Author response: Panproteome-wide analysis of antibody responses to whole cell pneumococcal vaccination (2018)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Pneumococcal whole cell vaccines (WCVs) could cost-effectively protect against a greater strain diversity than current capsule-based vaccines. Immunoglobulin G (IgG) responses to a WCV were characterised by applying longitudinally-sampled sera, available from 35 adult placebo-controlled phase I trial participants, to a panproteome microarray. Despite individuals maintaining distinctive antibody 'fingerprints', responses were consistent across vaccinated cohorts. Seventy-two functionally distinct proteins were associated with WCV-induced increases in IgG binding. These shared characteristics with naturally immunogenic proteins, being enriched for transporters and cell wall metabolism enzymes, likely unusually exposed on the unencapsulated WCV's surface. Vaccine-induced responses were specific to variants of the diverse PclA, PspC and ZmpB proteins, whereas PspA- and ZmpA-induced antibodies recognised a broader set of alleles. Temporal variation in IgG levels suggested a mixture of anamnestic and novel responses. These reproducible increases in IgG binding to a limited, but functionally diverse, set of conserved proteins indicate WCV could provide species-wide immunity. Clinical trial registration: The trial was registered with ClinicalTrials.gov with Identifier NCT01537185; the results are available from https://clinicaltrials.gov/ct2/show/results/NCT01537185. https://doi.org/10.7554/eLife.37015.001 eLife digest Streptococcus pneumoniae is a bug that causes pneumonia and meningitis, killing around a million people each year. Vaccines now exist to protect young children against these diseases, but they are expensive and do not work against all the strains of the bacteria. This is because these shots train the body's immune system to recognize and attack the bacterium's capsule, a layer of sugars that surrounds the microbe and is often different between strains. One possible solution could be a cheap, whole cell vaccine. These injections expose the body to genetically modified S. pneumoniae that do not carry the capsule. Such treatment has now been tested in a small number of people during a clinical trial. Here, Campo et al. use a technique known as panproteome array to scan samples collected during this trial, and identify which elements the body learns to recognize when it is exposed to the genetically manipulated strain of S. pneumoniae. The results show that when volunteers receive this vaccine, their body targets proteins that the capsule normally shields from the immune system. Many of these proteins are very similar across all strains of S. pneumoniae, which means that the whole cell vaccine could potentially better protect against a broad spectrum of bacteria. However, further studies are needed to assess whether this is the case, especially in infants. https://doi.org/10.7554/eLife.37015.002 Introduction Streptococcus pneumoniae (the pneumococcus), commonly carried in the nasopharynx, is an important respiratory pathogen capable of causing pneumonia, bacteraemia and meningitis. The earliest recorded pneumococcal vaccinations consisted of two doses of heat-killed pneumococci cultured from the sputum of pneumonia patients, which resulted in limited protection against pneumococcal infections for a few months after inoculation (Maynard, 1915). Later pneumococcal vaccines used purified capsule polysaccharides, of which there are almost 100 immunologically distinguishable variants (Bentley et al., 2006), termed 'serotypes'. These formulations expanded from a bivalent formulation in the 1930s (Ekwurzel et al., 1938) to include 23 capsular polysaccharides by the 1980s (Mufson et al., 1985). Such formulations afford little protection to infants, however, as polysaccharides are T-cell-independent antigens that are not efficiently recognised by the immature adaptive immune system (Stein, 1992). Therefore, the most commonly used pneumococcal vaccines at present are protein-polysaccharide conjugate vaccines (PCVs), currently containing up to 13 different polysaccharides, each attached to a carrier protein (Nunes and Madhi, 2011). These vaccines elicit protective immune responses, even in young children, which prevent nasopharyngeal carriage as well as disease (Lee et al., 2014). The intrinsic disadvantage of PCVs is the limited number of serotypes against which they provide protection, resulting in serotype replacement disease that reduces their impact (Weinberger et al., 2011). The further expansion of their valency is limited by the complexity of their manufacture, which also makes them costly (Miller et al., 2011; Ray, 2002). Therefore, efforts have continued to develop alternative vaccines that are cheaper, generate T-cell-dependent responses to non-capsular antigens, and afford protection against all pneumococci. Whole cell-based vaccines (WCVs) present a possible solution, as they can be relatively inexpensively manufactured and present an almost full