key: cord-0269494-rgf0p461 authors: Dias, M. H. F.; Guimaraes, L. F. F.; Barcelos, M. G.; Moreira, E. U. M.; Nascimento, M. F. A.; Souza, T. N.; Pires, C. V.; Monteiro, T. A. F.; Middeldorp, J. M. M.; Soares, I. S.; Ntumngia, F. B.; Adams, J. H.; Kano, F. S.; Carvalho, L. H. title: Impact of Epstein-Barr virus co-infection on natural acquired Plasmodium vivax antibody response date: 2022-03-10 journal: nan DOI: 10.1101/2022.03.08.22272037 sha: 32ef5b2cecd53efe937cfd9be96851888d7d16c4 doc_id: 269494 cord_uid: rgf0p461 Background: The simultaneous infection of Plasmodium falciparum and Epstein-Barr virus (EBV) could promote the development of the aggressive endemic Burkitt's Lymphoma (eBL) in children living in P. falciparum holoendemic areas. While it is well-established that eBL is not related to other human malaria parasites, the impact of EBV infection on the generation of human malaria immunity remains largely unexplored. Considering that this highly prevalent herpesvirus establishes a lifelong persistent infection on B-cells with possible influence on malaria immunity, we hypothesized that EBV co-infection could have impact on the naturally acquired antibody responses to P. vivax, the most widespread human malaria parasite. Methodology/Principal Findings: The study design involved three cross-sectional surveys at six-month intervals (baseline, 6 and 12 months) among long-term P. vivax exposed adults living in the Amazon rainforest. The approach focused on a group of malaria-exposed individuals whose EBV-DNA (amplification of balf-5 gene) was persistently detected in the peripheral blood (PersV DNA, n=27), and an age-matched malaria-exposed group whose EBV-DNA could never be detected during the follow-up (NegV DNA, n=29). During the follow-up period, the serological detection of EBV antibodies to lytic/latent viral antigens showed that IgG antibodies to viral capsid antigen (VCA-p18) were significantly different between groups (PersV DNA > NegV DNA ). A panel of blood-stage P. vivax antigens covering a wide range of immunogenicity confirmed that in general PersV DNA group showed low levels of antibodies as compared with NegV DNA. Interestingly, more significant differences were observed to a novel DBPII immunogen, named DEKnull-2, which has been associated with long-term neutralizing antibody response. Differences between groups were less pronounced with blood-stage antigens (such as MSP1-19) whose levels can fluctuate according to malaria transmission. Conclusions/Significance: In a proof-of-concept study we provide evidence that a persistent detection of EBV DNA in peripheral blood of adults in a P. vivax semi-immune population may impact the long-term immune response to major malaria vaccine candidates. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted March 10, 2022. ; https://doi.org/10.1101/2022.03.08.22272037 doi: medRxiv preprint 7 150 Northeast of Amazonas State in the Brazilian Amazon region. The study site and 151 malaria transmission patterns were described in detail elsewhere [29, 32] . In this 152 area, malaria transmission is considered hypo to mesoendemic, and most 153 residents were natives of the Amazon region. Inhabitants of the settlement live 154 on subsistence farming and fishing along the small streams. In the study area, P. 155 falciparum malaria incidence has decreased drastically in recent years, and P. 156 vivax is now responsible for all clinical malaria cases reported. 157 Study design and cross-sectional surveys A population-based open cohort study was initiated in November of 2008 159 and included three cross-sectional surveys carried at six-months interval 160 (baseline, 6 and 12-months), and distributed in periods of high and low malaria 161 transmission (S1 Fig. 1) , as previously reported [29] . Briefly, (i) interviews were 162 conducted through a structured questionnaire to obtain demographical, 163 epidemiological, and clinical data; (ii) physical examination, including body 164 temperature and spleen/liver size were recorded according to standard clinical 165 protocols; (iii) venous blood was collected for individuals aged five years or older 166 (EDTA, 5 mL); and (iv) examination of Giemsa-stained thick blood smears for the 167 presence of malaria parasites by conventional light microscopy, with P. vivax 168 infection confirmed later by a species-specific real-time PCR as described [33] . 169 The geographical location of each dwelling was recorded using a hand-held 12- is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted March 10, 2022. ; https://doi.org/10.1101/2022.03.08.22272037 doi: medRxiv preprint 224 (VCA and EBNA-1) reactivities were determined using commercial anti-human 225 IgM and IgG secondary antibodies conjugated to horseradish to peroxidase 226 (HRP) (Sigma-Aldrich). For each peptide, EBV seronegative (n=5) and positive 227 controls (n=8) were used on each experiment. Receiver-operating characteristic 228 (ROC) curves was used to determine optimal cutoff points for each peptide (S2 229 Fig.) . Based The PCR primers for this assay were previously selected in the BALF-5 gene is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted March 10, 2022. ; https://doi.org/10.1101/2022.03.08.22272037 doi: medRxiv preprint 272 parallel, we selected a group of 29 age-matched malaria-exposed individuals with 273 no detectable viral DNA in the peripheral blood (NegV DNA ) ( Table 1) . 