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. 2023 Apr 7;17(4):e0011229.
doi: 10.1371/journal.pntd.0011229. eCollection 2023 Apr.

Immunological characterization of a VIR protein family member (VIR-14) in Plasmodium vivax-infected subjects from different epidemiological regions in Africa and South America

Raianna F Fantin  1   2   3 , Camila H Coelho  1 , Anne D Berhe  1 , Luisa M D Magalhães  3 , Dhélio B Pereira  4 , Nichole D Salinas  1 , Niraj H Tolia  1 , Chanaki Amaratunga  1 , Seila Suon  5 , Issaka Sagara  6 , David L Narum  1 , Ricardo T Fujiwara  3 , Claudia Abejon  7 , Antonio Campos-Neto  7   8 , Patrick E Duffy  1 , Lilian L Bueno  3
Affiliations

Immunological characterization of a VIR protein family member (VIR-14) in Plasmodium vivax-infected subjects from different epidemiological regions in Africa and South America

Raianna F Fantin et al. PLoS Negl Trop Dis. .

Abstract

Plasmodium vivax is a major challenge for malaria control due to its wide geographic distribution, high frequency of submicroscopic infections, and ability to induce relapses due to the latent forms present in the liver (hypnozoites). Deepening our knowledge of parasite biology and its molecular components is key to develop new tools for malaria control and elimination. This study aims to investigate and characterize a P. vivax protein (PvVir14) for its role in parasite biology and its interactions with the immune system. We collected sera or plasma from P.vivax-infected subjects in Brazil (n = 121) and Cambodia (n = 55), and from P. falciparum-infected subjects in Mali (n = 28), to assess antibody recognition of PvVir14. Circulating antibodies against PvVir14 appeared in 61% and 34.5% of subjects from Brazil and Cambodia, respectively, versus none (0%) of the P. falciparum-infected subjects from Mali who have no exposure to P. vivax. IgG1 and IgG3 most frequently contributed to anti-PvVir14 responses. PvVir14 antibody levels correlated with those against other well-characterized sporozoite/liver (PvCSP) and blood stage (PvDBP-RII) antigens, which were recognized by 7.6% and 42% of Brazilians, respectively. Concerning the cellular immune profiling of Brazilian subjects, PvVir14 seroreactive individuals displayed significantly higher levels of circulating atypical (CD21- CD27-) B cells, raising the possibility that atypical B cells may be contribute to the PvVir14 antibody response. When analyzed at a single-cell level, the B cell receptor gene hIGHV3-23 was only seen in subjects with active P.vivax infection where it comprised 20% of V gene usage. Among T cells, CD4+ and CD8+ levels differed (lower and higher, respectively) between subjects with versus without antibodies to PvVir14, while NKT cell levels were higher in those without antibodies. Specific B cell subsets, anti-PvVir14 circulating antibodies, and NKT cell levels declined after treatment of P. vivax. This study provides the immunological characterization of PvVir14, a unique P. vivax protein, and possible association with acute host's immune responses, providing new information of specific host-parasite interaction. Trial registration: TrialClinicalTrials.gov Identifier: NCT00663546 & ClinicalTrials.gov NCT02334462.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Relative quantity of anti-PvVir14 IgG and IgM antibodies, and prevalence of IgG subclasses to PvVir14.
a) Subjects from the Brazilian Amazon (n = 121, Porto Velho–RO) and from Cambodia (n = 55) with an acute infection by P. vivax—IgG titers; Subjects from Western Africa (Mali) with an acute infection by P. falciparum (n = 28)–IgG titers; and subjects living in the Brazilian amazon (Porto Velho—RO), thus exposed to P. vivax but not infected at the moment of collection (n = 15). b) Subjects from the Brazilian Amazon (Porto Velho–RO) with an acute infection by P. vivax (n = 119)–IgM titers. For a and b, Y axis represents the mean reactivity index (RI) and the dotted line shows the seropositivity threshold (RI = 1). c) Frequency of detectable IgG subclasses against PvVir14 among infected Brazilian subjects. The radar charts are divided into nine lines, each of them representing a 5% value, data are presented (orange rhomboid) as the percentage of individuals (n = 121) with detectable levels for the four IgG subclasses against PvVir14.
Fig 2
Fig 2. Seroreactivity to PvVir14 versus other P. vivax candidate vaccine antigens (PvCSP, PvDBP-RII and Pvs230D1).
(a) IgG Reactivity Index against P. vivax antigens in P. vivax infected subjects. For (b-d), mean OD of anti-PvVir14 IgG versuis (b) mean OD of anti-PvCSP, (c) mean OD of anti-PvDBP-RII (d) mean OD of anti-Pvs230D1. The dotted line marks the minimum value of the mean OD for a subject to be considered above the level of detection (> 1, R.I) Data were analyzed considering a 99% confidence interval (CI). P < 0.05 was considered significant (* p<0.05; **p<0.01; ***p<0.001).
Fig 3
Fig 3. B cell phenotypic analysis by multiparametric flow cytometry.
(a) Unsupervised high dimensional analysis of flow cytometry data (tSNE) from combined data from all B cells populations. Activated (red), Atypical (green), Classical (light blue), and Naïve (purple). (b) Heatmap showing marker expression within each cluster previously identified among B cells. (c) Bar graphs shows percentage of CD19+ (d), Atypical Memory B cells CD19+CD27-CD21- (e), Activated Memory B cells CD19+CD27+CD21- (f), Classical Memory B cells CD19+CD27+CD21+ (g) and Naïve B cells CD19+CD27-CD21+ from total Live PBMC cells. (h) Unsupervised high dimensional analysis of flow cytometry data (tSNE) from combined data of acute infected subjects with or without antibody titers against PvVir14 gated on LiveCD19+.The darker the color (reddish), the greater the intensity of expression. (i) Diffusion map of CD21 (top) or CD27 (bottom) intensity of expression depicting each group (PvVir14+: n = 7, PvVir14-: n = 7): and merged plot. P < 0.05 was considered significant (* p<0.05; **p<0.01; ***p<0.001).