complement of antigens to the recipient's immune system. Rather than the historical precedent of killed clinical isolates, the WCV used in this study is a specifically engineered version of the unencapsulated strain S. pneumoniae RM200 (Lu et al., 2010b). A randomised double-blind phase I safety and immunogenicity trial of this WCV in 42 healthy U.S. adults was designed to compare four cohorts (Figure 1). One received placebo saline injections, while the other three received 100 μg, 300 μg or 600 μg doses of the pneumococcal WCV adsorbed to aluminium hydroxide adjuvant. Each individual was given three injections 28 days apart, and serum samples were taken pre-vaccination and at 28, 56 and 84 days subsequent to the first injection to assay for immunoglobulin G (IgG) responses. To quantify the potential multiplicity of immune responses, a set of available samples were analysed with a panproteome microarray including over 2100 probes (Croucher et al., 2017), most of which corresponded to full-length proteins, with others representing fragments of larger polypeptides. This provides information on responses to proteins encoded by the core and accessory genome, as well as multiple variants of diverse core loci (DCL), corresponding to genes that can be identified in almost all isolates based on their location in the chromosome and the domain structure of the translated protein, but which exhibit little detectable sequence similarity across the species. In S. pneumoniae, four such loci encode pneumococcal surface proteins A (PspA) and C (PspC), and the zinc metalloproteases A (ZmpA) and B (ZmpB). This study therefore aimed to identify the range and types of antigens to which IgG responses were mounted following WCV administration, and how these might vary between individuals. The availability of longitudinal samples from multiple trial participants also offered the opportunity to study differences between individuals' antibody repertoires. Finally, the trial structure allowed the kinetics of responses to successive doses of different concentrations to be to quantified. These analyses should provide information on whether the WCV is likely to consistently induce antibodies capable of recognising antigens conserved across S. pneumoniae isolates. Figure 1 with 1 supplement see all Download asset Open asset Flow chart describing the analysed samples. Forty-two healthy adult volunteers were randomly assigned to either a placebo control group, or one of three cohorts receiving different doses of the WCV, in a double-blind phase I trial. Serum samples were planned to be taken prior to the first dose, and 28 days after each of the three doses. Not all participants completed the trial, and therefore the set of available samples was limited. Those analysed in this study came from 35 of the trial participants, and are broken down by cohort and timepoint in this chart. https://doi.org/10.7554/eLife.37015.003 Figure 1—source data 1 Sample collection data. https://doi.org/10.7554/eLife.37015.005 Download elife-37015-fig1-data1-v2.tds Results Genomic analysis of the WCV formulation The WCV formulation contains chemically-killed S. pneumoniae RM200 cells (Malley et al., 2001), the genome sequence of which was aligned against the original progenitor, S. pneumoniae D39 (Ravin, 1959). Twenty recombinations distinguishing the pair were identified, one of which represented reversible inversion at a phase variable locus (Croucher et al., 2014). The generation of S. pneumoniae SIII-N, an intermediate genotype expressing the mucoid serotype three capsule (Ravin, 1959), likely accounts for eighteen of these recombinations, which span a total of 101 kb in the RM200 genome and include a recombination importing the serotype three capsule polysaccharide synthesis (cps) locus (Figure 1—figure supplement 1). RM200 was derived from Rx1, a spontaneous mutant of SIII-N that no longer expressed a capsule. Correspondingly, the cps locus contains two candidate mutations potentially responsible for this phenotype. An Arg320Cys substitution in the 6-phosphogluconate dehydrogenase protein replaces a catalytically-important arginine, which binds a pyrophosphate moiety, with a cysteine, which could interfere with the active site thiol group (Campbell et al., 2000). Similarly, the phosphoglucomutase protein has an Asn146Thr substitution that disrupts a Ser-His-Asn motif involved in divalent cation coordination, which is conserved across many orthologues (NCBI conserved domain cd05799). A subsequently-introduced recombination represents further engineering to replace the lytA gene with the Janus cassette (Sung et al., 2001), to reduce virulence and improve the yield of cells from culture (Berry et al., 1989; Lu et al., 2010b) (Figure 1—figure supplement 1). Alteration of the pneumolysin toxin gene was not associated with an inferred recombination, as only three bases were substituted, in order to remove the protein's cytolytic and complement-activating activity (Lu et al., 2010b). This change also resulted in insertion of the pDP28 shuttle vector (accession code