Dynamics of IgM and IgG responses to the next generation of engineered Duffy binding protein II immunogen: Strain-specific and strain-transcending immune responses over a nine-year period.

BACKGROUND: A low proportion of P. vivax-exposed individuals acquire protective strain-transcending neutralizing IgG antibodies that are able to block the interaction between the Duffy binding protein II (DBPII) and its erythrocyte-specific invasion receptor. In a recent study, a novel surface-engineered DBPII-based vaccine termed DEKnull-2, whose antibody response target conserved DBPII epitopes, was able to induce broadly binding-inhibitory IgG antibodies (BIAbs) that inhibit P. vivax reticulocyte invasion. Toward the development of DEKnull-2 as an effective P. vivax blood-stage vaccine, we investigate the relationship between naturally acquired DBPII-specific IgM response and the profile of IgG antibodies/BIAbs activity over time. METHODOLOGY/PRINCIPAL
FINDINGS: A nine-year follow-up study was carried-out among long-term P. vivax-exposed Amazonian individuals and included six cross-sectional surveys at periods of high and low malaria transmission. DBPII immune responses associated with either strain-specific (Sal1, natural DBPII variant circulating in the study area) or conserved epitopes (DEKnull-2) were monitored by conventional serology (ELISA-detected IgM and IgG antibodies), with IgG BIAbs activity evaluated by functional assays (in vitro inhibition of DBPII-erythrocyte binding). The results showed a tendency of IgM antibodies toward Sal1-specific response; the profile of Sal1 over DEKnull-2 was not associated with acute malaria and sustained throughout the observation period. The low malaria incidence in two consecutive years allowed us to demonstrate that variant-specific IgG (but not IgM) antibodies waned over time, which resulted in IgG skewed to the DEKnull-2 response. A persistent DBPII-specific IgM response was not associated with the presence (or absence) of broadly neutralizing IgG antibody response.
CONCLUSIONS/SIGNIFICANCE: The current study demonstrates that long-term exposure to low and unstable levels of P. vivax transmission led to a sustained DBPII-specific IgM response against variant-specific epitopes, while sustained IgG responses are skewed to conserved epitopes. Further studies should investigate on the role of a stable and persistent IgM antibody response in the immune response mediated by DBPII.

Introduction

Plasmodium vivax is characterized by dormant liver stage hypnozoite-parasites responsible for high frequency of relapses [1], which imposes a challenge for the current policies of malaria control and elimination. With great potential for transmission from first generation of blood-stage infection [2, 3] and lower levels of parasitemia often undetected by routine surveillance [4, 5], the proportion of malaria infections attributed to P. vivax has increased in areas of relatively low transmission [6].

Although there are major hurdles for vivax malaria elimination, clinical immunity to P. vivax is acquired much more rapidly than for P. falciparum [revised in [7, 8], even in low transmission settings, which make the development of an effective vaccine worth pursuing. Duffy binding protein II (DBPII) is a leading P. vivax malaria vaccine candidate that binds the Duffy antigen receptor for chemokines (DARC) on reticulocytes is critical for reticulocyte invasion [9, 10]. Although naturally acquired DBPII antibodies tend to be biased towards strain-specific responses [11-13], our project identified the epitope targets of protective neutralizing IgG antibody response to overlap conserved residues essential for receptor binding and DBP dimerization [12, 14–19]. Individuals able to produce these broadly binding-inhibitory antibodies (BIAb) to DBPII present reduced risk of clinical P. vivax malaria [20, 21]. In pursuing a structural vaccinology approach, our project created surface-engineered DBPII vaccine candidate, DEKnull-2, that retains the conserved functional epitopes needed for receptor binding and DBP dimerization but removed residues of variant nonfunctional epitopes associated with strain-specific immune responses [22]. Naturally-occurring protective immunity associated with induction of long-term memory IgG responses have anti-DBPII BIAb and DBPII reactive cells that are highly reactive with DEKnull-2.

While it is well established that naturally acquired IgG antibody responses are associated with protective clinical immunity to blood-stage malaria [23, 24], the role of IgM is not well defined [25, 26]. A recent study in a murine model of malaria demonstrated that Plasmodium-specific IgM memory B cells are somatically hypermutated, high-affinity, and dominate the early memory response to recurring malaria infections [27]. Likewise, the production of protective IgM antibodies during experimental malaria provides evidence of additional mechanisms by which the immune system controls Plasmodium infection [28, 29]. These results might explain recent data associating the depth and breadth of Plasmodium-specific IgM antibodies with genetic resistance to malaria infection [30], and with the reduced risk of clinical malaria in a cohort of children [31]. Taken together, we are in accordance with other that suggest that IgM antibodies seem to be much more than just an early responder to malaria infection [32], and should be investigated during the development of vaccines.

Therefore, the goal of this study is to understand the mechanisms that underlie the broader humoral immune responses against the novel engineered DBPII vaccine candidate, DEKnull-2 [22], including the IgM response. For that, we took advantage of the long-term follow-up study previously carried out in the Amazon rainforest, where different profiles of DBPII-specific IgG responders were identified [33]. We have examined the frequency and distribution of DBPII-specific IgM and IgG antibodies during a 9-years follow-up period. As IgM antibodies may be necessary to sustain an optimal long-term protective IgG response [34-36], we also investigate whether a stable DBPII-specific IgM response could interfere with the profile of antibodies able to block the interaction ligand-receptor.

Materials and methods

Study area and population

The study was carried-out in the agricultural settlement of Rio Pardo (1°46’S—1°54’S, 60°22’W—60°10’W), Presidente Figueiredo municipality, Northeast of Amazonas State in the Brazilian Amazon region. The study site and malaria transmission patterns were described in detail elsewhere [37-39]. In this area, malaria transmission is considered hypo to mesoendemic, and the majority of residents were natives of the Amazon region [37]. Inhabitants of the settlement live on subsistence farming and fishing along the small streams. In the study area, P. falciparum malaria incidence has decreased drastically in recent years, and P. vivax is now responsible for all clinical malaria cases reported (S1 Fig).