274 Demographic, parasitological, and epidemiological variables were comparable 275 between groups. Accordingly, most individuals were adults with similar proportion 276 of male: female, and their age basically corresponding to their lifetime exposure 277 to malaria in the Amazon area (medians of 32 and 33 years for PersV DNA and 278 NegV DNA , respectively). In this long-term malaria exposed individuals, few acute 279 malaria infections were detected during the follow-up period (all P. vivax, as 280 detected by microscopy and/or species-specific PCR assay) ( Table 1) . . CC-BY-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted March 10, 2022. ; https://doi.org/10.1101/2022.03.08.22272037 doi: medRxiv preprint 282 283 Table 1 . Demographic, epidemiological, and parasitological data of malaria-exposed individuals whose EBV-DNA could be detected (PersVDNA) or not (NegVDNA) in the peripheral blood during the 12-month is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted March 10, 2022. ; https://doi.org/10.1101/2022.03.08.22272037 doi: medRxiv preprint 295 NegV DNA groups (Figure 1) . At enrollment, while 89% of PersV DNA had a positive 296 IgG antibody response to VCA-p18, only 45% of NegV DNA had IgG VCA-p18 (chi-297 square test; p= 0.0006). Of interest, the difference in response between groups 298 remained constant throughout the follow-up period. The levels of antibodies to 299 VCA-p18 were also significantly different between groups (Mann-Whitney test), Fig.) . All raw data are available in the S1 313 Table. 314 315 316 317 Although IgM antibodies to EBV lytic antigens (VCA-p18, EAd-p45/52 and Zebra) 318 did not show any differences between the study groups, at the individual level, a 319 positive IgM response for one EBV antigen was related to positivity for the others 320 (Fig. S4) ; of note, this IgM response profile was sustained throughout the study . CC-BY-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted March 10, 2022. ; https://doi.org/10.1101/2022.03.08.22272037 doi: medRxiv preprint 321 period. Further, we compared the pattern of humoral antibody responses to all 322 EBV antigens between PersV DNA and NegV DNA (Figure 2) . In the PersV DNA group, 323 there was a tendency of positive correlation between EBV-specific antibodies, 324 specially to IgM antibodies. On the contrary, NegV DNA group was characterized 325 by a predominance of negative antibody correlations to several EBV antigens. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted March 10, 2022. ; https://doi.org/10.1101/2022.03.08.22272037 doi: medRxiv preprint 346 response to different P. vivax antigens were significantly different between 347 PersV DNA and NegV DNA . The antibody response to the engineered DBPII 348 immunogen (DEKnull-2) showed a trend towards lower antibody levels in 349 PersV DNA compared with NegV DNA (Figure 3) . Specifically, while DEKnull-2 350 serological reactivity index (RI) ranged from 0.59 to 0.95 for PersV DNA , RI values 351 ranged from 2.11 to 3 for NegV DNA group. Considering all samples assayed, the 352 frequency of positivity was 40% vs. 61% (chi-square test, p=0.0079) for PersV DNA 353 and NegV DNA samples , respectively. A similar pattern was observed with the 354 original DBPII protein (Fig. S5) . For P. vivax MSP1-19, the differences between 355 the groups was less pronounced (Figure 4) , with statistically significant 356 differences observed only during high transmission period at the study baseline 357 (Figure 4) . Circulating antibodies against AMA-1 showed a similar pattern of 358 response as MSP1-19 (Fig. S6) . is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted March 10, 2022. ; https://doi.org/10.1101/2022.03.08.22272037 doi: medRxiv preprint 371 calculated as described in methods and RI > 1 corresponded to an ELISA-372 positive response. Regardless, the detection of EBV-DNA in peripheral blood, antibodies 389 against P. vivax blood antigens did not correlate with EBV antibody pattern. In 390 both groups, antibody response to P. vivax blood-stage antigens showed overall 391 a weak correlation with IgG EBV pattern (sometimes negative associations were 392 also observed) (Fig. S7) . is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted March 10, 2022. ; https://doi.org/10.1101/2022.03.08.22272037 doi: medRxiv preprint In the Amazon rain forest both malaria and EBV infections are common, yet is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted March 10, 2022. ; https://doi.org/10.1101/2022.03.08.22272037 doi: medRxiv preprint 420 results suggested that long episodes of EBV-DNA detection may influence the 421 levels of both strain-specific and strain-transcending DBPII immune responses. With respect to more immunogenic blood stage P. vivax antigens, such as It is also worth mentioning that in this immunocompetent malaria-exposed is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted March 10, 2022. ; https://doi.org/10.1101/2022.03.08.22272037 doi: medRxiv preprint 494 in peripheral blood of an adult P. vivax semi-immune population may impact the 495 long-term malaria immune response to major malaria vaccine candidates. