key: cord-0794892-j7mgv5fi
authors: Nielsen, C. M.; Barrett, J. R.; Davis, C.; Fallon, J. K.; Goh, C.; Michell, A. R.; Griffin, C. L.; Kwok, A. J.; Loos, C.; Darko, S.; Laboune, F.; Silk, S. E.; Tekman, M.; Francica, J. R.; Ransier, A.; Payne, R. O.; Minassian, A. M.; Lauffenburger, D. A.; Seder, R. A.; Douek, D. C.; Alter, G.; Draper, S. J.
title: Delayed booster dosing improves human antigen-specific Ig and B cell responses to the RH5.1/AS01B malaria vaccine
date: 2022-04-27
journal: nan
DOI: 10.1101/2022.04.25.22274161
sha: d4afd62b732155d55e5422846210d0eaa3729514
doc_id: 794892
cord_uid: j7mgv5fi

Background: Antibodies are crucial for vaccine-mediated protection against many pathogens. Modifications to vaccine delivery that increase antibody magnitude, longevity, and/or quality are therefore of great interest for maximising efficacy. We have previously shown that a delayed fractional (DFx) dosing schedule (0-1-6mo) - using AS01B-adjuvanted RH5.1 malaria antigen - substantially improves serum IgG durability as compared to monthly dosing (0-1-2mo; NCT02927145). However, the underlying mechanism and whether there are wider immunological changes with DFx dosing was unclear. Methods: Immunokinetics of PfRH5-specific Ig across multiple isotypes were compared between DFx and monthly regimen vaccinees. Peak responses were characterised in-depth with a systems serology platform including biophysical and functional profiling. Computational modelling was used to define the humoral feature set associated with DFx dosing. PfRH5-specific B cells were quantified by flow cytometry and sorted for single cell RNA sequencing (scRNA-seq). Differential gene expression between DFx and monthly dosing regimens was explored with Seurat, DESeq2 and gene set enrichment analysis. Results: DFx dosing increases the frequency of circulating PfRH5-specific B cells and longevity of PfRH5-specific IgG1, as well as other isotypes and subclasses. At the peak antibody response, DFx dosing was distinguished by a systems serology feature set comprising increased FcRn-binding, IgG avidity, and proportion of G2B and G2S2F IgG Fc glycans, alongside decreased IgG3, antibody-dependent complement deposition, and proportion of G1S1F IgG Fc glycan. At the same time point, scRNA-seq of PfRH5-specific B cells revealed enriched plasma cell and Ig / protein export signals in the monthly dosing group as compared to DFx vaccinees. Conclusions: DFx dosing of the RH5.1/AS01B vaccine had a profound impact on the humoral response. Our data suggest plausible mechanisms relating to improved FcRn-binding (known to improve Ig longevity) and a potential shift from short-lived to long-lived plasma cells. Recent reports of the positive impact of delayed boosting on SARS-CoV-2 vaccine immunogenicity highlight the broad relevance of these data.

Vaccines are among public health's most effective tools for combatting infectious disease but 55 a poor understanding of the underlying immunological mechanisms frequently impedes 56 vaccine development. One of the greatest perennial issues for the vaccinology field is a lack 57 of knowledge of how to induce more durable immune responses in the target populations. 58 While many vaccine candidates generate encouraging peak antibody concentrations, these 59 often wane rapidly in the following months. This rapid decay can be highly problematic and 60 poses a particular issue for pathogens such as blood-stage malaria where the threshold 61 antibody concentration required for protection is high (1, 2). 62 63 Interestingly, in a clinical trial with the leading blood-stage malaria vaccine candidate (RH5.1/ 64 AS01B) we have recently shown that a delayed fractional booster 0-1-6-month schedule 65 ("DFx") induces higher magnitude vaccine-specific antibody as compared to a more typically 66 used 0-1-2-month "monthly" vaccination schedule (NCT02927145). Moreover, the DFx anti-67 PfRH5 serum IgG plateaus at a 10X higher concentration over the next 2 yearsan 68 unprecedented finding (1). While data from other malaria vaccine trialsand more recent 69 SARS-CoV-2 trials (3-5)are broadly supportive of a beneficial impact of delayed (fractional) 70 booster dosing on antibody-mediated immunity, there is yet to be any other demonstration of 71 a comparable impact of vaccine regimen on human antibody longevity or any analyses 72 involving the direct detection and isolation of antigen-specific B cells (6-9). 73 74 Here, using samples from the RH5.1/AS01B trial, we interrogate the PfRH5-specific antibody 75 and B cell responses in both DFx and monthly regimens ( Table 1) . Through a combination of 76 immunokinetic, systems serology, and single cell RNA sequencing (scRNA-seq) analyses, we 77 identify features of the PfRH5-specific Ig and B cell responses that discriminate between these 78 dosing regimens. These data are informative for understanding the potential underlying 79 mechanisms of DFx-mediated improvements in humoral immunity, and will be of great 80 5 86 87 Table 1 . Overview of vaccination regimen. 88 PfRH5.1 = full-length PfRH5 protein (1, 12). Vaccinee demographics relate only to those included in this study. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 27, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 Supplemental Figure 1A) . Frequencies of PfRH5-specific mBCs were compared between 152 (A) samples from a heterologous viral vector trial (ChAd63-MVA; ChAd63-PfRH5 prime, PfRH5 boost (10, 11) and the protein/AS01B trial, or (B) between monthly regimen vaccinees 154

and DFx vaccinees within the protein/AS01B trial. (C) Spearman correlation analysis was 155 performed between the frequency of PfRH5-specific mBCs 4-weeks after the final vaccination 156 and the concentration of anti-PfRH5 serum IgG at the same time point ( DFx within protein/AS01B trial: Pre-vacc. n = 15/3; 2-week n = 16/9; 4-week n = 19/10; 12-161

week n = 17/8. (A-B) Comparisons were performed by Mann-Whitney tests. Whiskers denote 162 5 th and 95 th percentiles. * p < 0.05, ** p < 0.01, **** p < 0.0001. (C) Sample size, Spearman 163 r, and p value are annotated on the graph. Each triangle represents one vaccinee and purple 164 triangles indicate DFx vaccinees. Anti-RH5.1 FL (full length RH5.1) serum Ig was assayed by 165 standardised ELISA to report (D) IgG1, (E) IgG2, (F) IgG3, and (G) IgG4 at key time points 166 (Days 0, 14, 28, 42, 56/182, 70/196, 84/210, 240/366, 696/822 Day 366). Monthly regimen/ DFx: n = 31/ 11 with the exception of IgG4 where n = 18 /9. 175