Study design and cross-sectional surveys

A population-based open cohort study was initiated in November of 2008, and included three cross-sectional surveys carried at six-months interval (baseline, 6 and 12-months) as previously reported [37, 39]. Briefly, (i) interviews were conducted through a structured questionnaire to obtain demographical, epidemiological, and clinical data; (ii) physical examination, including body temperature and spleen/liver size were recorded according to standard clinical protocols; (iii) venous blood was collected for individuals aged five years or older (EDTA, 5 mL), or blood spotted on filter paper (finger-prick) for those aged <5 years; and (iv) examination of Giemsa-stained thick blood smears for the presence of malaria parasites by light microscopy. The geographical location of each dwelling was recorded using a hand-held 12-channel global positioning system (GPS) (Garmin 12XL, Olathe, KS, USA) with a positional accuracy of within 15 m. Additional cross-sectional surveys were carried-out six (August 2014), seven (July 2015) and nine years later (July 2017) [22, 33]. During the long-term follow up study, the number of malaria cases fluctuated in the study area, reflecting period of high (I and III) and low (II) malaria transmission (S1 Fig). For the current study, the non-eligible criteria were (i) refusal to sign the informed consent; (ii) children, as clinical immunity is not prevalent in Amazon children [40]; (iii) pregnant women; (iv) any other morbidity that could be traced; and (v) individuals who were unable to be recruited during at least two consecutive cross-sectional surveys. The 163 participants who were eligible to the current study matched the original adult population (n = 300) for age, sex, malaria exposure [33]. Eighty-eight (54%) and 77 (47%) out of 163 eligible subjects could be recruited six and nine years later, respectively. Fifty-seven (35%) subjects presented consecutive samples throughout the 9-years follow-up period.

The ethical and methodological aspects of this study were approved by the Ethical Committee of Research on Human Beings from the René Rachou Institute (Reports No. 007/2006, No. 07/2009, No.12/2010, No. 26/2013 and CAAE 50522115.7.0000.5091), according to the Resolutions of the Brazilian Council on Health (CNS-196/96 and CNS-466/2012).

Laboratory diagnosis of malaria

At the time of blood collection, all individuals were submitted to a finger-prick for malaria diagnosis by light microscopy. The Giemsa-stained thick blood smears were prepared and examined by experienced local microscopists, according to the malaria diagnosis guidelines of the Brazilian Ministry of Health (2009) [41]. Species-specific PCR assays targeting different plasmodial targets (18S rRNA gene and non-ribosomal Pvr47/Pfr364 sequences) were carried-out essentially as previously described [42]. For this, genomic DNA was extracted from either whole blood samples collected in EDTA, or from dried blood spots on filter paper using the Puregene blood core kit B (Qiagen, Minneapolis, MN, USA) or the QIAmp DNA mini kit (Qiagen), respectively, according to manufacturers’ instructions.

Recombinant blood stage P. vivax proteins

DBPII-related antigens

Recombinant DBPII-related proteins included amino acids 243–573 of the Sal-1 reference strain, DBPII-Sal1 [43], and an engineered DBPII termed DEKnull-2 [22]. These proteins were expressed as a 39kDa 6xHis fusion protein, properly refolded, as previously described [16, 22].

Immunoglobulin (Ig) M and IgG detection assays

A conventional enzyme-linked immunosorbent assay (ELISA) for antigen-specific IgM and IgG antibody response was carried out as previously described [38], with plasma samples diluted at 1:100 (IgG) or 1:400 (IgM). Recombinant proteins were used at a final concentration of 3 μg/ml (DBPII and DEKnull-2). For each protein, the results were expressed as ELISA reactivity index (RI), calculated as the ratio of the mean optical density (OD at 492 nm) of each sample to the mean OD plus three standard deviations of samples from 20–30 unexposed volunteers. Values of RI > 1.0 were considered positive.

Anti-DBPII erythrocyte-binding-inhibitory antibodies

The functional proprieties of DBPII antibodies (binding-inhibitory activity, BIAbs) were performed on a subset of the study population comprising 57 individuals matched for age, sex and malaria exposure. Plasma samples were tested for inhibition of DBPII-erythrocyte binding at 1:40 dilution by the standard COS7 cell assay as described [44]. A pool of P. vivax immune serum able to inhibit erythrocyte binding, and naïve serum was used as positive and negative control respectively. Binding was quantified by counting rosettes observed in 10–20 fields of view (x200). Percent binding-inhibition was quantified by assessing the percentage of rosettes in wells of transfected cells in the presence of test plasma relative to rosettes in wells in the presence of negative control plasma sample. Plasma samples with more than 50% inhibition of DBPII-erythrocyte binding were considered inhibitory.

Statistical analysis

A database was created using Epidata software (http://www.epidata.dk). The graphics and the analysis were performed using GraphPad Prism version 7—GraphPad Software, La Jolla California USA, and R statistical software (version 3.3.3). Differences in proportions were evaluated by chi-square (Χ2) test or Fisher’s exact tests, as appropriate. The Shapiro-Wilk test was performed to evaluate normality of variables. Differences in means were tested using either the one-way ANOVA, with Turkey’s post hoc, or the Mann-Whitney test or Kruskal–Wallis tests, with Dunn’s post hoc test, as appropriate. Linear correlation between variables, such as levels of antibodies and recent episodes of malaria, was determined by using the Spearman’s correlation coefficient. Only variables associated with statistical significance at the 5% level were maintained in the final models.

Results

Subject characteristics and antibody profiles to DBPII-related antigens at enrollment

The median age of individuals included in the study was 42 years (IQR: 28–53) with a 1.1:1 proportion of male to female (Table 1). The age was significantly associated with a subject’s time of malaria exposure in the Amazon area (r = 0.75; p<0.0001, Spearman’s correlation test). At the time of their first blood collection, the overall prevalence of malaria was 13%, with all infections caused exclusively by P. vivax; 3% of infections were detected by conventional microscopy, and 10% by a species-specific Real-Time PCR.

