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RTS,S/AS01E has been tested in a phase 3 malaria vaccine study with partial efficacy in African children and infants. In a cohort of 1028 subjects from one low (Bagomoyo) and two high (Nanoro, Kintampo) malaria transmission sites, we analysed IgG plasma/serum concentration and avidity to CSP (NANP-repeat and C-terminal domains) after a 3-dose vaccination against time to clinical malaria events during 12-months. Here we report that RTS,S/AS01E induces substantial increases in IgG levels from pre- to post-vaccination ( p  < 0.001), higher in NANP than C-terminus (2855 vs 1297 proportional change between means), and higher concentrations and avidities in children than infants ( p  < 0.001). Baseline CSP IgG levels are elevated in malaria cases than controls ( p  < 0.001). Both, IgG magnitude to NANP (hazard ratio [95% confidence interval] 0.61 [0.48–0.76]) and avidity to C-terminus (0.07 [0.05–0.90]) post-vaccination are significantly associated with vaccine efficacy. IgG avidity to the C-terminus emerges as a significant contributor to RTS,S/AS01E-mediated protection. RTS,S/AS01E has been tested in a phase 3 malaria vaccine trial and has shown partial efficacy in children and infants. Here, the authors analyze IgG concentration and avidity to CSP in ~1000 participants and show that IgG avidity to the C-terminus of CSP is significantly associated with vaccine-mediated protection.

Infection with Plasmodium can elicit antibodies that inhibit parasite survival in the mosquito, when they are ingested in an infectious blood meal. Here, we determine the transmission-reducing activity (TRA) of naturally acquired antibodies from 648 malaria-exposed individuals using lab-based mosquito-feeding assays. Transmission inhibition is significantly associated with antibody responses to Pfs48/45, Pfs230, and to 43 novel gametocyte proteins assessed by protein microarray. In field-based mosquito-feeding assays the likelihood and rate of mosquito infection are significantly lower for individuals reactive to Pfs48/45, Pfs230 or to combinations of the novel TRA-associated proteins. We also show that naturally acquired purified antibodies against key transmission-blocking epitopes of Pfs48/45 and Pfs230 are mechanistically involved in TRA, whereas sera depleted of these antibodies retain high-level, complement-independent TRA. Our analysis demonstrates that host antibody responses to gametocyte proteins are associated with reduced malaria transmission efficiency from humans to mosquitoes. Naturally acquired antibodies may inhibit Plasmodium survival in mosquitoes, alter malaria transmission dynamics, and form the basis for transmission-blocking vaccines. Here, using sera from malaria-exposed individuals, Stone et al. reveal novel antibody correlates of transmission-reducing activity.

Characterizing the immune response to pneumococcal proteins is critical in understanding this bacterium’s epidemiology and vaccinology. Probing a custom-designed proteome microarray with sera from 35 healthy US adults revealed a continuous distribution of IgG affinities for 2,190 potential antigens from the species-wide pangenome. Reproducibly elevated IgG binding was elicited by 208 “antibody binding targets” (ABTs), which included 109 variants of the diverse pneumococcal surface proteins A and C (PspA and PspC) and zinc metalloprotease A and B (ZmpA and ZmpB) proteins. Functional analysis found ABTs were enriched in motifs for secretion and cell surface association, with extensive representation of cell wall synthesis machinery, adhesins, transporter solute-binding proteins, and degradative enzymes. ABTs were associated with stronger evidence for evolving under positive selection, although this varied between functional categories, as did rates of diversification through recombination. Particularly rapid variation was observed at some immunogenic accessory loci, including a phage protein and a phase-variable glycosyltransferase ubiquitous among the diverse set of genomic islands encoding the serine-rich PsrP glycoprotein. Nevertheless, many antigens were conserved in the core genome, and strains’ antigenic profiles were generally stable. No strong evidence was found for any epistasis between antigens driving population dynamics, or redundancy between functionally similar accessory ABTs, or age stratification of antigen profiles. These results highlight the paradox of why substantial variation is observed in only a subset of epitopes. This result may indicate only some interactions between immunoglobulins and ABTs clear pneumococcal colonization or that acquired immunity to pneumococci is an accumulation of individually weak responses to ABTs evolving under different levels of functional constraint.

