeneity of pTfh cells in healthy individuals and individuals with controlled human malaria infection using scRNAseq. We identified two, previously unidentified, pTfh1-like subsets with functional relevance, which can be defined based on CCR7 expression. CCR7pos pTfh1 cells have increased capacity to produce IL-21, whereas CCR7neg pTfh1 cells express markers of cytotoxicity. In controlled human malaria infection, we show that both CCR7pos and CCR7neg pTfh1 cells, along with Tfh2 cells, clonally expand, are transcriptionally and phenotypically activated, and are malaria specific. However, only CCR7pos pTfh1 and pTfh2 cells associated with antibody responses to infection. Our data expand our knowledge of Tfh cell diversity and function during human infection and resolve contradictions of the role of pTfh1 cells in antibody development targeting malaria. Data advance our knowledge of Tfh cell diversity and may inform approaches to target these key CD4 T cells during vaccination. ### Competing Interest Statement The authors have declared no competing interest.

In areas with low malaria transmission intensity, most infections during pregnancy are not detected by standard diagnostic tests. New data shows that these missed asymptomatic infections have substantial negative impacts for both the mother and the developing fetus.

RTS,S/AS01E is the first malaria vaccine implemented for young African children. However, it provides partial protection against Plasmodium falciparum (Pf) malaria, and a better understanding of the mechanisms and determinants of vaccine immunity will help develop second-generation improved vaccines. We measured IgG to vaccine target and Pf blood-stage off-target proteins before and after vaccination in 874 children aged 1–4 years in a phase 2b trial of RTS,S/AS02A in Mozambique. We found that naturally acquired PfCSP IgG levels pre-vaccination were positively associated with RTS,S immunogenicity. Increased levels of IgG to the C-terminus and NANP-repeat regions of PfCSP, and to PfMSP5 and PfMSP1 block 2, following vaccination, were significantly associated with a lower hazard of clinical malaria over 6 months. Thus, immune priming, anti-PfCSP C-terminus and off-target antibody responses contributed to malaria protection after adjusting for prior Pf exposure, and this could guide strategies for optimizing the immunogen and vaccine deployment.

Antibodies are important for protection against malaria. For optimal protective activity, it is thought that antibodies need to have high affinity. A longitudinal study conducted in Uganda followed newborn infants and their mothers for nine months. The study found that antibody affinity (here measured as dissociation rate constant, kd) against the merozoite antigens AMA1 and MSP2 decreased from birth to six months in the infants, then gradually increased to 9 months, but not reaching the level observed in the mothers. In contrast, affinity against the sporozoite antigen CSP, did not change throughout the study period. Among mothers, no significant changes in antibody affinity were observed for any antigen, which is consistent with expectations for adults in an endemic area. Comparing specific antibody affinities to total antibody levels revealed almost no correlations, indicating that antibody magnitude and affinity evolve differently during immune development. Significant correlations were observed between antibody affinities and some atypical memory B cells. In conclusion, our study shows that development of naturally acquired slowly dissociating (high affinity) antibodies against malaria can evolve separately across different antigens. This is important information for future vaccine development studies.

OBJECTIVES:Repeated COVID-19 mRNA vaccinations increase SARS-CoV-2 IgG4 antibodies, indicating extensive IgG class switching following the first booster dose. This shift in IgG subclasses raises concerns due to the limited ability of IgG4 to mediate Fc-dependent effector functions. METHODS:To assess the impact of IgG4 induction on protective immunity, we analyzed longitudinal SARS-CoV-2 IgG subclasses, C1q and FcγR responses, and neutralizing activity in a well-characterized cohort of healthcare workers in Spain. RESULTS:Elevated IgG4 levels and higher ratios of non-cytophilic to cytophilic antibodies after booster vaccination were significantly associated with an increased risk of breakthrough infections (IgG4 HR[10-fold increase]=1.8, 95% CI=1.2-2.7; non-cytophilic to cytophilic ratio HR[10-fold increase]=1.5, 95% CI=1.1-1.9). Moreover, an increased non-cytophilic to cytophilic antibody ratio correlated with reduced functionality, including neutralization. CONCLUSIONS:These findings suggest a potential association between IgG4 induction by mRNA vaccination and a higher risk of breakthrough infection, warranting further investigation into vaccination strategies to ensure sustained protection.