Table 1

Demographic, epidemiological and immunological characteristics of 163 individuals at enrolment.

Characteristics
Median age, years (IQR)		42 (28–53)
Gender, male: female		1.1: 1
Previous malaria self-reported episodes, median (IQR)		5 (3–15)
Years of residence in Amazon area, median (IQR)		35 (24–50)
Location of residence in Amazon area, riverine: non-riverine		1: 1
Acute P. vivax infection:
	Patent P. vivax infection1, n (%)	5 (3)
	Sub patent P. vivax infection2, n (%)	17 (10)
	Total, n (%)	22 (13)
Antibody response3, positive n (%):
IgM*	DBPII-Sal1	64 (39)
	DEKnull-2	37 (23)
IgG	DBPII-Sal1	75 (46)
	DEKnull-2	69 (43)

IQR = InterQuartile Range.

1Positive P. vivax infections detected by conventional light microscopy.

2Positive P. vivax infections detected by real-time PCR.

3Evaluated by conventional ELISA serology using recombinant proteins against P. vivax Duffy binding protein region II (DBPII).

*Statistically different (Chi Square test with Yates correction, X2 = 9.698; p = 0.018).

To determining whether DBPII antibody responses included both strain specific and broadly reactive antibodies, two different DBPII allelic variants were tested, Sal1, a common variant circulating in the study area and the DBPII reference strain, and the engineered DEKnull-2 whose antibody response target conserved DBPII epitopes. While 39% of the individuals enrolled in the study had IgM antibodies to DBPII-Sal1, only 23% had detectable IgM antibodies to the conserved DEKnull-2 (X2 = 9.698 p = 0.018). Considering IgG antibodies, a similar proportion of individuals (46–43%) responded to each of recombinant protein assayed (p>0.05).

Composition and dynamics of strain-specific and strain-transcending DBPII antibody repertoire over a nine-year period

IgM DBPII-related response

Over three cross-sectional surveys at 6-month intervals (high transmission, Phase I), between 40% and 37% of individuals had DBPII-Sal1 IgM antibodies, as detected by conventional serology (S2 Fig). Broadly reactive antibodies (DEKnull-2) were detected at significantly lower frequencies (24–16%). During all follow-up period DBPII-Sal1 IgM antibodies predominate over DEKnull-2 antibodies, and it was independent of the levels of malaria transmission in the study area (phase I, II and III). The levels of antibodies (evaluated here by medians of reactivity) showed a similar tendency towards strain-specific IgM antibodies (S2 Fig and S1 Table).

IgG DBPII-related response

The frequencies and levels of IgG antibody response to both DBPII proteins were similar over the first 12-months period (phase I) (S3 Fig). Despite of that, antibody levels varied among responders, including individuals with strong IgG antibody response (RI > 10) to both DBPII-related antigens. Of interest, the intensity of malaria transmission influenced in the proportion of Sal-1 versus DEKnull-2 immune responses. In the low transmission period (phase II), a significant decrease in strain-specific IgG antibodies was observed while the frequencies and levels of DEKnull-2 remained similar to the baseline (47% vs. 45–39%) (S3 Fig and S1 Table). The profile of DEKnull-2 over Sal1-specific IgG response was maintained until the end of the study (phase III, high transmission).

Ratio IgG to IgM antibodies

The ratio IgG/IgM to each recombinant protein confirmed that IgG but not IgM Sal1-antibodies were sensitive to malaria transmission intensity (Fig 1). Anti-Sal1 IgG antibodies decreased during a transmission period, and this profile remained until the end of the study. The low malaria transmission period strengthened DEKnull-2 antibodies, especially for IgG antibodies (Fig 1B).

Fig 1

Ratio of IgG to IgM antibody responses against DBPII-Sal1 and DEKnull-2 during the 9 years follow-up study period.

(A) The ratio between IgG and IgM response against DBPII-Sal1 (blue line) and DEKnull-2 (red line) for all subjects enrolled in the study was represented for each cross-sectional survey. The longitudinal study comprised six cross-sectional surveys, which included periods of high (phase I and III) and low (phase II) malaria transmission; the first three cross-sectional surveys were carried-out during the first year (baseline, 6 and 12 months), and three carried-out 6th, 7th and 9th years later. Individual responses towards IgG were represented on left (white) and towards IgM on right (grey). Individuals with equal IgG and IgM responses were show on central dotted line. (B) Pie charts representing the percentage of subjects with the following IgG and IgM response profiles: DBPII-Sal1 skewed, equal, and DEKnull-2 skewed during the three phases of transmission.

Influence of acute infection in the DBPII antibody repertoire

During the 9-years follow-up study period, 42 (26%) out of 163 studied individuals had a detectable P. vivax infection, most of them detected only during the first 12-month period (n = 36, Phase I). Although this subgroup was not differentiated from the study population by age (42 vs. 39 yrs-old), gender (1.3:1 vs. 1.1:1), or time of malaria-exposure (34 vs. 33 yrs), the majority of them were classified as riverine population (30 out of 42, 71%).

In these long-term exposed individuals, there was a predominance of sub patent (PCR-positive) over patent (microscopy-positive) P. vivax infections, and acute infection was not associated with the presence of either IgM or IgG antibody responses to any of the proteins, i.e., Sal1 or DEKnull-2 (Fig 2). Specifically, individuals with persistent malaria infection (for example, RP553 and RP555) did not present DBPII-related IgM antibodies, and vice-versa, a persistent IgM antibody response to both proteins (RP416 and RP516) was not associated with a potential booster by blood-stage infections. The absence of correlation between infection and antibody response was also observed for IgG antibodies (RP405, RP416 and RP433). This data is confirmed by the Spearman's correlation coefficient between IgG and IgM antibody responses (S4 Fig).

Fig 2

IgM and IgG antibody responses to DBPII-Sal1 and DEKnull-2 during P. vivax infections.