KJ395591) at the adjacent site (Figure 1—figure supplement 1). Other than these known alterations, there were no large structural changes to the RM200 genome. The cryptic pDP1 plasmid of S. pneumoniae D39 was retained (Oggioni et al., 1999), and the large adhesin PclA was still present, but the glycoprotein PsrP, both known pneumococcal pili, and degradative zinc metalloprotease ZmpC were absent (Croucher et al., 2017). None of the DCL were affected by the recombinations, and consequently they were all similar to the alleles in D39. However, there was a nonsense mutation in the pspC gene which removed six of the eight choline-binding domains (CBDs), which may reduce the proportion of this protein attached to the cell surface. Stable antibody 'fingerprints' of vaccinated individuals Overall, 130 samples were studied from 35 of the 42 trial participants: all four timepoints were analysed for 27 individuals, at least the initial and final samples were available for a further two people, and at least one timepoint was analysed for a further six people (Figure 1). Of these, 35 samples were from the placebo group, 32 from cohort 1 (100 μg dose), 29 from cohort 2 (300 μg dose), and 34 from cohort 3 (600 μg dose). Each sample was treated as a biological replicate. No technical replicates were included, as analysis of pilot data obtained using a smaller version of the array found reproducible differences between individuals (Figure 2—figure supplement 1). This suggested results were consistent between array assays of samples from the same individual, and that the study should span the maximal number of trial participants, as between-individual variation might be an important factor in understanding the response to the WCV. To visualise the relative importance of these differences between trial participants, the IgG binding across all probes for each sample was projected in two dimensions using t-distributed stochastic neighbor embedding (t-SNE; Figure 2), which clusters together similar sets of multidimensional data. This revealed individuals had a distinctive antibody 'fingerprint' that was generally preserved over the course of the trial, independent of vaccination dose. A similar pattern was observed when the DCL were excluded from the analysis (Figure 2—figure supplement 2), or when only probes from core proteins conserved across related streptococcal species were considered (Figure 2—figure supplement 3). Therefore, these fingerprints do not appear to reflect individuals' distinctive histories of exposure to variable proteins, but instead unique patterns of IgG responses to common antigens. These were sufficiently robust not to be disrupted by WCV administration. Figure 2 with 3 supplements see all Download asset Open asset Variation in the immune profiles of trial participants. The IgG-binding data for each sample were projected across two dimensions using t-distributed stochastic neighbour embedding. Points are coloured according to the cohort of the individual contributing the sample. The shape represents the day of the trial on which the sample was collected. Ellipses surround sets of samples taken from the same individual. The separation of each ellipse shows the distinctive immune 'fingerprint' of each individual, which is maintained over the course of the trial. https://doi.org/10.7554/eLife.37015.006 Figure 2—source data 1 Panproteome array IgG binding data. https://doi.org/10.7554/eLife.37015.010 Download elife-37015-fig2-data1-v2.tds Despite this starting variation between trial participants, there was no overall significant difference in individuals' median IgG binding to S. pneumoniae proteins between cohorts before vaccination (Figure 4—figure supplement 1A; Kruskal-Wallis test, N = 29, χ2 = 0.20, df = 3, p = 0.98). Changes in IgG binding between the start of the trial, day 0, and the end, day 84, were quantified as Δ0→84 (Figure 4—figure supplement 1B). The cohorts did not differ in the distribution of median Δ0→84 per individual (Kruskal-Wallis test, N = 29, χ2 = 3.17, df = 3, p = 0.37), indicating that the WCV did not raise IgG binding to a high proportion of S. pneumoniae proteins across all members of the vaccinated cohorts. Restricting this analysis to the antibody-binding targets (ABTs), defined as those proteins associated with high IgG binding in the pre-vaccination sample (Croucher et al., 2017), also failed to detect overall heterogeneity in the distribution of individuals' median Δ0→84 between the groups (Kruskal-Wallis test, N = 29, χ2 = 4.63, df = 3, p = 0.20; Figure 4—figure supplement 1C). However, an ANOVA test of the fits of linear mixed effects models of Δ0→84 across probes (considered fixed effects) and individuals (considered random effects) found a significant improvement when cohort was also included as a fixed effect (χ2 = 8.96, df = 3, p = 0.030; see Materials and methods). To test whether this reflected biologically uninformative changes in the responses to poorly immunogenic proteins, this analysis was repeated using only the 1,584 'immunoreactive' probes, to which an IgG binding