Recombinant Pvs48/45 antigen expressed in E. coli generates antibodies that block malaria transmission in Anopheles albimanus mosquitoes

Abstract

Transmission of malaria parasites from humans to Anopheles mosquitoes can be inhibited by specific antibodies elicited during malaria infection, which target surface Plasmodium gametocyte/gamete proteins. Some of these proteins may have potential for vaccine development. Pvs48/45 is a P. vivax gametocyte surface antigen orthologous to Pfs48/45, which may play a role during parasite fertilization and thus has potential for transmission blocking (TB) activity. Here we describe the expression of a recombinant Pvs48/45 protein expressed in Escherichia coli as a ∼60kDa construct which we tested for antigenicity using human sera and for its immunogenicity and transmission blocking activity of specific anti-mouse and anti-monkey Pvs48/45 antibodies. The protein reacted with sera of individuals from malaria-endemic areas and in addition induced specific IgG antibody responses in BALB/c mice and Aotus l. griseimembra monkeys. Sera from both immunized animal species recognized native P. vivax protein in Western blot (WB) and immunofluorescence assays. Moreover, sera from immunized mice and monkeys produced significant inhibition of parasite transmission to An. Albimanus mosquitoes as shown by membrane feeding assays. Results indicate the presence of reactive epitopes in the Pvs48/45 recombinant product that induce antibodies with TB activity. Further testing of this protein is ongoing to determine its vaccine potential.

Fig 1. Immunization Schedule.

For: A. Mice, animals were immunized four times at 30 days interval with 20 μm of Pvs48/45 formulated with Freund Adjuvant. Blood samples were taken on days 0, 30, 60 and 90 for ELISA testing and on day 120 for functional and biological assessing. B. Monkeys, two experimental animals were immunized with 50 μg of Pvs48/45, and one control group with PBS emulsified in 1:1 with Montanide ISA 51 Adjuvant. Protein was administrated subcutaneously in the back at 20 days intervals, blood samples were taken before each immunization, 15 days after the third immunization and then at 30 days intervals until day 150. EG: Experimental group; CG: Control group; CFA: Freund Complete Adjuvant; IFA: Freund Incomplete Adjuvant; EM: Experimental Monkeys, CM: Control Monkey, ISA 51: Montanide ISA 51 Adjuvant, Prot. Pvs48/45 protein.

Fig 2. Schematic diagram and amino acid sequence alignment of Pvs48/45 protein.

A. Schematic diagram of Pvs48/45; boxes in grey represent the three P48/45 domains and the black box at the C-terminal region represents the trans-membrane domain. B. Multiple alignments between primary sequences of P48/45 proteins using the software Geneious. Conserved regions are represented in black. Cysteines are shown in blue.

Fig 3. Pvs48/45 protein expression in E. coli.

A. 10% SDS-PAGE stained with Coomasie blue. Lane 1, molecular weight marker; lane 2, un-induced cells total extract; lane 3, purified Pvs48/45 under reducing conditions (10mM β-mercaptoethanol). B. Western blot of Pvs48/45. Lane 1, molecular weight marker; Lane 2, un-induced cells total extract under reducing conditions (10mM β-mercaptoethanol). Lane 3, Pvs48/45 revealed against anti-His antibody; Lane 4, Pvs48/45 revealed against hyper-immune monkey sera. Arrows indicate the expected weight. C. Mass spectrum of Pvs48/45.

Fig 4. Immune responses to rPvs48/45.

In A. BALB/c mice. Serum samples were collected pre-immunization on day 0 and post-immunization on days 30, 60, 90 and 150 and B. Aotus lemurinus griseimembra. Kinetics of antibody titers in Aotus during immunization and follow-up period. Experimental monkeys immunized with 50 μg rPvs48/45 formulated in Montanide ISA-51; Control monkey immunized with a mixture of Montanide ISA-51 and PBS 1X. Titers correspond to the last dilution of the test sera in which OD₄₀₅ values were above that of the cut-off. Cut-off value were defined as pooled naïve mouse or monkey sera, OD₄₀₅ plus 3SD. Serum samples were tested at two-fold serial dilutions (1x10²–2x10⁵); a pool serum from naïve mice was used as negative control.

Fig 5. Recognition of parasite Pvs48/45 protein by sera of immunized monkeys.

A) Western blot assay using antigen extracted from pellet after iRBCs lysis in reduced conditions (β-mercaptoethanol). Lane 1, antigen (non-diluted); lane 2, antigen 1:2 diluted; lane 3, antigen 1:4 diluted. B) Western blot assay using antigen extracted from supernatant after iRBCs lysis in reduced conditions (β-mercaptoethanol). Lane 1, antigen (non-diluted); lane 2, antigen 1:2 diluted; lane 3, antigen 1:4 diluted. Triangles indicate increased concentration. C) Monkey IgG was reactive with Pvs48/45 on acetone-fixed smears of sexual blood stages of P. vivax with monkey sera containing antibodies to rPvs48/45. From left to right: parasite in light (left), parasite in epifluorescence (right).