Each line represents one subject who had detectable P. vivax blood stage infection at any time of the follow-up study. The longitudinal study comprised six cross-sectional surveys, which included periods of high (I and III) and low (II) malaria transmission; the first three cross-sectional surveys were carried-out during the first year (zero, 6 and 12 months; 0-12m), and three carried-out 6th, 7th and 9th years later (6-9y). Forty-two subjects were positive for P. vivax infections and classified into sub patent and patent infection accordingly to PCR or conventional microscopy diagnosis, respectively (red color variation). In the right panel, the green color variation in the heatmap shows IgM and IgG responses for each cross-sectional survey. ELISA antibody responses were expressed as Reactivity Index (RI) calculated by dividing the mean optical density (OD at 492 nm) of each sample to the mean OD plus three standard deviations of samples from unexposed volunteers. Values of RI > 1.0 were considered positive (on a green scale).

IgG and IgM antibodies repertoire according to the immunological background

Plasma samples were screened for anti-DBPII BIAbs to investigate the relationship between IgM/ IgG antibody responses and the profile of BIAbs responders’ DBPII inhibitory immune responses. These were classified as persistent non-responders (NR) characterized by the absence of BIAbs antibodies; temporary responders (TR) whose BIAbs response alternated between positive/negative, and persistent responders (PR) whose BIAbs were stable throughout the study (Fig 3). During the low transmission period (Phase II), IgG responses were consistently detected against both recombinant proteins (DBPII-Sal1 and DEKnull-2) for the persistent BIAbs responders and, less with the temporary BIAbs responders. All but one (RP416) of PR subgroup (93%, n = 15) had DEKnull-2 IgG antibodies, while only 7 out of 13 (54%) of TR reacted to DEKnull-2. As expected, the majority of NR did not have detectable long-term IgG. IgM antibody responses were not associated with any profile of BIAbs responders, with some individuals with broadly and long-term IgM responses detected in all subgroups (Fig 3).

Fig 3

Profile of IgM and IgG antibody responses of Rio Pardo subjects previously classified according to the DBPII Binding Inhibitory Antibodies (BIAbs).

According to their BIAbs response, malaria-exposed individuals (n = 57) were previously characterized as [22]: i) Persistent responder (PR), who had BIAbs response during the 9 years of follow-up; ii) Temporary responders (TR), who had variable BIAbs response during those cross-sectional surveys and iii) Persistent non-responders (NR), who had no BIAbs detected any time in the study. Each line represents individual IgM and IgG antibody responses against DBPII-Sal1 or DEKnull-2, in each cross-sectional survey. Colored symbols indicate positive antibody response at ELISA (IR > 1) and white symbols negative response at ELISA (IR ≤ 1). ELISA results were expressed as Reactivity Index (IR), with Reactivity Index (RI) > 1 considered positive.

Discussion

In an Amazonian community exposed to low levels intermittent malaria transmission, we sought to investigate the relationship between anti-DBPII IgM and IgG antibody responses reactive with strain-specific (Sal1) or strain-transcending (DEKnull-2) immune responses [22]. In general, the frequencies and levels of IgM antibodies showed a tendency towards strain-specific antibodies (Sal1). Interestingly, the response profile of Sal1 over DEKnull-2 IgM antibodies was sustained throughout the 9-years observation period, including in consecutive years in which malaria transmission dropped drastically in the study area (Phase II). Although the majority of high IgM responses (RI> 3) were no longer detected in the low transmission period, the results suggest that individuals in P. vivax-endemic Amazonian communities were able to sustain their DBPII-specific IgM antibody responses. These results may explain recent findings showing that IgM-expressing memory B cells are expanded in malaria patients living in endemic areas [27], and confirm data from other studies, which suggest that IgM antibodies may play an underappreciated role in immune response against malaria infections [29-31]. Although scant longitudinal data are available about secondary IgM responses in P. vivax-exposed populations, a recent prospective study undertaken in a low transmission area of Western India demonstrated that P. vivax alters peripheral B‐cell profiles and induces parasite-specific IgM that persisted post recovery [45]. While the results from India are in concordance with a persistent IgM response, antigen-specific antibody responses were evaluated only over a short period-of time, i.e., during acute infection and upon 30 days post‐treatment. Here, we demonstrated that long-term P. vivax exposure to low and unstable levels of malaria transmission can lead to a sustained DBPII-specific IgM response. At this time, it is not possible to define if this persistent DBPII-related IgM response could indicate that IgM-experienced B cells needs to be constantly activated or if this response is rather associated with a bona-fide memory [29, 46]. Of relevance, P. falciparum-specific IgM antibodies were detected for more than 6 months in Australians returning from malaria endemic areas [31]. Together, the findings of long-term IgM response emphasize the need to understand the role of the IgM specific antibodies in both natural infection and vaccine antigens.

Our results showing an absence of correlation between antigen-specific IgM antibodies and acute malaria infection are intriguing, considering that in response to infection the antibodies made initially are usually IgM confined to the intravascular pool [26, 47]. Although unknown, we hypothesize that antigenically distinct DBPII variants could be responsible to new blood-stage infections, including relapses. Still, this hypothesis may not explain many of the acute infections since Sal1 was the most prevalent DBPII variant circulating at that time in the study area [37]. As sub patent infections predominate in the study area (77% by PCR vs. 23% by microscopy), one may speculate that these low parasite densities were not sufficient to provide a booster of IgM antibody response. However, recent findings in experimental malaria infections demonstrated that secondary IgM response was not affected by either challenge dose or the time of rechallenge [27]. Perhaps a more plausible and not mutually exclusive explanation for why IgM antibodies were not associated with peripheral blood stage infections is the relative underrepresentation of P. vivax asexual stages in patient blood [48, 49]. Nowadays, a considerable body of evidence indicates a tissue reservoir, such as bone marrow, where most P. vivax parasite burden resides [50]. Consequently, we cannot exclude that P. vivax-specific IgM production is dependent upon tissue parasite persistence, a phenomenon that needs to be investigate. In different experimental models, low-level of chronic infection may provide sufficient antigen to maintain IgM plasmablasts in the bone marrow either through inflammation [51] or antigenic stimulation [52].