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted March 10, 2022. ; https://doi.org/10.1101/2022.03.08.22272037 doi: medRxiv preprint . CC-BY-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted March 10, 2022. ; https://doi.org/10.1101/2022.03.08.22272037 doi: medRxiv preprint For the current study, we focused on two sub-groups of malaria-180 exposed individuals (i) individuals whose EBV-DNA could be persistently 181 detected from peripheral blood (PersV DNA ) and (ii) an age-matched subgroup 182 whose viral DNA was undetected throughout the 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 186 Formal written consent was obtained 189 from all participants, which was also obtained from the next of kin, caregivers DBPII-Sal1, a recombinant Duffy binding protein region 194 II (DBPII) including amino acids 243-573 of the Sal-1 reference strain [35], and 195 recombinant DEKnull-2, an engineered DBPII immunogen MSP1-19 antigen. The 19-kDa C-terminal region of the Merozoite Surface Protein-1 of P. vivax (MSP1-19), which represents amino 199 acids 1616-1704 of the full-length MSP-1 polypeptide The ectodomain of P. vivax Apical Membrane 201 Antigen-1 (AMA-1, encompassing amino acids 43 to 487 Linked Immunoassays (ELISA) for P. vivax IgG antibodies was carried out 206 using P. vivax blood-stage recombinant proteins as previously described All synthetic peptides were kindly provided by For the 220 assessment of the levels of antibodies to lytic (VCA-p18, EAd and Zebra) and 221 latent EBV antigens (EBNA1), we used synthetic peptide-based ELISA assays 222 as described Briefly, each peptide was used at final concentration of 1 Zebra and EAd) and IgG 445 much broader EBV antibody response than the NegVDNA group, with a individual EBV serological markers, only IgG VCA-p18 448 distinguished PersV DNA and NegV DNA groups during the follow-up study 38-45%, respectively). Interestingly, measuring VCA-IgG antibodies seems 450 to be a best single test to indicate a previous EBV infection IgG-VCA antibodies tended to be associated with IgM 452 antibodies to lytic antigens (VCA-p18, EAd-p45/52 and Zebra). Intriguingly, EBVruled out, the tendency of correlation with EBV-DNA may reflect constant 459 EBV activation-triggering of B-cells; in fact, EBV persistently infecting naive or 460 anti-EBV B-cells leading to it is important 461 to clarify about the high sensitivity and specificity of the EBV-peptides used in our 462 ELISA assays. 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A Literature-Based Study by the RIAL-CYTED. Cancers 801 (Basel) Quantitative cytokine level 804 of TNF-alpha, IFN-gamma, IL-10, TGF-beta and circulating Epstein-Barr virus DNA 805 load in individuals with acute Malaria due to P. falciparum or P. vivax or double infection 806 in a Malaria endemic region in Indonesia The 820 current study included three cross-sectional surveys at six-month intervals 821 (Baseline, Bs; 6-and 12-months latter). Malaria cases were based on results of 822 conventional microscopy provided by the National Malaria Surveillance System 823 Registry (SIVEP-Malaria) Two-graph receiver operating characteristic curves (TG-ROC) to 827 EBV proteins. For ELISA-detected antibodies [IgM (VCA-p18, Zebra and EAd-828 p45/52) and IgG (VCA-p18 and EBNA-1) Frequency of anti-EBV antibodies according to the number of 833 serological markers recognized by individuals whose EBV-DNA could be 834 detected (PersV DNA ) or not (NegV DNA ) over the follow-up period. For each 835 group, results were presented as the proportion of responders for one (1), two 836 (2), three (3), four (4) or five (5) EBV serological markers EAd-p45/52 and EBNA-1) during the cross-sectional 842 surveys. Heatmaps illustrated individual antibody response of each malaria-843 exposed individuals classified according to the detection (PersV DNA ) or not 844 (NegV DNA ) of EBV-DNA over the follow-up period. According to EBV antibody 845 response, individuals were categorized as non-responder (negative) or 846 responders (stratified as low, medium or high, according to EBV antibody region II (DBPII) in individuals with (PersV DNA ) or without (NegV DNA ) 851 persistent viral DNA over the follow-up period AMA-1), in individuals with (PersV DNA ) or without (NegV DNA ) 866 persistent viral DNA over the follow-up period. In A, results are shown by 867 cross-sectional surveys (baseline, 6-and 12-month) PersV DNA ) or without (NegV DNA ) persistent viral DNA during 881 the cross-sectional surveys. Clustering was based on the Spearman 882 correlation coefficient for assays measuring anti-EBV antibodies in serum. Matrix 883 heatmaps were shown for each cross-sectional survey (baseline, 6-and 12-884 months), with top and bottom panels representing Pers V DNA an Neg V DNA groups, 885 respectively. Positive correlations shown in blue and negative correlations shown 886 in orange IgM and IgG antibody response against Epstein-Barr virus peptides 889 in Amazonian children over time. For each EBV-peptide (VCA-p18 EAd-p45/52 and EBNA-1) antibody response was represented by frequency of 891 responders (bar) and magnitude of response (optical density-OD median, lines) The results represented three cross-sectional surveys carried-out at 6-month 893 intervals, i.e., at enrollment (0m) and six (6m) and 12-month latter (12m)