Comparisons were performed between DFx and monthly regimens with Mann-Whitney tests. 176

Bars denote medians and error bars denote interquartile ranges. * p < 0.05, ** p < 0.01, *** p 177 < 0.001, **** p < 0.0001. Full kinetics for IgA, IgA1, IgA2 and IgM, as well a stratified version 178

of panel H are shown in Supplemental Figure 2 . 179 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted April 27, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 Systems serology feature set associated with DFx dosing includes increased 180 FcRn-binding, IgG avidity, and proportion of bi-galactosylated IgG Fc glycans 181 Our stratified antibody isotype and subclass immunokinetic data suggest a significant impact 182 of DFx dosing on humoral immunogenicity. We therefore next extended these analyses using 183 a systems serology pipeline to integrate data on Fc biophysical and functional characteristics, 184 measuring a total of 49 parameters (see Methods (13, 14) ). As previously described (1), in 185 addition to quantification of post-vaccination plasma levels of PfRH5-specific antibodies of 186 each major isotype and subclass (with a Luminex bead-based assay rather than ELISA as 187 above) this approach extends the biophysical analyses to include characterisation of the 188 glycosylation profile of the anti-PfRH5 IgG Fc domains, which is known to influence these Fc-189 mediated functions (15). To assess Fc-mediated functionality of PfRH5-specific antibodies, 190 the systems serology platform incorporates evaluation of their capacity to bind Fc receptors 191 (FcRs), and to activate monocytes, neutrophils, natural killer (NK) cells, as well as the 192 complement cascade. Here, in initial univariate analyses, we observed regimen-dependent 193 differences in Fc-mediated activation with intriguing trends towards reduced functionality in 194

DFx as compared to the monthly-low or monthly-medium vaccinees (Figure 2) . Specifically, 195 PfRH5-specific Ig in plasma samples 2-weeks following the final vaccination initiated 196 decreased antibody-dependent complement deposition (ADCD; Figure 2A ), antibody-197 dependent neutrophil phagocytosis (ADNP; Figure 2B ) and NK cell cytokine production 198 (MIP1β or IFNγ; Figure 2C ). No differences were observed in NK cell degranulation as 199 measured by CD107a expression (Figure 2C ) or antibody-dependent cellular (monocyte) 200 phagocytosis (ADCP; Figure 2D ). It was of interest to note that in each instance samples from 201 monthly-high vaccinees performed comparably to DFx vaccinees. This suggested that there 202 was possibly an impact of the 'high' 50µg first and second doses (shared by these two groups) 203 on Fc-mediated functionality, independent from any effect of a DFx final booster. 204 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted April 27, 2022. Plasma from pre-vaccination and 2-weeks following the final vaccination was assessed for the capacity of anti-PfRH5 Ig to induce Fc-mediated 208 innate immune activation following incubation with PfRH5-coupled beads. The Fc-mediated functionality of post-vaccination anti-PfRH5 Ig was 209 compared between dosing regimen with respect to (A) antibody-dependent complement deposition (ADCD), (B) antibody-dependent neutrophil 210 phagocytosis (ADNP), (C) NK cell activation, and (D) antibody-dependent cellular (monocyte) phagocytosis (ADCP bead-positive cells) x (gMFI of bead-positive cells) / (10 x gMFI of the first bead-positive peak). (C) NK cells were purified from buffy coats and 215 incubated with antigen-coated ELISA plates, then stained to define NK cells as CD56+CD3-cells. Activation was measured as the percentage 216 of NK cells expressing MIP1β, CD107a, or IFN-γ as detected by fluorescent antibodies. (D) The functional capacity of pre-/post-vaccination 217 plasma to induce antibody-dependent monocyte phagocytosis was compared based on the capacity of anti-PfRH5-bound beads to induce 218 phagocytosis by the THP-1 (monocyte) cell line. Phagocytic score of each sample = (% bead-positive cells) x (geometric median fluorescence 219 intensity [MFI]) / (10x MFI of first bead-positive peak). Plasma was available from all vaccinees for inclusion in these analyses in technical 220

duplicates. Pre-vaccination/ post-vaccination: Monthly-low n = 12/12; monthly-medium n =11/11; DFx n = 11/11; monthly-high n = 9/9. 221

Comparisons between groups were performed by Kruskal-Wallis test with Dunn's correction for multiple comparisons. Whiskers denote 5 th and 222 95 th percentiles. * p < 0.05, ** p < 0.01, **** p < 0.0001. 223

To deconvolute these data, we took three approaches with our subsequent computational 224 analyses of the complete systems serology datasets. First, we focused on our original 225 research question by comparing all monthly versus DFx regimens (Figure 3A-C) . Next, we 226 limited this analysis to a direct comparison between the DFx and monthly-high vaccinees, 227 these differ only in their final vaccination and are thus optimal comparators (DFx versus 228 monthly-high; Figure 3D The first analysis indicates a feature set able to discriminate between DFx and monthly 240 regimens: FcRn-binding, IgG avidity, proportion of three different Fc glycans (G1FBG2, G2S2, 241 G2S2F), IgG4 (all higher in DFx vaccinees); and IgG3, FcαR-binding, ADCD, and ADCP, and 242 G1S1F glycan (all higher in monthly regimen vaccinees; Figure 3B ; Supplemental Figure  243 3). The co-correlate network additionally detects a significant negative association between 244 G2S2 and both NK cell (MIP1β and IFNγ) and complement cascade activation ( Figure 3C) . 245

