PvDBP gene amplification is associated with functional immune evasion by Plasmodium vivax in vivo

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.