Immunization through repeated direct venous inoculation of Plasmodium falciparum (Pf) sporozoites (PfSPZ) under chloroquine chemoprophylaxis, using the PfSPZ Chemoprophylaxis Vaccine (PfSPZ-CVac), induces high-level protection against controlled human malaria infection (CHMI). Humoral and cellular immunity contribute to vaccine efficacy but only limited information about the implicated Pf-specific antigens is available. Here, we examined Pf-specific antibody profiles, measured by protein arrays representing the full Pf proteome, of 40 placebo- and PfSPZ-immunized malaria-naïve volunteers from an earlier published PfSPZ-CVac dose-escalation trial. For this purpose, we both utilized and adapted supervised machine learning methods to identify predictive antibody profiles at two different time points: after immunization and before CHMI. We developed an adapted multitask support vector machine (SVM) approach and compared it to standard methods, i.e. single-task SVM, regularized logistic regression and random forests. Our results show, that the multitask SVM approach improved the classification performance to discriminate the protection status based on the underlying antibody-profiles while combining time- and dose-dependent data in the prediction model. Additionally, we developed the new f E ature di S tance ex P lainabilit Y (ESPY) method to quantify the impact of single antigens on the non-linear multitask SVM model and make it more interpretable. In conclusion, our multitask SVM model outperforms the studied standard approaches in regard of classification performance. Moreover, with our new explanation method ESPY, we were able to interpret the impact of Pf-specific antigen antibody responses that predict sterile protective immunity against CHMI after immunization. The identified Pf-specific antigens may contribute to a better understanding of immunity against human malaria and may foster vaccine development.

There is no vaccine to protect from cryptosporidiosis, a leading cause of diarrhea in infants in low- and middle-income countries. Here, we comprehensively identified parasite antigens associated with protection from reinfection. A Cryptosporidium protein microarray was constructed by in vitro transcription and translation of 1,761 C. parvum, C. hominis, or C. meleagridis antigens, including proteins with a signal peptide and/or a transmembrane domain. Plasma IgG and/or IgA from Bangladeshi children longitudinally followed for cryptosporidiosis from birth to 3 years of age allowed for identification of 233 seroreactive proteins. Seven of these were associated with protection from reinfection. These included Cp23, Cp17, Gp900, and 4 additional antigens - CpSMP1, CpMuc8, CpCorA and CpCCDC1. Infection in the first year of life, however, often resulted in no detectable antigen-specific antibody response, and antibody responses, when detected, were specific to the infecting parasite genotype and decayed in the months after infection. In conclusion, humoral immune responses against specific parasite antigens were associated with acquired immunity. While antibody decay over time and parasite genotype-specificity may limit natural immunity, this work serves as a foundation for antigen selection for vaccine design.

Background The RTS,S/AS01E vaccine provides partial protection against malaria in African children, but immune responses have only been partially characterized and do not reliably predict protective efficacy. We aimed to evaluate comprehensively the immunogenicity of the vaccine at peak response, the factors affecting it, and the antibodies associated with protection against clinical malaria in young African children participating in the multicenter phase 3 trial for licensure. Methods We measured total IgM, IgG, and IgG 1–4 subclass antibodies to three constructs of the Plasmodium falciparum circumsporozoite protein (CSP) and hepatitis B surface antigen (HBsAg) that are part of the RTS,S vaccine, by quantitative suspension array technology. Plasma and serum samples were analyzed in 195 infants and children from two sites in Ghana (Kintampo) and Mozambique (Manhiça) with different transmission intensities using a case-control study design. We applied regression models and machine learning techniques to analyze immunogenicity, correlates of protection, and factors affecting them. Results RTS,S/AS01E induced IgM and IgG, predominantly IgG1 and IgG3, but also IgG2 and IgG4, subclass responses. Age, site, previous malaria episodes, and baseline characteristics including antibodies to CSP and other antigens reflecting malaria exposure and maternal IgGs, nutritional status, and hemoglobin concentration, significantly affected vaccine immunogenicity. We identified distinct signatures of malaria protection and risk in RTS,S/AS01E but not in comparator vaccinees. IgG2 and IgG4 responses to RTS,S antigens post-vaccination, and anti-CSP and anti- P. falciparum antibody levels pre-vaccination, were associated with malaria risk over 1-year follow-up. In contrast, antibody responses to HBsAg (all isotypes, subclasses, and timepoints) and post-vaccination IgG1 and IgG3 to CSP C-terminus and NANP were associated with protection. Age and site affected the relative contribution of responses in the correlates identified. Conclusions Cytophilic IgG responses to the C-terminal and NANP repeat regions of CSP and anti-HBsAg antibodies induced by RTS,S/AS01E vaccination were associated with malaria protection. In contrast, higher malaria exposure at baseline and non-cytophilic IgG responses to CSP were associated with disease risk. Data provide new correlates of vaccine success and failure in African children and reveal key insights into the mode of action that can guide development of more efficacious next-generation vaccines.

