The Acquisition of Human B Cell Memory in Response to Plasmodium Falciparum Malaria

Immunity to Plasmodium falciparum (Pf), the most deadly agent of malaria, is only acquired after years of repeated infections and appears to wane rapidly without ongoing exposure. Antibodies (Abs) are central to malaria immunity, yet little is known about the B‐cell biology that underlies Pf‐specific humoral immunity. To address this gap in our knowledge we carried out a year‐long prospective study of the acquisition and maintenance of long‐lived plasma cells (LLPCs) and memory B cells (MBCs) in 225 individuals aged two to twenty‐five years in Mali, in an area of intense seasonal transmission. Using protein microarrays containing approximately 25% of the Pf proteome we determined that Pf‐specific Abs were acquired only gradually, in a stepwise fashion over years of Pf exposure. Pf‐specific Ab levels were significantly boosted each year during the transmission season but the majority of these Abs were short lived and were lost over the subsequent six month period of no transmission. Thus, we observed only a small incremental increase in stable Ab levels each year, presumably reflecting the slow acquisition LLPCs. The acquisition Pf‐specific MBCs mirrored the slow step‐wise acquisition of LLPCs. This slow acquisition of Pf‐specific LLPCs and MBCs was in sharp contrast to that of tetanus toxoid (TT)‐specific LLPCs and MBCs that were vi vi rapidly acquired and stably maintained following a single vaccination in individuals in this cohort. In addition to the development of normal MBCs we observed an expansion of atypical MBCs that are phenotypically similar to hyporesponsive FCRL4+ cells described in HIV‐infected individuals. Atypical MBC expansion correlated with cumulative exposure to Pf, and with persistent asymptomatic Pf‐infection in children, suggesting that the parasite may play a role in driving the expansion of atypical MBCs. Collectively, these observations provide a rare glimpse into the process of the acquisition of human B cell memory in response to infection and provide evidence for a selective deficit in the generation of Pf‐specific LLPCs and MBCs during malaria. Future studies will address the mechanisms underlying the slow acquisition of LLPCs and MBCs and the generation and function of atypical MBCs

Bar charts showing peak gamma frequency (a), M100 Latency (b), and baseline gamma (c), beta (d) and alpha amplitudes (e).

<p>(*<i>p</i><.05, **<i>p</i><.01, ***<i>p</i><.001, bars represent SEM).</p

Summary of total (evoked plus induced) amplitude differences in the experiment.

<p>a) Grand-averaged source localisation of gamma oscillations (40–80 Hz) for awake and sedated states respectively. Units are <i>t</i> statistics. The peak source location for the gamma band was at MNI co-ordinate [15–95 7] for awake and [17 97 1] for sedated (adjacent SAM voxels). b) Grand-averaged time-frequency spectrograms showing source-level oscillatory amplitude (evoked+induced) changes following visual stimulation with a grating patch (stimulus onset at time = 0) during awaked and sedated states. Spectrograms are displayed as percentage change from the pre-stimulus baseline and were computed for frequencies from 5 up to 150 Hz but truncated here to 100 Hz for visualisation purposes. c) Envelopes of oscillatory amplitude for the gamma (40–80 Hz) and alpha (8–15 Hz) bands respectively. Time-periods with significant differences between the awake and sedated states are indicated with a black bar (*<i>p</i><.05, **<i>p</i><.01, ***<i>p</i><.001, shaded areas represent SEM).</p

Summary of evoked amplitude differences in the experiment.

<p>a) Grand-averaged time-frequency spectrograms showing source-level oscillatory amplitude changes for the evoked response. b) Evoked amplitude spectra for the 0–0.2 s time period. c) Source-level time-averaged evoked responses for awake and sedated states. Significant differences were seen in the amplitude of the M100 and M150 responses (*<i>p</i><.05, **<i>p</i><.01, ***<i>p</i><.001, shaded areas represent SEM).</p

Experiment 4 – ERF results.