Latent cytomegalovirus (CMV) infection has widely reported immunomodulatory effects on the immune system, leading to altered responses to vaccination or infection with other pathogens. However, the impact of CMV on the immune response to the malaria parasite Plasmodium falciparum is unknown. Here we assessed antibody and cellular responses in malaria-naive volunteers during Controlled Human Malaria Infection (CHMI) with blood stage P. falciparum parasites. Latent CMV infection was associated with reduced induction of parasite specific IgG, IgG1, and functional antibodies following infection. Within the T-follicular helper (Tfh) cell compartment, CMV was associated with a skew towards Tfh1 cell subsets before and after malaria, and reduced activation of Tfh2 cells which have been previously associated with antibody induction during malaria. Taken together, CMV latent infection impacts the phenotype of Tfh cells required for robust antibody induction to malaria and may have particular importance in malaria endemic countries where CMV infection is almost universal and acquired early in life. ### Competing Interest Statement The authors have declared no competing interest.

Abstract Pathogen-specific antibodies that recruit complement have been associated with protection in several diseases, including malaria. Antibodies bind to C1q via their Fc-region, initiating the classical complement cascade. This involves the sequential recruitment of serum complement proteins including C3, which is cleaved to form the opsonising fragment C3b, and C5, 6, 7, 8 and 9, which form a membrane attack complex and lyse parasites. This protocol describes two methods to measure complement-fixing antibodies against Plasmodium falciparum parasites, specifically antibodies targeting P. falciparuminfected red blood cell surface expressed antigens during the trophozoite stage of development (e.g. PfEMP1). Briefly, parasites are incubated with antibodies and a source of complement (e.g. fresh normal human serum) to allow antibody binding/opsonization and resultant complement-fixation to the antigen-antibody complex. Antibody-bound complement is detected using complement-specific antibodies via either flow cytometry or Western blot. This assay can be used to specifically measure C1q-recruitment by antibodies, or downstream complement activation by assessing C3 or C5 – 9 components.

Background: The integration of maternal and child health services (MCH) with routine immunization is an important global health strategy, particularly in low- and middle-income countries (LMICs). However, evidence is lacking regarding the best practices for service integration and the effect of integration on immunization and linked health service outcomes. Methods: We searched publication databases and gray literature for articles published between 2011 and 2020 that include approaches to integrating MCH services with immunizations during the first two years of life in LMICs. Abstracts and full-text articles were screened for eligibility. For the included articles, data extraction and analysis examined the descriptive characteristics of studies, outcomes, and implementation considerations. Results: Among the 16,578 articles screened, 44 met the criteria for inclusion, representing 34 studies, of which 29 were from Africa. The commonly linked MCH services were family planning (24%), human immunodeficiency virus (HIV) diagnosis or care (21%), and malaria prevention or control (21%). Multiple integration strategies were typically used; the co-location of linked services (65%), the provision of extra services by immunization staff (41%), and/or the provision of extra information by immunization staff (41%) were the most common. In general, integration improved MCH service outcomes (76%) and was either beneficial (55%) or neutral for immunization (35%), with some examples in family planning, malaria, and HIV where integrated services were not beneficial. Important implementation considerations included the careful matching of target populations in service re-design, ensuring support from policy, logistics, and information systems, the provision of adequate training and support of staff to avoid overload, clear client communication regarding service integration, and the need to address community concerns. Conclusions: Integrating MCH services with routine immunization can expand linked services and improve immunization coverage. This study has identified key implementation considerations relevant to both childhood and adult vaccination programs. More research is needed regarding costs and client preferences.