Streptococcus pneumoniae (pneumococcus) is a nasopharyngeal commensal and respiratory pathogen. This study characterises the immunoglobulin G (IgG) repertoire recognising pneumococci from birth to 24 months old (mo) in a prospectively-sampled cohort of 63 children using a panproteome array. IgG levels are highest at birth, due to transplacental transmission of maternal antibodies. The subsequent emergence of responses to individual antigens exhibit distinct kinetics across the cohort. Stable differences in the strength of individuals’ responses, correlating with maternal IgG concentrations, are established by 6 mo. By 12 mo, children develop unique antibody profiles that are boosted by re-exposure. However, some proteins only stimulate substantial responses in adults. Integrating genomic data on nasopharyngeal colonisation demonstrates rare pneumococcal antigens can elicit strong IgG levels post-exposure. Quantifying such responses to the diverse core loci (DCL) proteins is complicated by cross-immunity between variants. In particular, the conserved N terminus of DCL protein zinc metalloprotease B provokes the strongest early IgG responses. DCL proteins’ ability to inhibit mucosal immunity likely explains continued pneumococcal carriage despite hosts’ polyvalent antibody repertoire. Yet higher IgG levels are associated with reduced incidence, and severity, of pneumonia, demonstrating the importance of the heterogeneity in response strength and kinetics across antigens and individuals. By combining genome sequencing and antibody binding (to all common pneumococcal proteins) data, Croucher et al. present a high-resolution analysis of the emergence of immune responses in children that can protect against pneumonia.

Naturally acquired immunity (NAI) to malaria is mainly mediated by IgG antibodies but the subclasses, epitope targets and effector functions have not been unequivocally defined. Dissecting the type and specificity of antibody responses mediating NAI is a key step toward developing more effective vaccines to control the disease. We investigated the role of IgG subclasses to malaria antigens in protection against disease and the factors that affect their levels, including vaccination with RTS,S/AS01E. We analyzed plasma and serum samples at baseline and 1 month after primary vaccination with RTS,S or comparator in African children and infants participating in a phase 3 trial in two sites of different malaria transmission intensity: Kintampo in Ghana and Manhiça in Mozambique. We used quantitative suspension array technology (qSAT) to measure IgG responses to 35 pre-erythrocytic and blood stage antigens. Our results show that the pattern of IgG response is predominantly IgG1 or IgG3, with lower levels of IgG2 and IgG4. Age, site and RTS,S vaccination significantly affected antibody subclass levels to different antigens and susceptibility to clinical malaria. Univariable and multivariable analysis showed associations with protection mainly for cytophilic IgG3 levels to selected antigens, followed by IgG1 levels and, unexpectedly, also with IgG4 levels, mainly to antigens that increased upon RTS,S vaccination such as MSP5 and MSP1 block 2, among others. In contrast, IgG2 was associated with malaria risk. Stratified analysis in RTS,S vaccinees pointed to novel associations of IgG4 responses with immunity mainly involving pre-erythrocytic antigens upon RTS,S vaccination. Multi-marker analysis revealed a significant contribution of IgG3 responses to malaria protection and IgG2 responses to malaria risk. We propose that the pattern of cytophilic and non-cytophilic IgG antibodies is antigen-dependent and more complex than initially thought, and that mechanisms of both types of subclasses could be involved in protection. Our data also suggests that RTS,S efficacy is significantly affected by NAI, and indicates that RTS,S vaccination significantly alters NAI.

