Naturally Acquired Antibody Response to Malaria Transmission Blocking Vaccine Candidate Pvs230 Domain 1

Abstract

Plasmodium vivax malaria incidence has increased in Latin America and Asia and is responsible for nearly 74.1% of malaria cases in Latin America. Immune responses to P. vivax are less well characterized than those to P. falciparum, partly because P. vivax is more difficult to cultivate in the laboratory. While antibodies are known to play an important role in P. vivax disease control, few studies have evaluated responses to P. vivax sexual stage antigens. We collected sera or plasma samples from P. vivax-infected subjects from Brazil (n = 70) and Cambodia (n = 79) to assess antibody responses to domain 1 of the gametocyte/gamete stage protein Pvs230 (Pvs230D1M). We found that 27.1% (19/70) and 26.6% (21/79) of subjects from Brazil and Cambodia, respectively, presented with detectable antibody responses to Pvs230D1M antigen. The most frequent subclasses elicited in response to Pvs230D1M were IgG1 and IgG3. Although age did not correlate significantly with Pvs230D1M antibody levels overall, we observed significant differences between age strata. Hemoglobin concentration inversely correlated with Pvs230D1M antibody levels in Brazil, but not in Cambodia. Additionally, we analyzed the antibody response against Pfs230D1M, the P. falciparum ortholog of Pvs230D1M. We detected antibodies to Pfs230D1M in 7.2 and 16.5% of Brazilian and Cambodian P. vivax-infected subjects. Depletion of Pvs230D1M IgG did not impair the response to Pfs230D1M, suggesting pre-exposure to P. falciparum, or co-infection. We also analyzed IgG responses to sporozoite protein PvCSP (11.4 and 41.8% in Brazil and Cambodia, respectively) and to merozoite protein PvDBP-RII (67.1 and 48.1% in Brazil and Cambodia, respectively), whose titers also inversely correlated with hemoglobin concentration only in Brazil. These data establish patterns of seroreactivity to sexual stage Pvs230D1M and show similar antibody responses among P. vivax-infected subjects from regions of differing transmission intensity in Brazil and Cambodia.

Keywords: Plasmodium vivax; Pvs230; malaria; seroreactivity; transmission-blocking vaccine.

Figure 1

Study sites in (A) Rondônia state, Brazil, South America (pink), and (B) Pursat province, Cambodia, Asia (green). Samples were collected in Brazil in 2011 and 2014 and in Cambodia during 2010.

Figure 2

IgG response against Pvs230D1M in P. vivax-infected subjects, determined by ELISA. The seroprevalence of antibodies with specificity for Pvs230D1M in Brazil was 27.1% (19/70 subjects), and in Cambodia 26.6% (21/79 subjects). Cut-off value (1.18) was calculated based on mean control + 3 standard deviations. One-Way ANOVA followed by multiple comparisons was used for this analysis and results are displayed as mean ± SD.

Figure 3

Immunoglobulin G subclass responses to Pvs230D1M in Brazil and Cambodia. Healthy malaria-naïve donor sera were used to define the background for each subclass, and cut-off was calculated based on mean control + 3 standard deviations. IgG3 and IgG1 responses were predominant with limited IgG2 and no IgG4 response. One-Way ANOVA followed by multiple comparisons was used for this analysis and results are displayed as mean ± SD.

Figure 4

Pvs230D1M IgG responses do not correlate with age in Cambodia but increase significantly within age strata. (A) Pearson correlation and Linear regression, comparing Pvs230D1M antibody titers and age. (B) Seroprevalence for Pvs230D1M IgG (mean ± SD) was increased with age strata in Cambodian subjects: Pvs230D1M IgG response detected in 1.3% of 1–9 year old children (N = 8), 6.3% in 10–19 year old children (N = 26), and 19% in adults 20 years and older (N = 45). Kruskal-Wallis statistic test was used for the comparisons.

Figure 5

Parasitemia levels in comparison to Pvs230D1M IgG titers in (A) Brazil and (B) Cambodia. Kruskal-Wallis analysis revealed no significant difference between the groups. Detailed information on parasitemia levels for each subject is reported in Table S1.

Figure 6

Pvs230D1M-specific antibody responses inversely correlate with hemoglobin levels in Brazil but not in Cambodia.

Figure 7

IgG ELISA levels against PvDBP-RII, PvCSP, and Pfs230D1M in P. vivax-infected subjects. The seroprevalence of IgG antibodies with specificity for (A) PvDBP-RII in Brazil was 67.1% (47/70 subjects) and in Cambodia 48.1% (38/79 subjects); (B) PvCSP in Brazil was 11.4% (8/70 subjects) and in Cambodia 41.8% (33/79 subjects); (C) Pfs230D1M in Brazil was 7.2% (5/69 subjects; the volume of one sample was insufficient for assay) and in Cambodia 16.5% (13/79). The cut-off levels for detection (1.44, 0.97, and 1.16 respectively for PvDBP-RII, PvCSP, and Pfs230D1M ELISA) were based on control mean + 3SD calculation. One-Way ANOVA followed by multiple comparisons was used for this analysis and results are displayed as mean ± SD.

Figure 8

Correlation between antibodies elicited in response to blood-stage antigen (PvDBP-RII) or sporozoite-stage antigen (PvCSP) and hemoglobin levels in Brazil and Cambodia.

Figure 9

ELISA on Pvs230D1M-depleted sera. Pvs230D1M IgG levels were gradually reduced in sera by depletion assay (Figure S2), and then samples were submitted to ELISA against Pfs230D1M. Specificity of depletion was confirmed measuring antibody titers to PvDBP-RII in sera depleted for Pvs230 IgG (Figure S3).