Conversely, ADCD and ADCP are associated with the monthly regimens, as suggested by the 246 univariate data (Figure 2) . 247

With respect to the second analysis, direct comparison of the DFx regimen with only the 249 monthly-high regimen showed that increased FcRn-binding, IgG avidity, G2S2F and G2B 250 glycans, alongside decreased IgG3, ADCD, and G2S1F glycan, were able to significantly 251 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 27, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 discriminate DFx vaccinees ( Figure 3E; Supplemental Figure 3 ). The corresponding co-252 correlate network also identified positive correlations between FcRn-binding and FcR2B-or 253

FcR3AV-binding, as well as correlations between G2S2F and G2S2 (positive) and FcR3AV-254 binding (negative). 255 256 Finally, the computational analyses identified increased FcRn-binding, IgG2 and IgG4, in 257 addition to decreased IgM, ADCD, FcαR-binding, and G2F glycan as the significant feature 258 set to discriminate the 'high' dose vaccinees ( Figure 3H ). To note, the FcRn-binding signature 259 in this analysis is attributable only to the DFx samples: FcRn-binding levels separate DFx vs 260 monthly-high ( Figure 3E ) and removing FcRn-binding from the feature set, considered when 261 separating lower vs higher dose groups, does not impact separation (data not shown). The 262 selected features truly elevated in 'high' dosing are thus IgG2 and IgG4. This is consistent with 263 the divergent IgG subclass immunokinetics analysed by ELISA described above ( Figure 1D -264 G). No further correlations were identified in the co-correlates model with any of the three 265 parameters elevated in the 'high' dose groups ( Figure 3I) . 266 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. vaccinees were included in this analysis. Following normalisation of data, we identified the ten 308 genes with the highest variance across the entire sample set: MZB1, KNG1, LEO1, EAF2, 309 UBXN8, P2RY12, ZNF234, MRPL35, TNFRSF17, IGKV1-39. Two of these genes are 310 noteworthy for their associations with plasma cells that could be highly relevant for 311 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 27, 2022. ; https://doi.org/10.1101/2022.04.25.22274161 doi: medRxiv preprint understanding our humoral data set: MZB1 and TNFRSF17. MZB1also known as Plasma 312 cell-induced ER protein (pERp1)is a key effector of the transcription factor B that regulates 313 terminal plasma cell differentiation (Blimp1) and is of particular interest due to its reported 314 roles in antibody secretion, as well as plasma cell differentiation and migration to the bone 315 marrow (17-24). TNFRSF17also known as BCMAis the APRIL and BAFF receptor which 316 is restricted to mature B cells and plasma cells (including both short-lived and long-lived) and 317 required for survival of LLPCs in the bone marrow (25-27). 318 319 Next, we ran a PCA to identify the number of principal components appropriate for 320 downstream clustering and visualisation with UMAP ( Figure 4B ). Running UMAP on these 321 principle components, we observe phenotypically distinct populations of cells ( Figure 4C Table 1 ). The other clusters are comprised more evenly of 327 monthly-high regimen and DFx cells with the top genes for each as follows: MS4A1 (CD20) 328 and YPEL5 for Cluster 0; GAREML and PSD for Cluster 1; LDHA and CCR7 for Cluster 2; 329 and, MT-RNR2 and MUC3A for Cluster 3 (top 5 genes for each shown in Supplemental Table  330 1). While these gene rankings don't suggest specific B cell subsets for clusters 0-3, the 331 prevalence of Ig genes in cluster 4 suggest this may be an (antibody-producing) plasma cell 332 population. A heat map visualisation of the top 50 most differentially expressed genes between 333 the clusters additionally highlights increased expression among nearly all cells in cluster 4 of 334 further genes with potential relevance to Ig production or plasma cell phenotype. In addition 335 to MZB1 and TNFRSF17, this group of genes includes multiple encoding proteins with 336 functions related to the secretory pathway or protein production and trafficking e.g. PPIB, 337 ARF3, FKBP11, SEC11C, SSR4, BLOC1S5, and TXNDC5 ( Figure 4E) . 338 339 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 27, 2022. ; https://doi.org/10. 1101 /2022 To further probe this potential cluster 4 plasma cell phenotype, we plotted several of the genes 340 of interest as flagged above that should have clear positive or negative expression in a plasma 341 cell population: MS4A1 (CD20), CCR7, MZB1 and TNFRSF17. CD20 and CCR7 are both 342 downregulated during plasma cell differentiation (20, 28-30) and indeed here we see 343 decreased expression in cluster 4 as compared to clusters 0-3 ( Figure 4F-G) . Given that the 344 absence of CD20 expression on a B cell is the canonical definition of an antibody-secreting 345 cell (though other marker combinations can be used e.g. CD27hiCD38hi (28)), this is strong 346 evidence that cluster 4 is a plasma cell population. CD20 expression has also been used 347 previously to differentiate antibody-secreting ( ("TARTE_PLASMA_CELLS_VS_B_LYMPHOCYTE_UP" (32)) also yields a strong 353 statistically significant difference between cluster 4 and the other clusters ( Figure 4J) . 354 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 27, 2022. ("TARTE_PLASMA_CELLS_VS_B_LYMPHOCYTE_UP" (32)) was also compared between cluster 4 and the other clusters by Kruskal-Wallis 367 test (J). Finally, gene set enrichment analysis with the TARTE_PLASMA_CELLS_VS_B_LYMPHOCYTE_UP gene set was run with the 5,115 368 significant genes from DESeq2 analyses comparing gene expression in PfRH5-specific CD19+IgG+ B cells from monthly-high and DFx regimen 369 vaccinees (top twenty genes shown in Table 2 ). NES: normalised enrichment score; Leading Edge: subset of genes from gene set contributing 370 most to the enrichment signal. 371