Reducing variability of quantitative suspension array assays is key for multi-center and large sero-epidemiological studies. To maximize precision and robustness of an in-house IgG multiplex assay, we analyzed the effect of several conditions on variability to find the best combination. The following assay conditions were studied through a fractional factorial design: antigen-bead coupling (stock vs. several), sample predilution (stock vs. daily), temperature of incubation of sample with antigen-bead (22°C vs. 37°C), plate washing (manual vs. automatic) and operator expertise (expert vs. apprentice). IgG levels against seven P. falciparum antigens with heterogeneous immunogenicities were measured in test samples, in a positive control and in blanks. We assessed the variability and MFI quantification range associated to each combination of conditions, and their interactions, and evaluated the minimum number of samples and blank replicates to achieve good replicability. Results showed that antigen immunogenicity and sample seroreactivity defined the optimal dilution to assess the effect of assay conditions on variability. We found that a unique antigen-bead coupling, samples prediluted daily, incubation at 22°C, and automatic washing, had lower variability. However, variability increased when performing several couplings and incubating at 22°C vs. 37°C. In addition, no effect of temperature was seen with a unique coupling. The expertise of the operator had no effect on assay variability but reduced the MFI quantification range. Finally, differences between sample replicates were minimal, and two blanks were sufficient to capture assay variability, as suggested by the constant Intraclass Correlation Coefficient of three and two blanks. To conclude, a single coupling was the variable that most consistently reduced assay variability, being clearly advisable. In addition, we suggest having more sample dilutions instead of replicates to increase the likelihood of sample MFIs falling in the linear part of the antigen-specific curve, thus increasing precision.

BACKGROUNDThe use of high-throughput technologies has enabled rapid advancement in the knowledge of host immune responses to pathogens. Our objective was to compare the repertoire, protection, and maternal factors associated with human milk antibodies to infectious pathogens in different economic and geographic locations.METHODSUsing multipathogen protein microarrays, 878 milk and 94 paired serum samples collected from 695 women in 5 high and low-to-middle income countries (Bangladesh, Finland, Peru, Pakistan, and the United States) were assessed for specific IgA and IgG antibodies to 1,607 proteins from 30 enteric, respiratory, and bloodborne pathogens.RESULTSThe antibody coverage across enteric and respiratory pathogens was highest in Bangladeshi and Pakistani cohorts and lowest in the U.S. and Finland. While some pathogens induced a dominant IgA response (Campylobacter, Klebsiella, Acinetobacter, Cryptosporidium, and pertussis), others elicited both IgA and IgG antibodies in milk and serum, possibly related to the invasiveness of the infection (Shigella, enteropathogenic E. coli \"EPEC\", Streptococcus pneumoniae, Staphylococcus aureus, and Group B Streptococcus). Besides the differences between economic regions and decreases in concentrations over time, human milk IgA and IgG antibody concentrations were lower in mothers with high BMI and higher parity, respectively. In Bangladeshi infants, a higher specific IgA concentration in human milk was associated with delayed time to rotavirus infection, implying protective properties of antirotavirus antibodies, whereas a higher IgA antibody concentration was associated with greater incidence of Campylobacter infection.CONCLUSIONThis comprehensive assessment of human milk antibody profiles may be used to guide the development of passive protection strategies against infant morbidity and mortality.FUNDINGBill and Melinda Gates Foundation grant OPP1172222 (to KMJ); Bill and Melinda Gates Foundation grant OPP1066764 funded the MDIG trial (to DER); University of Rochester CTSI and Environmental Health Sciences Center funded the Rochester Lifestyle study (to RJL); and R01 AI043596 funded PROVIDE (to WAP).