Variable immunogenicity of a vivax malaria blood-stage vaccine candidate

Abstract

Relapsing malaria caused by Plasmodium vivax is a neglected tropical disease and an important cause of malaria worldwide. Vaccines to prevent clinical disease and mosquito transmission of vivax malaria are needed to overcome the distinct challenges of this important public health problem. In this vaccine immunogenicity study in mice, we examined key variables of responses to a P. vivax Duffy binding protein vaccine, a leading candidate to prevent the disease-causing blood-stages. Significant sex-dependent differences were observed in B cell (CD80+) and T cell (CD8+) central memory subsets, resulting in significant differences in functional immunogenicity and durability of anti-DBP protective efficacy. These significant sex-dependent differences in inbred mice were in the CD73+CD80+ memory B cell, H2KhiCD38hi/lo, and effector memory subsets. This study highlights sex and immune genes as critical variables that can impact host responses to P. vivax antigens and must be taken into consideration when designing clinical vaccine studies.

Keywords: Malaria; Memory B cells; Plasmodium vivax; Sex as a biological variable; Vaccine.

Fig. 1:

Kinetics of humoral immune responses in mice immunized with P. vivax DBPII-Sal1. (a) IgG antibody titers in inbred BALB/c mice (n=5) and (b) outbred Swiss ND4 mice (n=5). (c and d) Inhibition of P. vivax DBPII transiently expressed on surface of COS7 cells from binding to DARC on erythrocyte surface (COS7 cell assay) by serum from inbred and outbred mice respectively. (e and f) Competition between mouse immune sera from inbred and outbred mice respectively and human mAb 092096 for binding to rDBPII. Chart depicts percent inhibition of DBPII binding to human mAb 092096 by mouse immune serum. Mice are defined as I = inbred, O = outbred, F = female, M = male, while groups are defined as: 1 (immunized with rDBPII, CpG and alum), 2 (immunized with rDBPII and alum) and 3 (immunized with CpG and alum only). Results are expressed as mean IgG antibody titers± SEM, mean percent inhibition relative to mAb 092096± SEM and mean percent inhibition± SEM. After normality testing, a Kruskal-Wallis with Dunn’s corrections were performed. Significant differences are noted on the graph: *p<0.05; **p<0.01;***p<0.001.

Fig. 2:

Heatmap showing Spearman’s correlation coefficient (rho - ρ). The heatmap depicts the correlation (rho - ρ) between the IgG titers from the IgG ELISAs (IgG), the inhibition data from Cos7 assays (COS7), and the competition data from competitive ELISAs (CE). Data from each of these assays were pooled by group, with all four time points (14, 35, 56, and 77 days post-immunization) being included to ensure that all ρ values remained consistent over time. All ρ values were positive, indicating that the data were positively correlated with each other. Darker shades represent stronger associations between data points, while lighter shades represent weaker associations. A ρ value of approximately 0.5 corresponds to a p-value below 0.05, so all pairwise associations with a correlation coefficient above 0.5 were deemed significant. Note that I = inbred BALB/c mice, O = outbred Swiss ND4 mice, F = female, M = male, 1 = group 1: mice (n=5) immunized with DBPII, CpG and alum, 2 = group 2: mice (n=5) immunized with DBPII and alum, 3 = group 3: mice (n=5) immunized with CpG and alum only.

Fig. 3:

Frequency of memory B cells defined using surface markers CD73 and CD80 from spleen and blood samples obtained from groups 1, 2 and 3 inbred (BALB/c) and outbred (SwissND4) mice on day 80 post immunization, (a and d) CD73+ CD80− memory B cell subsets in (a) BALB/c and (d) Swiss ND4 mice; (b and e) CD73+ CD80+ memory B cells in (b) BALB/c and (e) Swiss ND4 mice, and (c and f) CD73− CD80+ memory B cell subsets in (c) BALB/c and (f) Swiss ND4 mice. I = inbred BALB/c mice, O = outbred Swiss ND4 mice, F = female, M = male, 1 = group 1: mice (n=5) immunized with DBP II, CpG and alum; 2 = group 2: mice (n=5) immunized with DBPII and alum; 3 = group 3: mice (n=5) immunized with CpG and alum only. Results are expressed as mean ± SEM. Significant differences are noted on the graph: *p<0.05; **p<0.01; ***p<0.001.

Fig. 4:

Variation in the antigen binding and presentation profile. Frequency of activated MHC class I (H2K) and class II (I-A/I-E) were defined using surface markers H2K, I-A/I-E and CD38 from spleen samples obtained from group 1, 2 and 3 inbred (BALB/c) and outbred (SwissND4) mice (n=3). Activated lymphocyte percentages were expressed as (a) h2khiCD38hi/lo and (b) I-A/I-EhiCD38hi/lo from each of the CD8+ and CD4+ T cell plots. Results are expressed as mean ± SEM. Significant differences are noted on the graph as *p<0.05; **p<0.01; ***p<0.001. I = inbred BALB/c mice, O = outbred Swiss ND4 mice, F = female, M = male, 1 = group 1: mice (n=3) immunized with DBP II, CpG and alum; 2 = group 2: mice (n=3) immunized with DBPII and alum; 3 = group 3: mice (n=3) immunized with CpG and alum only.

Fig. 5:

Frequency of memory T cells were defined using surface markers CD44 and CD62l on activated lymphocytes from spleen samples obtained from group 1, 2 and 3 inbred (BALB/c) and outbred (SwissND4) mice (n=3). Percentages of memory T cell subsets are defined as (a and b) T central memory (CM T) or CD44hiCD62hi and (c and d) T effector memory (EM T) or CD44hiCD62llo populations, gated from each of the CD4+ and CD8+ populations. I = inbred BALB/c mice, O = outbred Swiss ND4 mice, F = female, M = male, 1 = group 1: mice (n=3) immunized with DBP II, CpG and alum; 2 = group 2: mice (n=3) immunized with DBPII and alum; 3 = group 3: mice (n=3) immunized with CpG and alum only. Results are expressed as mean ± SEM. Significant differences are noted on the graphs as *p<0.05; **p<0.01; ***p<0.001.