We next proceeded to validate the indication from this single cell Seurat analysis that there 372 was a discrepancy in the proportion of plasma cells (cluster 4) within PfRH5-specific B cells 373 by dosing regimen using differential gene expression analyses between monthly-high and DFx 374 regimen vaccinees. A total of 5,115 genes were differentially expressed (adjusted p value 375 (padj) <0.05); the 30 significant genes with the greatest fold change in expression are 376 presented in Table 2 . Genes with increased expression in cells from monthly-high as 377 compared to DFx vaccinees include MZB1, IGJ (also known as J Chain and associated with 378 plasma cells (33) . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 27, 2022. and monthly-high dosing vaccinees. 391 Differentially expressed genes between DFx and monthly dosing samples were identified by 392

DESeq2 and filtered to remove pseudogenes, LincRNA, IncRNA, and non-significant genes 393 as defined as padj > 0.05. Genes were then ranked by fold change to show the top thirty genes 394 differentially expressed between monthly-high regimen and DFx. 395

Log2(FC): log fold change between monthly-high regimen and DFx; padj: adjusted p value 396

after Benjamini-Hochberg correction for multiplicity of testing. 397 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 27, 2022. ; https://doi.org/10. 1101 /2022 Further exploration of the significantly differentially expressed genes by gene set enrichment 398 analysis (GSEA) with the Kegg mSigDB gene sets indicated significant enrichment 399 (padj<0.05) in 10 pathways, including: cell adhesion molecules, protein export, and intestinal 400 immune network for IgA production (Supplemental Table 2 ). Protein secretion and mTORC1 401 signalling (also related to protein secretion) were similarly flagged during Hallmark GSEA but 402 no pathway reached statistical significance with an adjusted p value (Supplemental Table 3) . 403

A third GSEA with the plasma cell gene set used in Figure 4J here run on all significantly 404 differentially expressed genes rather than just cluster 4also showed a significant enrichment 405 (p<0.0001; NES=2.24; Figure 4K) . 406 407 Initially, the remaining disease-related pathways (Supplemental Table 2 (1), we were also interested to interrogate the BCR repertoire of the circulating 423

PfRH5-specific B cells to determine if there were differences in percentage germline mutation 424 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 27, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 or other divergent trends in clonotypes. Following analysis with the MiXCR pipeline to extract 425 CDR3 sequences from the scRNA-seq data set, we compared CDR3 length as well as heavy 426 and light chain CDR3 V-(D)-J percentage mutation from germline between monthly-high and 427

DFx vaccinees. Here, we observe minimal differences in CDR3 length (Figure 5A ), but 428 increased somatic hypermutation in the DFx regimen PfRH5-specific cells as compared to 429 those from the monthly-high regimen (Figure 5B-C) . Analysis of heavy chain V gene usage 430 showed substantial variation between individuals (Supplemental Figure 4) but broadly similar 431 patterns when comparing monthly-high regimen and DFx vaccinee groups (Figure 5D-E) . antibody-dependent NK cell activation as compared to monthly-low / monthly-medium. 488

Systems serology computational analyses indeed identified IgG2 and IgG4 as components of 489 the feature set that was associated with 'high' DFx / monthly-high. 490 491 An increase in antigen-specific IgG4 following DFx has been observed previously with the 492

PfCSP-based pre-erythrocytic malaria vaccine RTS,S (0-1-7mo in this instance; Fx017M) 493 when compared to a monthly 0-1-2mo regimen, as well as an IgG4-related decrease in ADCP 494 (38). In the context of PfCSP-mediated vaccines it appears that IgG4 inhibition of 495 opsonophagocytosis has a positive impact on protection, potentially related to a sporozoite 496 immune escape mechanism, but whether this would also be true for blood-stage parasites is 497 unknown. The authors comment that the prevailing view of chronic exposure to antigen as a 498 driver of IgG4 (reviewed in (39, 40)) seems to conflict with their data, whereas the relevance 499 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 27, 2022. ; https://doi.org/10.1101/2022.04.25.22274161 doi: medRxiv preprint appears clearer in our PfRH5 analyses whereby IgG4 is increased in both the DFx and 500 monthly-high groups primed with the highest 50 µg dose of RH5.1 vaccine. To the best of our 501 knowledge, there are no published data on the impact of relatively 'high' protein vaccine dosing 502 on induction of an IgG2 response and the interpretation of these results is not clear. 503

With respect to the DFx regimen specifically, the associated systems serology feature set, as 505 compared to all monthly regimen groups, consisted of increased FcRn-binding, IgG avidity, 506

IgG4, and proportions of three galactosylated Fc glycans (G1FBG2, G2S2, G2S2F) and 507 decreased IgG3, ADCD, ADCP, FcαR-binding, and G1S1F galactosylated Fc glycan. This 508 was refined when analyses were limited to a DFx versus monthly-high regimen comparison: 509 elevated FcRn-binding, IgG avidity, G2S2F and G2B Fc glycans, and decreased ADCD, IgG3, 510 and G1S1F glycan. Our data therefore suggest both a negative impact of 'high' first and 511 second doses (e.g. reduced capacity to induce ADCD) and a positive impact of the delayed 512 fractional final dose (e.g. increased avidity and FcRn-binding) on the antigen-specific Ig 513 response with the DFx regimen. This concept of a potential detrimental impact of excessive 514 antigen or antigen saturation on vaccine immunogenicity has previously been proposed by 515 others and merits further investigation, as do DFx regimens using lower priming doses (9, 41). 516

The avidity signal for DFx bolsters the conclusions of our previously published trial report with 518 a different assay (1), while the glycan, subclass and FcRn-binding data provide novel insights 519 into the profound modulation of the humoral response that can be achieved through 520 modification of dosing regimen. The importance of improved avidity in the specific context of 521