of at least one (i.e. twice the background level) was detected across the dataset. This found a more significant effect of including cohort as a fixed effect (χ2 = 11.8, df = 3, p = 0.0081), indicating there was a statistically, and potentially biologically, significant difference in the distribution of Δ0→84 between cohorts. Hence the WCV did have a detectable effect on vaccinated cohorts when considering per-probe, rather than per-individual, data. Combined with the observation that unique antibody fingerprints were maintained throughout the trial, this suggests the WCV either elevated responses to only a subset of proteins, or uniformly boosted multiple anamnestic responses in only a subset of individuals. WCV elicits elevated IgG to a specific minority of proteins Comparing the within-cohort medians of pre-vaccine IgG binding and Δ0→84 across all probes provided a simple approach to judging the relative contributions of these alternative explanations. If there were a broad response in only some individuals, the median Δ0→84 values would not be expected to vary much across probes, except for a general rise with higher pre-vaccine antibody binding, if this correlated with the strength of immunological memory. Alternatively, a strong response to only a subset of probes across individuals should result in much greater variation in within-cohort median Δ0→84. The plots revealed a statistically significant positive correlation between pre-vaccine-binding strength and post-vaccine rise for the three vaccinated groups (Pearson correlation coefficient, p < 10−16 for each), as expected for a uniform rise in IgG binding if many anamnestic responses were upregulated. By contrast, a negative correlation was evident in the placebo recipients, possibly representing regression to the mean (Figure 3A–D). Yet these general increasing trends for the vaccinated cohorts were small compared to the large increases in IgG binding to a particular subset of antigens. This indicates the post-vaccine changes primarily represented strong responses to a subset of proteins. Figure 3 Download asset Open asset Scatterplots showing the within-cohort median change in IgG binding to each probe over the course of the trial (Δ0→84) relative to the within-cohort median pre-vaccination IgG binding. (A–D) This relationship is shown for each probe on the proteome array using data from the 29 individuals with samples taken at the start and end of the trial. These are split into (A) the placebo group, (B) cohort 1 (100 μg doses), (C) cohort 2 (300 μg doses), and (D) cohort 3 (600 μg doses). The horizontal dashed line indicates the empirically-determined threshold for defining probes having undergone a substantial change in IgG binding during the trial (Figure 4—figure supplement 2). The Pearson's correlation statistics and gradient of the best-fitting linear model are annotated on each panel. (E–F) The relationship between the median Δ0→84 for each probe is shown for (E) a comparison of cohorts 1 and 3, and (F) a comparison of cohorts 2 and 3. https://doi.org/10.7554/eLife.37015.011 ABTs are enriched among immunogenic WCV proteins These plots indicated many probes associated with larger increases in IgG binding were already strongly recognised by adaptive immunity pre-WCV. This suggested WCV antigens, which were defined as being associated with Δ0→84 above the empirically-derived threshold Δ0→84 of 0.2 (Figure 4—figure supplement 2), would be enriched for ABTs, those proteins to which IgG binding was already high pre-WCV. ABT probes for 23 of probes above the threshold in cohort 1 a test, N = p = of probes in cohort 2 a test, N = p = and of probes in cohort 3 a test, N = p < These consistently ABTs were associated with IgG binding the indicate many ABTs did not a substantial The between those proteins to which there was little and those large increases in IgG binding was generally consistent both cohorts and between vaccinated cohorts (Figure these specific to ABTs were not by variation between individuals' antibody These results may WCV-induced responses being limited to those of the panproteome that are similar to proteins expressed by To test this an sequence threshold of at least between array and RM200 proteins was from the distribution of sequence (Figure 4—figure supplement all DCL proteins to could be to RM200 proteins with a similarity above the while proteins to a in these two to present and absent from the WCV a comparison of the median Δ0→84 using a test found no significant difference in the changes in IgG binding in cohort three between these sets of probes (Figure test, N = = p = However, an test to ABTs found antibody (Δ0→84) associated with the ABTs with a in RM200 to relative to the ABTs absent from the strain to Figure test, N = = p = This suggests a IgG response that primarily targets naturally immunogenic proteins expressed by the WCV, some ABTs present in the WCV still did not elicit antibody binding. Figure with supplements see all Download asset Open asset of WCV antigens from changes in IgG binding. (A) plots showing the