From high to low malaria incidence in two consecutive years of the study was critical to elucidate the relative contribution of IgG versus IgM antibodies to DBPII immune response. Variant-specific IgG but not IgM antibodies waned over time, which resulted in IgG skewed reactivity to the DEKnull-2 antigens lacking the variant epitopes but retaining the conserved epitopes. This profile of IgG response was not unexpected as we previously demonstrated that a significant number of long-term malaria-exposed individuals mount a strong and stable IgG response toward conserved DEKnull-2 epitopes [22, 33]. Of further interest, the levels of IgM antibodies were not related to IgG antibodies, and the lack of correlation occurred to both DBPII-related antigens. It has been proposed that the correlation (or not) between IgG/IgM in malaria may reflect inherent structural differences between antigens, including the relative conservation of the epitopes that are targeted; for example, strong correlations between IgG and IgM was observed to MSP2, whereas this was not seen with MSP1-19 and AMA-1 [53]. In the case of P. vivax MSP1, antibody response to different regions of the protein appeared distinct, with the N-terminal portion predominantly associated with IgM antibodies and C-terminal with IgG response [54-57]. In the case of DBPII, several studies have mapped functional immunoreactive B cell epitopes associated with broadly neutralizing IgG antibody response [12, 14, 15, 18], but no data is available about IgM response.

It has been shown that IgM antibodies may be necessary to sustain an optimal long-term protective IgG response [34-36]. Assuming that functionally acquired IgG antibodies able to broadly inhibit DBPII-DARC interaction (BIAbs) are associated with a reduced risk of clinical P. vivax malaria [20, 21], we sought to investigate whether a stable DBPII-specific IgM response could interfere with the profile of BIAbs responder. It is particularly relevant as we demonstrated that long-term DEKnull-2 responders with high levels of IgG antibodies are able to produce a persistent BIAbs response [22]. Stratification of the DBPII BIAbs responders (persistent, temporary and no-responder) showed no association (positive or negative) between antigen-specific IgM response and the profile of BIAbs. Additional investigation may help define the contribution (if any) of anti-DBPII IgM antibodies in the immune response mediated by P. vivax.

The present study has limitations that must be considered when interpreting the results. As DBPII sequences from all cross-section surveys were not available, antibody response against a DBPII variant circulating in the study area (Sal1) was used to characterize species-specific immune response. Our previous studies confirm Sal1 as major local DBPII variant [37, 58, 59], whose antibody response is highly prevalent in Amazonian exposed-individuals [33, 38]. Consequently, we are confident that Sal1 is a key DBPII variable for assessing the species-specific immune response in the study area. Taken together, our results demonstrated that IgG (but not IgM) variant-specific DBPII antibodies were poorly sustained at low transmission period, which confirms that IgM antibodies may be more indicative of continuous exposure to malaria, whereas epitope-conserved IgG antibodies are relatively stable and associated with BIAbs response. The reason for the persistence of the IgM response in our cohort merits further investigations.

Temporal distribution of malaria cases in the agricultural settlement of Rio Pardo (Amazonas, Brazil) during 9 years follow-up study.

P. vivax (blue) and P. falciparum (red) microscopy diagnosed case report data in Rio Pardo were provided by the National Malaria Surveillance Information System (SIVEP-Malaria) and plotted per month. The longitudinal study comprises six cross-sectional surveys during 2008–2017, which includes periods of high (dark-grey, phase I and III) and low (light-grey, phase II) malaria transmission; the first three cross-sectional surveys were carried-out during the first year (baseline, 6 and 12 months); three carried-out 6th, 7th and 9th years later. Modified from Pires et al., 2018 [33].

(TIF)

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The levels of IgM antibody response against DPBII-Sal1 and DEKnull-2 during the study period.

The IgM responses were expressed as Reactivity Index (RI), with Reactivity Index (RI)>1.0 considered positive. The individual values are represented by blue (DBPII-Sal1) and red (DEKnull-2) open circles. Transversal lines indicate medians and interquartile ranges. The cross-sectional surveys were carried-out as described in legend to S1 Fig. The frequency of seropositive subjects (Pos (%)) on each cross-sectional survey is represented below each graphic. Different number of asterisks indicate the variation on p value (*p< 0.05 to **p<0.0001; Fisher’s exact test), for significance differences between the frequency of DBPII-Sal1 and DEKnull-2 response.

(TIF)

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The levels of antibodies IgG response against DPBII-Sal1 and DEKnull-2 during the study period.

The IgG responses were expressed as Reactivity Index (RI), with Reactivity Index (RI)>1.0 considered positive. The individual values are represented by blue (DBPII-Sal1) and red (DEKnull-2) open circles. Transversal lines indicate medians and interquartile ranges. The cross-sectional surveys were carried-out as described in legend to S1 Fig, with IgG original data obtained from Pires et al., 2018 [33]. The frequency of seropositive subjects (Pos (%)) on each cross-sectional survey is represented below each graphic. Different number of asterisks indicates the variation on p value (*p< 0.05 to **p<0.0001; Fisher’s exact test), for significance differences between the frequency of DBPII-Sal1 and DEKnull-2 response.

(TIF)

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Spearman correlation between IgM and IgG antibody responses against DBPII-Sal1 (red) and DEKnull-2 (blue) of subjects with or without acute P. vivax infections.

The correlation between IgM and IgG antibodies response were performed separately to subjects with acute P. vivax infections (closed triangle) and non-infected individuals (open circles) to each protein.

(TIF)

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Levels of IgM and IgG antibodies response against P. vivax DBPII-proteins during the 9 years follow-up study.