PfRH5-based vaccines is uncertain given previous work with mAbs (derived from samples 522 from the viral vector trial) indicated that the speed of antibody-binding, rather than avidity, is 523 more relevant for in vitro anti-parasitic functionality (1, 42). However, improved antibody avidity 524 is associated with increased protection against other pathogens (including pre-erythrocytic P. 525 falciparum malaria (7, 43-46)) and thus these DFx data may be of great interest to other 526 vaccine development programmes. Importantly, this serum avidity improvement is supported 527 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 27, 2022. ; https://doi.org/10.1101/2022.04.25.22274161 doi: medRxiv preprint by CDR3 sequence analysis of single cell sorted PfRH5-specific B cells where greater somatic 528 hypermutation was observed in DFx vaccinees as compared to monthly regimen vaccinees. 529

Further studies are also underway to interrogate the binding kinetics of mAbs derived from 530 monthly regimen and DFx RH5.1/AS01B vaccinees and the relationship to the in vitro 531 functional correlate of protection (growth inhibitory activity [GIA] (1)). 532

The biological implication of increased capacity of post-vaccination Ig to bind to FcRn is 534 noteworthy due to the central role of FcRn-binding in antibody longevity through promoting 535 recycling rather than lysosomal degradation of IgG in circulation; durability of mAbs can be 536 enhanced by modifying the Fc to improve FcRn-binding (47, 48). FcRn-binding can also be 537 ameliorated with more highly galactosylated Fc glycans (15) and, consistent with this, the 538 systems serology signature associated with the DFx samples included increased proportions 539 of two bi-galactosylated glycan moieties (G2B and G2S2F). Similarly, and consistent with the 540 avidity data, is evidence from influenza vaccine antibody analyses demonstrating increases in 541 sialylation (e.g. as in G2S2F) correlated with generation of higher affinity antibodies (15, 49). 542

It is therefore possible that in DFx vaccinees there is an increase in expression of the 543 glycosyltransferases responsible for adding galactose (B4GALT1) and sialic acid (ST6GAL1) 544 with functional significance for FcRn-binding / longevity (galactose) and avidity (sialic acid). 545

These hypotheses require further interrogation however, especially given the glycosylation 546 changes are more specific than increases in all bi-galactosylated or bi-sialylated moieties. To 547 note, although adjuvant selection can demonstrably impact peak antibody responses and 548 antibody quality (including Fc glycans), evidence of any effect on antibody long-term 549 maintenance is thus far limited to non-human primates (50, 51). 550 551 These changes in antigen-specific Ig quantity and quality thus suggest fundamentally different 552 B cell responses following the third dose in DFx versus monthly regimens. Based on observed 553 total IgG kinetics, we have previously hypothesised that the DFx regimen seeds a higher 554 frequency of LLPCs in the bone marrow (1) but empirical data on direct detection of antigen-555 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 27, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 specific B cells (of any phenotype) in circulation or in lymphoid tissue have not been reported. 556

We therefore first measured the frequency of peripheral antigen-specific cells within the 557 memory IgG+ B cell population. Here, we showed that the frequency of circulating antigen-558 specific B cells was significantly higher in DFx vaccinees at all time points measured. 559 However, we do not expect that a higher magnitude B cell response alone could be 560 responsible for the range of qualitative differences in the humoral response detailed above. 561

We therefore proceeded to scRNA-seq as an agnostic approach to qualitative antigen-specific 562 CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 27, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 At first glance, these scRNA-seq results suggesting greater IgG+ plasma cell activation in 583 monthly-high than DFx are surprising, especially when anti-PfRH5 serum IgG levels are 584 comparable between the two regimens at the peak time point used for this analysis (2-weeks 585 post final vaccination (1)) and subsequently higher in DFx vaccinees long-term. However, 586 selection of a single time point provides only a plasma cell "snapshot" rather than longitudinal 587 kinetics; it is entirely feasible that the DFx response actually peaked between 2-and 4-weeks 588 following final vaccination versus between 1-and 2-weeks with monthly-high. Furthermore, 589

LLPCs exit later than mBCs in germinal centre development and, while this has been 590 documented from 2-weeks post-vaccination onwards, it is possible that our scRNA-seq time 591 point is better suited to mBC and SLPC detection as compared to (later) LLPCs (53-55). 592

