Abstract
The key ligand involved in Plasmodium vivax reticulocyte invasion is the Duffy Binding Protein (PvDBP) which binds the Duffy receptor on reticulocytes. Anti-PvDBP human monoclonal antibodies can inhibit PvDBP-Duffy receptor binding and neutralize reticulocytes invasion in vitro . However, parasites with multiple copies of the pvdbp gene are protected in vitro against neutralization. Here, we evaluated whether this gene amplification also protects parasites in vivo. We hypothesized that: (i) multi- pvdbp copy parasites are more frequent in areas with a high P. vivax prevalence, (ii) individuals with naturally acquired binding inhibitory anti-PvDBP Abs (BIAbs) are predominantly infected over time by multi-copy parasites and (iii) multi-copy parasites infect asymptomatic carriers more frequently than symptomatic individuals. We analyzed samples from a 2019-2020 longitudinal cohort of individuals living in nine villages in Eastern Cambodia with low (~5%) to high (~30%) P. vivax prevalence. Using a PCR assay targeting the boundaries of the pvdbp duplication, we estimated the frequency of multi-copy parasites over time. Then, using a flow cytometry assay, we determined the presence of naturally acquired BIAbs in 657 participants' plasma. Finally, we compared parasite gene copy number over the 21-month follow-up in cohort participants according to the presence of BIAbs at the start of the study. In parallel, we determined the frequency of pvdbp duplication in samples collected among symptomatic treatment-seeking patients in the same area over the same period. We compared the frequency of infection with multi-copy parasites between asymptomatic cohort members and symptomatic patients. We found a significant association between P. vivax prevalence and the proportion of multi-copy parasites, which ranged from 35% in low prevalence villages to 47% in high prevalence villages (p=0.0246). We also observed that the more inhibitory the Abs in the hosts' plasma, the higher the proportion of multi-copy parasites: 87% (40/46) from individuals with high BIAbs while 38% (193/514) from individuals without any BIAbs (p<0.0001). This association between immunity and infection by multi-copy parasites remained consistent over the 21-month longitudinal follow-up. Finally, we found that the frequency of multi-copy parasites was higher in asymptomatic carriers than in symptomatic individuals. Overall, these results indicate that pvdbp duplication helps the parasites to avoid the hosts' anti-PvDBP immunity in vivo . It warrants further investigations to determine if immunization with a PvDBP vaccine could overcome this immune evasion mechanism.