(DOCX)

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26 Mar 2020

PONE-D-20-02118

Dynamics of IgM and IgG responses to the next generation of engineered Duffy binding protein II immunogen: strain-specific and strain-transcending immune responses over a nine-year period

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1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Medeiros et al. have studied the dynamics of IgM and IgG responses against P. vivax Duffy binding protein region II (PvDBPII) in cross-sectional surveys in a P. vivax endemic region in the Brazilian Amazon. They have used recombinant PvDBPII SalI and PvDBPII DEKnull-2 proteins for both ELISA and binding inhibition assays with endemic sera to study development of strain-specific and strain-transcending antibodies against PvDBPII respectively. The authors demonstrate for the first time the presence of long-lasting IgM antibodies directed against PvDBPII, which recognize PvDBPII SalI better than PvDBPII DEKnull-2 and also inhibit RBC binding to PvDBPII SalI better than PvDBPII DEKnull-2. In contrast IgG show the reverse specificity suggesting IgGs more commonly recognize strain-transcending epitopes in PvDBPII. This is the first study to systematically analyse IgM and IgG against PvDBPII and although the mechanism or reasons for these differences in specificity are not known and the implications for protective immunity are not understood, the descriptive analysis presented here is interesting and important. It emphasizes the importance and need to study both IgM and IgG responses against parasite antigens that are leading vaccine candidates. The study is technically sound and the analysis provides interesting insights into development of antibody responses against PvDBPII following natural exposure to P. vivax.

The authors have responded to the queries raised earlier adequately.

Reviewer #2: This manuscript presents to investigate the detail of IgM & IgG responses against DBPII-Sal1 & DEKnull-2 during the cross-sectional surveys study over nine-year period in the Amazon region. The authors argue that long term exposure to low levels of P. vivax transmission led to a sustained variant-specific IgM responses (DBPII-Sal1 over DEKnull-2), while sustained IgG responses are skewed to conserved epitopes (DEKnull-2 over DBPII-Sal1). Overall, this manuscript is of interest, clearly written some points, informative and statistical analysis has been properly done.

However, some of these findings had been reported using same field study from same group. And then it is required the authors to present these data with clearly and carful editing to avoid misleading as a redundant publication. The major IgG response against DBPII-Sal1 & DEKnull-2 was reported on Ref 35, fig 3. And then it may consider the major finding on this manuscript is IgM response against these antigens during long term field survey. Although the major purpose of this study has not been fully justified yet, especially it is still unclear the focus on IgM responses against DBPII-Sal1 & DEKnull-2 such a long term survey. And also a significant problem of this manuscript is figure 1 has been used on previous article, and it should be removed from main MS or move it on to supplemental figure.

Careful editing will greatly strengthen the manuscript, making it more clear and concise. Specific comments are as follows:

1. The authors has to justify the major purpose of this study, why focus on IgM response against DBPII-Sal1 & DEKnull-2 during such a long 9 year period? In generally, IgM antibodies appear early in the course of an infection and usually reappear, to a lesser extent, after further exposure. And it often found to bind to specific antigens, even in the absence of prior immunization. And also it was reported IgM as a rapid and early responder in Malaria (Ref 27). And also it is not justified yet to select DBPII-Sal1 & DEKnull-2 antigens for this study due to the authors mentioned these sentence as below (Ref 35) “Thus, while the DEKnull-2 antibody response is not a useful tool for serological evaluation of malaria transmission, it seems to be appropriate to evaluate naturally-acquired immunity to P. vivax blood-stage infections. At the individual level, DEKnull-2 immune responses confirmed the highest acquired immunity of HR group as compared with IR or LR group; all HR subjects were positive to DEKnull-2, and remained seroreactive until the end of the 9 year follow-up study. Despite of that, the levels of antibodies reacting to DEKnull-2 declined during the study, including in the high transmission phase III.”

Why did authors focus on IgG and IgM responses against DBPII-Sal1 and DEKnull-2? And what is the major new finding on the current study?

2. It is required the authors that ref 35 has to refer in the introduction on this manuscript, especially it is very important to mention it what they found it before. I assume this MS is a kind of retrospective serological follow up study, due to this, it has to be refereed well all the detail for them previous work. And also they need to explain the advantage of BIAbs assay due to clarify for the difference from Ref 35.

3. Fig 1 had been used on them previous article (Ref 35, fig1), and then it has to remove from main MS or move it on to supplemental figure. (Such a minor change will not be called as a “modified”)

4. Fig 2 and 3 have to remove from main MS or move it on to supplemental figure. These figures are just raw data sets for fig 4.

5. The information of right panel of fig 5 is not founded on the current manuscript. What is this meaning? What is the IR? The reviewer would not be founded any advantage points about this figure except left part. Moreover it is useful to combine the Pv-infected information (fig 5 left panel) with figure 6. Taken together, it would be getting better to merge fig 5 and 6.

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Reviewer #1: No

Reviewer #2: No

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2 Apr 2020

RESPONSES TO REVIEWERS

REVIEWER # 1

Major comment. (…) The authors demonstrate for the first time the presence of long-lasting IgM antibodies directed against PvDBPII (…) This is the first study to systematically analyze IgM and IgG against PvDBPII (…)the descriptive analysis presented here is interesting and important. (… )the analysis provides interesting insights into development of antibody responses against PvDBPII following natural exposure to P. vivax. The authors have responded to the queries raised earlier adequately.

AUTHORS: We thank the reviewer for the careful examination of the manuscript and rebuttal letter, and to consider our work well carried-out and relevant for this field of investigation.

REVIEWER # 2

Major comment

Rev#2 (…) Overall, this manuscript is of interest, clearly written some points, informative and statistical analysis has been properly done (…). However, some of these findings had been reported using same field study from same group. And then it is required the authors to present these data with clearly and carful editing to avoid misleading as a redundant publication (…). The major IgG response against DBPII-Sal1 & DEKnull-2 was reported on Ref 35, fig 3.