Accordingly, we propose that the Day 70 plasma cell signal in monthly-high vaccinees is 593 derived predominantly from mBCs that have differentiated into short-lived plasma cells 594 (SLPCs) following antigen re-exposure, rather than those that have returned to a draining 595 lymph node and differentiated into LLPCs via germinal centre reactions. It is also likely that 596 monthly regimen vaccinees still possessed PfRH5-specific germinal centres from the second 597 vaccination at the time of the third and final booster whichalongside higher concentrations 598 of anti-PfRH5 serum IgG at the time of vaccinationcould have dampened new germinal 599 centre responses (9, 56, 57) . This is consistent with HIV vaccinology data from non-human 600 primates where longer intervals between doses were associated with increased germinal 601 centre B cell responses (58). Germinal centre-independent SLPC differentiation in monthly-602 high vaccinees would also be consistent with the observed lower CDR3 somatic 603 hypermutation (and reduced serum IgG avidity) as compared to DFx vaccinees. 604 605 Another factor relevant for reconciling the serum IgG and plasma cell data is possible variation 606 in CD19 expression by different plasma cell populations. CD19 is a common marker for 607 identifying human B cells, but it has also been reported to be downregulated on terminally 608 differentiated bone marrow LLPCs as well as on a minority of circulating plasma cells following 609 vaccination (59-62). If peripheral LLPCs or LLPC-precursors downregulate CD19 they would 610 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 27, 2022. ; https://doi.org/10.1101/2022.04.25.22274161 doi: medRxiv preprint not have been captured for the scRNA-seq pipeline by the CD19+IgG+ gating strategy if truly 611 negative for CD19 (rather than CD19lo). This would again mean that the plasma cell 612 population identified in our analyses is skewed to SLPCs, while any differences in germinal 613 centre LLPC/precursor output between the two platforms are obscured. However, the 614 significance or likelihood of circulating CD19-LLPCs and LLPC-precursors is unclear, given 615 other reports of a putative LLPC-precursor population within the circulating CD19+ 616 compartment (63) and timing of CD19 downregulation only once cells reached the bone 617 marrow (59). 618 619 Future clinical trials should therefore seek to more precisely define this plasma cell population 620 kinetics with DFx versus monthly boosting and to confirm our hypothesised skew towards 621 LLPC and SLPC subsets, respectively. This distinction is currently difficult given the lack of 622 clear markers to resolve SLPCs from LLPCs / LLPC precursors in humans (reviewed in (64)), 623 although data suggest the transcription factor Zbtb20 may be used to define LLPCs in mice 624 ((65) no increased expression was observed in our putative SLPC population). Moving forward 625 will rely on more frequent venous sampling following the final vaccination, ideally coupled with 626 analysis of draining lymph node aspirates to directly monitor germinal centre formation / 627 longevity and define peripheral biomarkers of plasma cell output (57, 66, 67) . Bone marrow 628 aspirates may also be of use to confirm a link between higher serum anti-PfRH5 IgG 629 maintenance and presumed higher LLPC seeding in the DFx regimen, as well as with putative 630 LLPC precursor populations. 631 632 It will also be of great interest to better understand the discrepancy between the results 633 observed with DFx RH5.1/AS01B vaccination and similar Fx017M dosing with the RTS,S/AS01 634 which gave greater protection than the monthly regimen in controlled human malaria infection 635 (CHMI) studies (6-8, 41). Like with DFx RH5.1 dosing, the Fx017M RTS,S regimen increased 636 IgG avidity and (bulk plasmablast, i.e. SLPC) B cell somatic hypermutation after final 637 vaccinationwhich correlated with protectionbut not magnitude of the IgG response as 638 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 27, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 compared to the monthly 0-1-2-month regimen (7). However, there was no apparent Fx017M-639 mediated benefit to Ig longevity and thus durability of vaccine-mediated protection (7). This is 640 a critical distinction to understand in order to ensure relevance of DFx dosing to other antigens 641 and pathogens. However, the failure of Fx017M to improve Ig serum maintenance could be 642 related not to characteristics of the antigen, but of the platform; CSP is arrayed on a VLP which 643 could potentially form immune complexes affecting LLPC development (68) In conclusion, the data presented here support the DFx regimen as a more promising dosing 650 schedule as compared to more routine monthly dosing for optimising the humoral response 651 against difficult pathogens like blood-stage malaria that require high, sustained titres for 652 protection. The impact appears to be largely related to IgG1 but more subtle effects on other 653 isotypes and subclasses are also present. Two hypotheses regarding the underlying 654 mechanism of the improved plasma antibody longevity in the DFx schedule merit further 655 exploration: increased recycling through enhanced FcRn-binding, and a potential shift in B cell 656 fate from SLPC to LLPC following the delayed final dose. The mechanism for improved avidity 657 with DFx is also likely linked to germinal centre kinetics, potentially related to competition for 658 antigen which others have speculated is linked to the fractionation of the final dose rather than 659 the delay (7, 41, 71, 72). Further clinical trials will therefore be needed to directly compare the 660 impact of delayed boosting to the fractionation of the final dose, and alsofor antigens other 661 than PfRH5 such as PfCSPto delineate the possible roles of antigen and vaccine delivery 662 platform. 663 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Table 1 . Antibody and Tfh cell responses to the different 684 regimens have already been reported elsewhere (1, 10). Vaccinee age and sex were 685 comparable between regimens and are summarised in Table 1 is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 27, 2022. ; https://doi.org/10.1101 /2022 (10, 11) . This trial was also approved by the 691 CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 27, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 Memory PfRH5-specific IgG+ B cells identified as live PfRH5/APC+PfRH5/PE+ lymphocytes unless otherwise specified (Supplemental Figure 1 ) 719 and acquired on a Fortessa X20 flow cytometer with FACSDiva8.0 (both BD Biosciences). 720

Samples were analysed using FlowJo (v10; Treestar). Samples were excluded from analysis 721 if <100 cells in the parent population. (2020-04). 50µL of the secondary antibody dilution was added to each well of the plate and 741 incubated for 1 h at 37°C. Plates were developed using PNPP alkaline phosphatase substrate 742 (N2765, Sigma-Aldrich) for 1-4 h at 37°C. Optical density at 405 nm was measured using an 743 ELx808 absorbance reader (BioTek) until the internal control reached an OD405 of 1. The 744 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 27, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 reciprocal of the internal control dilution giving an OD405 of 1 was used to assign an AU value 745 of the standard. Gen5 ELISA software v3.04 (BioTek) was used to convert the OD405 of test 746 samples into AU values by interpolating from the linear range of the standard curve fitted to a 747 four-parameter logistics model. Any samples with an OD405 below the linear range of the 748 standard curve at the minimum dilution tested were assigned a minimum AU value according 749 to the lower limit of quantification of the assay. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) Computational analysis of systems serology data 800 Multivariate analysis of the systems serology data was performed in R with the following 801 approach. Features with missing measurements for more than 50% of subjects were removed 802 from this analysis. Missing values were then imputed using k-nearest neighbors (k = 3, R 803 package 'DMwR' v.0.4.1), and all data were mean-centred and variance-scaled ("z-scored"). 804