<p>A. The group averaged event related field (ERF) over the course of ‘seen’ and ‘unseen’ correct trials across TMS conditions. Data was band pass filtered (1 to 40 Hz). Shaded areas corresponded to standard error across subjects. B. Topographic representation of ERF distribution and channel section. The combined (across ‘seen’ and ‘unseen’ correct trials) data was used for channel selection, where the channels which expressed greatest deflection from baseline (−0.5 to 0 sec) in 0.1 to 0.4 second critical period following stimuli presentation were used.</p

Experimental Design.

<p>A. Example of arrow stimuli, noise (stimulus-absent) and task questions. The questions presented on every trial were ‘Was the arrow pointing left or right?’ denoted by ‘L R’ and ‘Did you see the arrow? Yes or No’ denoted by ‘Y N’. B. Time course of each trial. Fixation was followed by noise alternating at 50 Hz with a stimulus frame (20 ms) displayed at 800 ms on half of the trials. Responses to questions followed after a further 400 ms of noise and were not speeded. Questions commenced with the ‘L R?’ decision. C. Time course of the experiment. Behavioural (and MEG acquisition, see Experiment 4) blocks of eight minutes were collapsed into sixteen-minute analysis blocks, to align with the acquisition of MRS (see Experiment 3) and phosphene threshold data (see Experiment 2) acquisitions. Pre-TBS blocks were used to baseline the data. Active and control TMS were applied in separate sessions.</p

Experiment 4 – effects of cTBS on oscillatory responses.

<p>A. Time frequency induced responses to the presence of stimuli following cTBS and control stimulation. Data concatenated across post-TBS blocks. Highlighted regions indicate data used to derive dependent measures. B. Line plots illustrating change from pre-TBS baseine in the γ, α and β dependent measures under cTBS and control conditions. No statistically significant effects of the TMS were observed, but a trend for potentiated β ERD was observed which is consistent with previous research. γ cTBS <i>vs</i>. control <i>p</i> = 0.93, B_{(γ cTBS>sham)} = 0.44, α cTBS <i>vs</i>. control <i>p</i> = 0.28, B_{(α cTBS = 0.96, β cTBS <i>vs</i>. control <i>p</i> = 0.12, B_{(β cTBS = 1.85. Error bars are ±1 SEM.}}</p

Results of Experiment 3.

<p>A) Illustration of model fitting applied to MEGA-PRESS edited spectra that allowed for quantification of GABA concentration. Units are parts per million (ppm) of proton frequency. Glx is the combined glutamate and glutamine peak. NAA is the peak caused by N-acetyl aspartate. B) Illustration of the typical MRS voxel placement used here, as shown in the sagittal and axial view of one participant. C) Average change in GABA concentration followed occipital stimulation. The ordinate indicates change in GABA in institutional units (i.u) relative to the pre-TBS baseline, plotted according to the TMS condition (active cTBS <i>vs</i>. Sham control) and time after stimulation (mins). cTBS <i>vs</i>. control <i>p</i> = 0.034, B_(cTBS>sham) = 5.60. Error bars are ±1 SEM.</p

Experiment 4 – oscillatory results independent of TMS.

<p>A. Amplitude frequency distribution of combined (across ‘seen’ and ‘unseen’ correct) data sets under stimulus-present conditions, collapsed across time. Points highlighted are the initial frequency band pairings used for channel selection applied to the combined data set, the range of frequencies over which the ‘seen’ <i>vs</i>. ‘unseen’ correct contrast was applied, and the final set of band pairs upon which the dependent measures, applied to the TMS contrast, were based. B. Topographic distribution of synchrony levels and channels selected to be used in the ‘seen’ <i>vs</i>. ‘unseen’ correct contrast. Data used in the production of these plots was from the combined data set and averaged the oscillatory amplitude across the 0.1 to 0.4 second temporal epoch. Scales correspond to oscillatory amplitude in Tesla. Topographic plots are separated by the frequency bands over which mean amplitude was taken for channel section: i) γ ERS 61–71 Hz, ii) α ERD 8–14.5 Hz, and iii) β ERD 16.5–24 Hz.</p

Results of Experiment 2.

<p>The change in phosphene threshold from pre-TBS levels, following occipital cTBS and control stimulation. cTBS <i>vs</i>. control <i>p</i> = 0.04, B_(cTBS>sham) = 4.47. Error bars are ±1 SEM.</p