AUTHORS: First of all, we thank the reviewer for the careful examination of our manuscript and consider the results relevant for this field of investigation. Regarding the concern of redundant publication, we must clarify that the MS from Pires et al (ref 35) was design to evaluate the seroconversion rates as a tool for a rapid assessment of P.vivax malaria transmission intensity. For that, we include blood-stage antigens covering a wide range of immunogenicity such as MSP1-19, AMA-1 and DBPII-related proteins. An unsupervised learning algorithm such as k-means clustering allowed categorizing individuals based on the magnitude and breadth (specificities for multiple antigens including AMA-1, DBPs and MSP-1) of their conventional antibody response. In that MS (ref 35), we demonstrate that IgG antibodies against AMA-1/MSP1-19 were much more appropriate to detect temporal fluctuation of P.vivax transmission that the less immunogenic DBPII-related proteins. Consequently, in the previous MS we investigated which antigen should be most appropriate for detecting fluctuations in P.vivax malaria transmission.

Aiming towards universal strain-transcending DBPII immunity, in the current MS, we evaluate for the first time the long-term DBPII-specific IgM response and the relationship of IgM antibodies with (i) conventional IgG response and their (ii) ability to blocking the interaction ligand-receptor (binding inhibitory activity/BIAbs, as detected by COS-7 assays). Consequently, there is no overlap between the current MS and ref 35. As scant longitudinal data are available about secondary IgM responses in P. vivax-exposed populations, we are confident that our findings fill a gap in the literature for providing the first description of the of the dynamic of IgM versus IgG responses to a novel surface-engineered P.vivax DBPII-based vaccine (the relevance of studying IgM antibodies was included below; Major comment/Specific comment 1).

Finally, we would like to emphasize that long-term follow-up studies are expensive, time consumer, and offer an exceptional opportunity to investigate unappreciated topics. Consequently, to focus on DBPII-specific IgM responses, we took advantage of the long-term follow-up study previously carried out in the Amazon rainforest, where different profiles of DBPII-specific IgG responders were identified (as described in ref 35). We are confident that the results presented here will contribute to further optimize the design of new DBPII immunogens such as DEKnull-2.

In the revised version of the MS, we clarified this concern raised by the reviewer (please see track changes copy, for example, in the last paragraph of introduction). Also we explained in the legend of Fig 3 (now S3 Fig, as requested by the reviewer) that IgG original data were obtained from Pires et al., 2018 (previous ref 35, and in the revised version ref 33).

Rev#2: Major comment -Rev#2: (…)it is still unclear the focus on IgM responses against DBPII-Sal1 & DEKnull-2 such a long term survey. (…)

Specific comment -1 1. The authors have to justify (…), why focus on IgM response against DBPII-Sal1 & DEKnull-2 during such a long 9 year period? (…)Why did authors focus on IgG and IgM responses against DBPII-Sal1 and DEKnull-2? And what is the major new finding on the current study? (…) In generally, IgM antibodies appear early in the course of an infection and usually reappear, to a lesser extent, after further exposure.(…) And also it is not justified yet to select DBPII-Sal1 & DEKnull-2 antigens for this study due to the authors mentioned these sentence as below (Ref 35) “Thus, while the DEKnull-2 antibody response is not a useful tool for serological evaluation of malaria transmission, it seems to be appropriate to evaluate naturally-acquired immunity

AUTHORS: Currently, a significant body of evidence point to an underappreciated role for IgM in protection against infectious disease including malaria; for example:

(1) In experimental malaria, mice lacking specific-IgG antibodies but with strong anti-parasite IgM antibody responses develop long-lasting non-sterile immunity and survive lethal parasite challenge (Borges da Silva et al., 2018; PMID: 30148845);

(2) Somatically hypermutated Plasmodium-specific IgM+ MBCs proliferated and gave rise to antibody-secreting cells that dominated the early secondary response to parasite rechallenge (Krishnamurty et al., 2016; PMID:27473412);

(3) IgM antibodies (but in much less extension IgG) to a broad array of P. falciparum antigens was associated to genetic resistance to malaria (Arama et al., 2015; PMID: 26361633);

(4) In P.falciparum malaria, merozoite-specific IgM is an important functional and long-lived antibody response targeting blood-stage malaria parasites that contributes to malaria immunity (Boyle et al., 2019; PMID: 31579826);

(5) IgM antibodies may be necessary to sustain an optimal long-term protective IgG response (Harte et al., 1983; PMID: 6835362; Boes et al., 1998; PMID: 9590224),

Taken together, we are in accordance with other that suggest that IgM antibodies seem to be much more than just an early responder to malaria infection (Boonyaratanakornkit & Taylor, 2019; PMID: 31603585), and should be investigated during the development of vaccines.

In pursuing a P.vivax DBPII structural vaccinology approach, we created DEKnull-2 antigens lacking the variant epitopes but retaining the conserved protective functional epitopes, which are essential for binding its cognate reticulocyte receptor. As quoted above, DEKnull-2 should be used to evaluate natural acquired immunity and not fluctuation of P.vivax transmission (ref. 35). Considering the new paradigm of the relevance of IgM response in malaria (as summarized above), we sought to investigate here the relationship between the IgM and IgG antibody responses against conserved (DEKnull-2) and variable DBPII epitopes circulating in the area. In the current study, a comparison between conserved and variable epitopes was critical, as DBPII-based vaccine should target strain-transcending immune responses. As IgM antibodies may be necessary to sustain an optimal long-term protective IgG response (PMID: 6835362; PMID: 9590224), we also investigate whether a stable DBPII-specific IgM response could interfere with the profile of functional antibodies, i.e., antibodies able to block the interaction ligand-receptor (BIAbs).

Here, we demonstrated for the first time that long-term exposure to low and unstable levels of P. vivax transmission lead to a sustained DBPII-specific IgM response toward variant-specific epitopes while sustained IgG responses are skewed to conserved epitopes involved in protection (DEKnull-2 over Sal-1). Although a persistent DBPII-specific IgM response was not associated with broadly neutralizing IgG antibody response, future studies should investigate on the role of IgM in the DBPII acquired immune response (perhaps to sustain IgG response). We are confident that these findings will contribute to the current stage of development of DBP-based vaccines that focus broad protective immune response.

In the revised version of the MS we clarify this topic, as requested to the reviewer (see MS with track changes). More specifically, although most of the references on the importance of studying IgM antibodies were already included in the penultimate paragraph of the introduction, we reinforced this topic in the final paragraphs of the introduction and included additional references.