Univariate differences were assessed with Kruskal-Wallis tests, using the Benjamini-Hochberg 805 multiple hypothesis correction. Significant differences were then assessed in a pairwise 806 manner with Mann-Whitney U tests. Partial least-squares discriminant analysis (PLS-DA) was 807 performed using the 'ropls' (v.1.22.0) and 'systemsseRology' (v1.0) packages of R for model 808 building and cross-validation/visualization respectively. Significant features were chosen via 809 the LASSO feature selection algorithm, which was run 100 times on the entire dataset using 810 the function 'select_lasso' from the 'systemsseRology' R package (https://github.com/LoosC/ 811 systemsseRology). Features chosen in at least 80% of repetitions were used to build PLS-DA 812 classifiers. PLS-DA model performances were then assessed using a five-fold cross validation 813 approach and reported cross-validation accuracy is the mean of 10 rounds of 5-fold cross 814 validation, which includes 100 repeats of feature selection per fold per round. To assess the 815 importance of selected features, negative control models were built both by permuting group 816 labels and by selecting random, size-matched features in place of true selected features. 10 817 rounds of cross-validation with 5 permutation and 5 random-feature trials per round were 818 performed (again with 100 repeats of feature selection per fold per round for the permutation 819 trials), and exact p values were obtained from the tail probability of the generated null 820 distribution. Correlation networks were built to reveal additional serology features significantly 821 associated with the selected features. Serology features significantly (p < 0.05, after a 822

Benjamini-Hochberg correction) correlated (Spearman rs >|0.7|) via Spearman correlation 823 were selected as co-correlates. Correlation coefficients were calculated using the 'correlate' 824 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted April 27, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 Pathway analysis was also performed using Fgsea ( . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted April 27, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 Poulton. We also acknowledge technical laboratory support from David Ambrose, Andrew 876

Worth and Julie Furze, sequencing analysis advice from Jennifer Hillman-Jackson and Björn 877

Grüning. Finally, assistance with pilot work from Sarah Hamilton, and Marc Lievens, Danielle 878

Morelle and GSK for participation in the study design and supply of the AS01B adjuvant. Research (BMBF grants 031 A538A/A538C RBC, 031L0101B/031L0101C de.NBI-epi, 895 031L0106 de.STAIR (de.NBI)).The opinions expressed herein are those of the authors and 896 do not necessarily reflect the views of the US Agency for International Development. 897

GlaxoSmithKline Biologicals SA was provided the opportunity to review a preliminary version 898 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted April 27, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 

anti-human IgG-BB515 (564581), FVS780 (565388

as well as two fluorophore-conjugated PfRH5 probes (see flow cytometry 773 section for more details on probe production (10)). PfRH5-specific B cells identified as live 774

CD19+IgG+PfRH5/APC+PfRH5/PE+ lymphocytes were single cell sorted on a BD FACS Aria 775 (BD Biosciences) into Buffer TCL (1031576, Qiagen) with 1% 2-mercaptoethanol

mRNA from thawed single B cells was purified with RNAClean XP beads (A63987

Coulter) and converted to cDNA using dT30VN and TSO oligonucleotides and SMARTScribe 780 reverse transcriptase (639538, Clontech) with a modified Smart-Seq v4 for Ultra Low Input 781 RNA protocol (Takara Bio). Both steps were done in the presence of a recombinant RNase 782 inhibitor (2313B, Takara Bio). cDNA was then amplified with SeqAmp DNA

AC-60050, MagBio) were used to purify cDNA prior to quantification 786 with Qubit dsDNA HS Assay Kit (Q32854, Life Technologies) and cDNA normalisation

Sequencing libraries were created using the Nextera Index Kit v2

Reduced blood-stage 917 malaria growth and immune correlates in humans following RH5 vaccination

Malaria Vaccines: 920 Recent Advances and New Horizons

Immunogenicity of 922 standard and extended dosing intervals of BNT162b2 mRNA vaccine

Reactogenicity and 924 immunogenicity after a late second dose or a third dose of ChAdOx1 nCoV-19 in the UK: a substudy 925 of two randomised controlled trials (COV001 and COV002)

Single-dose 927 administration and the influence of the timing of the booster dose on immunogenicity and efficacy 928 of ChAdOx1 nCoV-19 (AZD1222) vaccine: a pooled analysis of four randomised trials

A preliminary 931 evaluation of a recombinant circumsporozoite protein vaccine against Plasmodium falciparum 932 malaria

Fractional Third 934 and Fourth Dose of RTS,S/AS01 Malaria Candidate Vaccine: A Phase 2a Controlled Human Malaria 935 Parasite Infection and Immunogenicity Study

Controlled Human Malaria Infection and Immunogenicity Study of RTS

Delayed Fractional Dose Regimens in Malaria-Naive Adults

A delayed fractionated 940 dose RTS,S AS01 vaccine regimen mediates protection via improved T follicular helper and B cell 941 responses

Protein/AS01B 943 vaccination elicits stronger, more Th2-skewed antigen-specific human T follicular helper cell 944 responses than heterologous viral vectors

Human vaccination against 946 RH5 induces neutralizing antimalarial antibodies that inhibit RH5 invasion complex interactions

Production, 949 quality control, stability, and potency of cGMP-produced Plasmodium falciparum RH5.1 protein 950 vaccine expressed in Drosophila S2 cells

Systems serology: profiling vaccine induced humoral immunity against 954 HIV

Transfer of maternal immunity and 956 programming of the newborn immune system

Reduced blood-stage malaria growth and 958 immune correlates in humans following RH5 vaccination

Mzb1 protein 960 regulates calcium homeostasis, antibody secretion, and integrin activation in innate-like B cells

Proteome Profiling Reveals Common Prevalence of MZB1-Positive Plasma B Cells in Human Lung and 967 Skin Fibrosis

Cochaperone 969 Mzb1 is a key effector of Blimp1 in plasma cell differentiation and beta1-integrin function

Increase of 972 MZB1 in B cells in systemic lupus erythematosus: proteomic analysis of biopsied lymph nodes

MZB1 folding and unfolding the role of IgA

Proteomics and 977 functional study reveal marginal zone B and B1 cell specific protein as a candidate marker of multiple 978 myeloma

Role of Polymeric Immunoglobulin Receptor in IgA and IgM Transcytosis. Int 980

BCMA is 982 essential for the survival of long-lived bone marrow plasma cells

Targeting B Cell Maturation Antigen (BCMA) in Multiple 984 Myeloma: Potential Uses of BCMA-Based Immunotherapy

Survival of Long-Lived Plasma Cells (LLPC): Piecing Together 986 the Puzzle

Challenges and 988 Opportunities for Consistent Classification of Human B Cell and Plasma Cell Populations

A coordinated change in 991 chemokine responsiveness guides plasma cell movements

Multifunctional role of 993 the transcription factor Blimp-1 in coordinating plasma cell differentiation

Defining antigen-996 specific plasmablast and memory B cell subsets in human blood after viral infection or vaccination

Gene expression profiling of plasma cells 999 and plasmablasts: toward a better understanding of the late stages of B-cell differentiation

Putting J chain back on the map: how might its expression define 1002 plasma cell development?