median Δ0→84 cohort three for array probes, split by whether they proteins with orthologues in the WCV strain RM200 as or The shows the same but further the to probes representing the proteins as ABTs based on high pre-vaccination IgG binding. The the probes corresponding to ABTs with orthologues in RM200 by whether the protein on the array was also conserved with sequence in S. and S. 1). The number of probes in each is shown at the of the The results of and are annotated on each panel. (B) showing the and biological of changes in IgG binding following WCV in cohort 3. The horizontal shows the change in IgG binding between the day and day 84 samples from cohort three on a two The shows the B from the representing the of the of IgG binding between cohort three and the placebo Points corresponding to array probes with a p are coloured they a of or (C) of antigens. The distribution of ABTs, defined as high IgG in the pre-vaccination samples (Croucher et al., 2017), and the WCV antigens identified by either the or analyses are compared across different changes are not of carriage or array A possible factor in this analysis is the higher of sequence expected for those proteins that are similar in RM200 and the This may mean these greater Δ0→84 values could be the result of the array being more to IgG to recognising antigens. However, a comparison of Δ0→84 and sequence between the WCV and proteins on the array failed to of a general correlation between Δ0→84 and sequence between the array proteins and RM200 (Figure 4—figure supplement responses to more conserved proteins could also result from with the related Streptococcus and Streptococcus However, the overall distribution of Δ0→84 for ABTs conserved in these other species was not higher than to those ABTs greater (Figure was also no significant difference in the pre-vaccination IgG binding to ABTs in the WCV test, = p = to those conserved in S. and S. test, = p = Figure 4—figure supplement immune carriage of naturally unencapsulated is to a in the immunity by WCV. of antigens by analyses Rather than on a threshold of two commonly used in the analysis of data were to identify which proteins statistically significant IgG responses. the probes were included in to analysis of changes to have biological an which relative to a prior distribution from the was to of Δ0→84 across proteins in each of the vaccinated cohorts against the same in the placebo By only using two data from 29 individuals could be A for with an expected of 2), identified probes as having significant Δ0→84 values in the comparison of the placebo group with cohort 3, received the of the WCV. The of Δ0→84 in this cohort are compared with the of this change relative to the placebo group in Figure the for multiple no probes were associated with significant changes in IgG binding in the of the placebo group with cohorts 1 or a linear mixed effects model was used to the IgG binding at each timepoint as a linear response to random corresponding to the different trial participants, and two sets of fixed corresponding to the of vaccine and number of injections This the information from the longitudinal but was limited to the individual trial participants in the three vaccinated cohorts for these data were were used to identify proteins that IgG responses that with or with number of a for multiple of the proteins a significant This is likely to the of cohort in which the overall changes in IgG were smaller than those for cohort 1 (Figure 3). By contrast, probes a significant change in IgG binding with repeated with all but one of these probes IgG binding over the course of the trial 2). Of these, were also significant in the analysis of Δ0→84 values (Figure 4—figure supplement Therefore, this of using all individuals for initial and final samples were as well as the full longitudinal data for vaccinated individuals, identified a total of probes to which IgG binding 1). These were consistent with the probes identified by applying the empirically-derived threshold to Δ0→84 values from cohort three in Figure (Figure 4—figure supplement of the probes represented DCL to test whether the of this variation affected the model or multiple the and analyses were repeated using the This found of the probes were still associated with significant in IgG binding if the DCL were excluded from the with no being indicating these results were generally robust to changes in the set of probes on the array 2 and Figure 4—figure supplement for those probes that represented different of large proteins, the probes came from distinct protein of which were identified by both and (Figure 4—figure supplement Hence the analyses provide a consistent of the most immunogenic proteins in the WCV. 1 of WCV proteins, represented by probes associated with IgG binding following WCV by either the or linear mixed effects analyses 2). Each protein is by common or a and the sequence for the in S. pneumoniae it could be IgG-binding values were across all probes corresponding to the protein that were associated with a significant are as a with the range in