Fig 4
Fig 4. T cell phenotypic analysis by multiparametric flow cytometry.
Bar graphs show percentage of CD3+ T cells (a) CD3+CD56-CD4+ T cells (b) and CD3+CD56-CD8+ T cells (c) from total Live PBMCs. Bar graphs show Mean Intensity Fluorescence (MFI) of CD27+ in CD4+ (d) or CD8+ (e). (f) tSNE from combined data of PvVir14+ (green) or−(red) subjects gated on LiveCD3+CD56- T cells. (g) Expression map of CD27+ cells from F depicting PvVir14- (left), PvVir14+ (middle) and Merged (right). The increasing intensity of red color indicates higher CD27 signal, and the increasing intensity of blue color indicates decreasing CD27 signal. Grey bars represent Healthy Donors (HD), red bars represent PvVir14- and green bars represent PvVir14+ subjects. P <0.05 was considered significant (* p<0.05; **p<0.01; ***p<0.001).
Fig 5
Fig 5. NK and NKT cell population dynamics by multiparametric flow cytometry.
(a) tSNE from combined data of PvVir14+ or PvVir14–subjects gated on CD19-CD14-CD56+ NK (purple) and NKT cells (green). (b) tSNE from (a) depicting PvVir14+ (red) and PvVir14- (blue) cells. (c) NK cells (CD3-CD19-CD14-CD56+) as a percentage of live cells, by group. (d) NKT cells as a percentage of live cells, by group. (e and f) Expression levels of distinguishing markers of NK and NKT cells. P <0.05 was considered significant (* p<0.05; **p<0.01; ***p<0.001).
Fig 6
Fig 6. Drug-treated subjects’ profile for antibody titers, B cells, and NK and NKT cells.
(a) Sample collection and therapeutic scheme. Created with BioRender.com. (b) Total IgG levels from two different points in time: acute infection and post-drug treatment. Serum samples were paired and belonged to the same subjects (acute and post-treatment samples). Two subjects, represented in purple, were also infected at the follow-up 40 days after primary acute infection. (c) tSNE from combined data of PvVir14+ during acute (red) or post treatment (blue) gated on Live CD19+ B cells. (d) tSNE from (c) depicting B cell populations Atypical Memory B cells CD19+CD27-CD21- (green), Activated Memory B cells CD19+CD27+CD21- (red), Classical Memory B cells CD19+CD27+CD21+ (light blue) and Naïve B cells CD19+CD27-CD21+ (purple). (e) Expression plot of distinguish markers CD27 (left) and CD21 (right). The arrows indicate populations with higher CD21 expression. (f) tSNE from combined data of PvVir14+ during acute (left plot) or post treatment (right plot) gated on LiveCD19-CD14-CD56+ showing NK CD3-CD56+ (purple) and NKT CD3+CD56+ (green) cells. (g) Percentage of NKT cells among live cells in Acutely infected and Drug-treated subjects. (h) Percentage of NK cells among live cells in Acutely infected and Drug-treated subjects. The data were analyzed considering a 99% confidence interval (CI). P < 0.05 was considered significant. (* p<0.05; **p<0.01; ***p<0.001).
Fig 7
Fig 7. B cell receptors: CDR3 length, isotypes and V gene usage.
(a) Experimental design of the sorting process. Single CD27+ memory B cells of two P.vivax-infected subjects with the highest anti-PvVir14+ IgG titers, and two malaria-naïve U.S. subjects, were sorted into a 96-well plates and had V gene usage analyzed. Created with BioRender.com. (b) Length of CDR3 sequences of two P. vivax-infected subjects (P. vivax 1 and P. vivax 2) from the Brazilian Amazon and two healthy donors (HD1 and HD2) from the U.S. The y axis represents the absolute number of amino acids in individual B cells as well as the mean for all BCR sequenced from each subject. (c) Heavy chain isotype of BCR sequenced from two P.vivax-infected subjects, presented in pie charts as proportions. (d) Heavy chain isotypes of BCR sequenced from two HD donors, presented in pie charts as proportions. (e) Heavy chain isotyping of all four subjects analyzed (two P. vivax-infected and two HD). The y axis represents the percentage of BCR belonging to specific antibody classes. (f) Heavy chain gene usage of BCR sequenced for two P. vivax-infected subjects. (g) Heavy chain gene usage of BCR sequenced for two HD subjects. The y axis presents the frequency of individual V genes as a percentage of all BCR sequenced in the two group. Open bars indicate V genes unique to a group; colored bars indicate V genes shared between the groups, and the colors correspond to the same V gene in each group.
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Grants and funding

This work was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases (NIAID)/National Institutes of Health (NIH) (ZIA AI001134-10 to PED); Coordination for the Improvement of Higher Education Personnel (CAPES) (Finance Code 001 to RFF), Fundação de Amparo a Pesquisa do Estado de Minas Gerais/FAPEMIG, Brazil (Grant# CBB APQ-00766-18 to LLB), the Brazilian National Research Council (CNPq) (Grant# 421392/2018-5 and Grant# 302491/2017-1 to LLB), MCV, RCR, RTF, and LLB are Research Fellows from the Brazilian National Research Council (CNPq). The funders had no role in study design, data collection and analysis, the decision to publish, or the preparation of the manuscript.
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