B is for 'Big Mac': GCs crave a high-fat diet

Germinal center B cells 1006 selectively oxidize fatty acids for energy while conducting minimal glycolysis

Human circulating specific antibody-forming cells after systemic and mucosal immunizations: 1010 differential homing commitments and cell surface differentiation markers

Delayed fractional 1016 dose regimen of the RTS,S/AS01 malaria vaccine candidate enhances an IgG4 response that inhibits 1017 serum opsonophagocytosis

IgG subclasses and allotypes: from structure to effector 1019 functions

A Temporal Model of Human IgE and IgG Antibody Function

Optimizing RTS,S Vaccination Strategies: Give It Your Best 1023 Parting Shot

Human Antibodies that Slow Erythrocyte Invasion Potentiate Malaria-Neutralizing Antibodies

Avidity as a determinant of the protective efficacy of human 1028 antibodies to pneumococcal capsular polysaccharides

Lack of 1030 antibody affinity maturation due to poor Toll-like receptor stimulation leads to enhanced respiratory 1031 syncytial virus disease

Antibodies with high avidity 1033 to the gp120 envelope protein in protection from simian immunodeficiency virus SIV(mac251) 1034 acquisition in an immunization regimen that mimics the RV-144 Thai trial

The potential significance of high avidity immunoglobulin G (IgG) for protective 1037 immunity towards SARS-CoV-2

Enhancing 1041 durability of CIS43 monoclonal antibody by Fc mutation or AAV delivery for malaria prevention

Drive B Cell Affinity Selection and Determine Influenza Vaccine Efficacy

Innate transcriptional 1046 effects by adjuvants on the magnitude, quality, and durability of HIV envelope responses in NHPs

Antibody 1049 avidity, persistence, and response to antigen recall: comparison of vaccine adjuvants. NPJ Vaccines

Single-cell analysis of 1052 human B cell maturation predicts how antibody class switching shapes selection dynamics

Short-lived and long-lived bone marrow plasma cells 1055 are derived from a novel precursor population

A Temporal Switch in the 1057

Germinal Center Determines Differential Output of Memory B and Plasma Cells

Cell fate dynamics and genomic programming of plasma cell 1060 precursors

Slow 1062 Delivery Immunization Enhances HIV Neutralizing Antibody and Germinal Center Responses via 1063 Modulation of Immunodominance

SARS-CoV-2 mRNA 1065 vaccines induce persistent human germinal centre responses

Elicitation of Robust Tier 2 Neutralizing Antibody Responses in Nonhuman Primates by HIV Envelope 1068 Trimer Immunization Using Optimized Approaches

A unique population of 1070 IgG-expressing plasma cells lacking CD19 is enriched in human bone marrow

Plasma Cells Are Contained within the CD19(-)CD38(hi)CD138(+) Subset in Human Bone Marrow

Early 1076 Emergence of CD19-Negative Human Antibody-Secreting Cells at the Plasmablast to Plasma Cell 1077 Transition

Differential 1079 transcriptome and development of human peripheral plasma cell subsets

Low CD21 expression defines a 1081 population of recent germinal center graduates primed for plasma cell differentiation

Cell fate dynamics and genomic programming of plasma cell 1084 precursors

The BTB-ZF transcription 1086 factor Zbtb20 is driven by Irf4 to promote plasma cell differentiation and longevity

Human germinal centres 1089 engage memory and naive B cells after influenza vaccination

The germinal centre B cell response to SARS-CoV-2

FcgammaRIIb 1093 controls bone marrow plasma cell persistence and apoptosis

Modelling the roles of antibody titre and avidity in protection from Plasmodium falciparum malaria 1096 infection following RTS,S/AS01 vaccination

Delayed fractional dosing with 1098

AS01 improves humoral immunity to malaria via a balance of polyfunctional NANP6-and Pf16-1099 specific antibodies

Safety and 1101 efficacy of the RTS,S/AS01E candidate malaria vaccine given with expanded-programme-on-1102 immunisation vaccines: 19 month follow-up of a randomised, open-label, phase 2 trial

Germinal center selection and the development of memory B and 1105 plasma cells

A library of 1107 functional recombinant cell-surface and secreted P. falciparum merozoite proteins

The Galaxy platform for 1110 accessible, reproducible and collaborative biomedical analyses: 2020 update

User-friendly, 1113 scalable tools and workflows for single-cell RNA-seq analysis

Reference-based 1115 RNA-Seq data analysis 2021

RNA-sequencing training and analysis suite using the Galaxy framework

Trimmomatic: a flexible trimmer for Illumina sequence data

STAR: ultrafast universal 1127 RNA-seq aligner

featureCounts: an efficient general purpose program for assigning 1129 sequence reads to genomic features

StringTie enables 1131 improved reconstruction of a transcriptome from RNA-seq reads

Integrated 1133 analysis of multimodal single-cell data

Fast, sensitive and 1135 accurate integration of single-cell data with Harmony

MiXCR: 1137 software for comprehensive adaptive immunity profiling

1139 the international ImMunoGeneTics information system

We thank the volunteers and clinical staff for participating in and running the clinical trials 874 essential for this study, especially Fay Nugent, Yrene Themistocleous, Alison Lawrie, and Ian