Rev#2: And also a significant problem of this manuscript is figure 1 has been used on previous article, and it should be removed from main MS or move it on to supplemental figure.

AUTHORS: the original Fig 1 was included in the Methods to clarify the study design and the long-term follow up cohort that was carried before (ref35); otherwise, the readers will need to access the ref 35. However, as requested to reviewer, Fig. 1 was included in the revised version of the MS as supplementary (now Fig 1S).

Rev#2: Careful editing will greatly strengthen the manuscript.

AUTHORS: The MS was revised by Dr. John H Adams, South Florida University, USA (https://health.usf.edu/publichealth/overviewcoph/faculty/john-adams). Dr. Adams is also a co-author of the MS.

Specific point 2. It is required the authors that ref 35 has to refer in the introduction on this manuscript, especially it is very important to mention it what they found it before. I assume this MS is a kind of retrospective serological follow up study, due to this, it has to be refereed well all the detail for them previous work. And also they need to explain the advantage of BIAbs assay due to clarify for the difference from Ref 35.

AUTHORS: As we clarified above (please see major comments) the MS from Pires et al (ref 35) was design to evaluate seroconversion rates as a tool for assess trends in P.vivax malaria transmission; in that study to evaluate the breadth of antibody response was critical to included antigens covering a wide range of immunogenicity (MSP1-19, AMA-1 and DBPIIs). On the other hand, the current MS was design to get insides in the acquired immune response to a novel engineered vaccine candidate - DEKnull-2. As quoted before, we have included in the last paragraph of the introduction that we took advantage of the long-term follow-up study previously carried out in the Amazon rainforest, where different profiles of DBPII-specific IgG responders were identified (Pires et al., 2018). In the current MS, it was critical to evaluate BIABs response because a strong naturally acquired BIAb response is associated with a reduced risk of clinical P. vivax malaria (King et al., 2008; PMID: 18523022; Nicolete et al., 2016; PMID: 27578850). All these references were included in the MS (please see highlighted text in the 2nd paragraph of the introduction, track changes MS).

Specific point 3. Fig 1 had been used on them previous article (Ref 35, fig1), and then it has to remove from main MS or move it on to supplemental figure. (Such a minor change will not be called as a “modified”)

AUTHORS: Okay, it was done. In the revised version of the MS, Fig 1 was included as supplementary Fig S1

Specific point 4. Fig 2 and 3 have to remove from main MS or move it on to supplemental figure. These figures are just raw data sets for fig 4.

AUTHORS: Okay, it was done. Figures were adjusted as suggested (Fig S2 and S3)

Specific point 5. The information of right panel of fig 5 is not founded on the current manuscript. (…)

AUTHORS: Perhaps the results in Fig. 5 have led to a misunderstanding. More specifically, Fig 5 shows every single individual who had blood-stage infection at any time of the follow-up study (left panel), and in the right panel (the heatmap) their correspondent antibody responses to DBPII-related antigens, including both IgM (1st part of the right panel, i.e., the first part of the heatmap) and IgG antibodies (2nd part of heatmap). At the top of heat map, IgM and IgG antibodies were identified in bold. The results showed no correlation between acute infection and either antigen-specific IgM or IgG antibodies. As example, in the text, we have identified some specific individuals (by code) and comments about their results in the MS. At this time, we should clarify that all data from the original Fig. 5 (now Fig. 2 as requested by the reviewer) were included in the text and/or in the legend of the figure (please see highlighted text in the track changes copy).

Rev#2: (…) What is the IR? (…)

In the figure, the results of antibody response were expressed as the ELISA reactivity index (IR) that was calculated as described in the methods (please see highlighted text in the item IgM and IgG detection assays). Briefly, the ELISA reactivity (RI) was calculated as the ratio of the mean optical density (OD at 492 nm) of each sample to the mean OD plus three standard deviations of samples from 20-30 unexposed volunteers. Values of RI > 1.0 were considered positive (on a green scale). In the revised version of the MS, we included RI definition in the legend of Fig 2 (ex-Fig 5).

Rev#2:The reviewer would not be founded any advantage points about this figure except left part.

AUTHORS: We are confident about the relevance of the data presented in the figure 5, particularly, due to the absence of correlation between antigen-specific IgM antibodies and acute malaria infection. These findings are intriguing as the antibodies made initially against infection are usually IgM. Perhaps a plausible explanation is the relative underrepresentation of P. vivax asexual stages in patient blood as a considerable body of evidence indicates a tissue reservoir, such as bone marrow, where most P. vivax parasite burden resides (please see 2nd paragraph of discussion). Consequently, we decided to keep figure 5 (now retitled as Fig. 2).

Rev #2: Moreover it is useful to combine the Pv-infected information (fig 5 left panel) with figure 6. Taken together, it would be getting better to merge fig 5 and 6.

AUTHORS: While the figure 5 shows the absence of correction between antigen-specific antibody responses and acute malaria infection, Fig 6 shows the IgM/IgG antibody responses according to the profile of binding inhibitory activity (BIAbs) as detected by functional assays (COS-7 transfected cells). Consequently, it is not possible to merge results that are unrelated.

We would like to thank deeply the reviewer for his time and effort, and we hope that the MS is now suitable for publication.

Submitted filename: Medeiros_etal_Response_to_Reviewers.docx

Click here for additional data file.

22 Apr 2020

Dynamics of IgM and IgG responses to the next generation of engineered Duffy binding protein II immunogen: strain-specific and strain-transcending immune responses over a nine-year period

PONE-D-20-02118R1

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Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: The authors have been addressed all reviewer's comments.

The current MS is suitable for publication.

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Reviewer #2: No

24 Apr 2020

PONE-D-20-02118R1

Dynamics of IgM and IgG responses to the next generation of engineered Duffy binding protein II immunogen: strain-specific and strain-transcending immune responses over a nine-year period

Dear Dr. Carvalho:

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