Extending the range of Plasmodium falciparum transmission blocking antibodies

Abstract

Recent work demonstrating that asymptomatic carriers of P. falciparum parasites make up a large part of the infectious reservoir highlights the need for an effective malaria vaccine. Given the historical challenges of vaccine development, multiple parasite stages have been targeted, including the sexual stages required for transmission. Using flow cytometry to efficiently screen for P. falciparum gamete/zygote surface reactivity, we identified 82 antibodies that bound live P. falciparum gametes/zygotes. Ten antibodies had significant transmission-reducing activity (TRA) in a standard membrane feeding assay and were subcloned along with 9 nonTRA antibodies as comparators. After subcloning, only eight of the monoclonals obtained have significant TRA. These eight TRA mAbs do not recognize epitopes present in any of the current recombinant transmission-blocking vaccine candidates, Pfs230D1M, Pfs48/45.6C, Pf47 D2 and rPfs25. One TRA mAb immunoprecipitates two surface antigens, Pfs47 and Pfs230, that are expressed by both gametocytes and gametes/zygotes. These two proteins have not previously been reported to associate and the recognition of both by a single TRA mAb suggests the Pfs47/Pfs230 complex is a new vaccine target. In total, Pfs230 was the dominant target antigen, with five of the eight TRA mAbs and 8 of 11 nonTRA gamete/zygote surface reactive mAbs interacting with Pfs230. Of the three remaining TRA mAbs, two recognized non-reduced, parasite-produced Pfs25 and one bound non-reduced, parasite-produced Pfs48/45. None of the TRA mAbs bound protein on an immunoblot of reduced gamete/zygote extract and two TRA mAbs were immunoblot negative, indicating none of the new TRA epitopes are linear. The identification of eight new TRA mAbs that bind epitopes not included in any of the constructs currently under advancement as transmission-blocking vaccine candidates may provide new targets worthy of further study.

Keywords: Malaria transmission; Monoclonal antibody screen; Mosquito membrane feed; Plasmodium falciparum gametes; Vaccines.

Figure 1

Monoclonal (mAb) selection schematic. Mice were immunized with whole gametes/zygotes, gamete/zygote membranes, or material immunoprecipitated with Pfs230-specific mAb 1B3 or Pfs25-specific mAb 4B7. The latter two immunogens induced more potent transmission-reducing activity (TRA) and splenocytes from 3 mice in each of these groups were used to generate hybridomas. The IgG+ hybridoma supernatants were screened for gamete/zygote surface reactivity using flow cytometry and the surface reactive IgGs were tested for TRA using a standard membrane feed assay. All the TRA and selected nonTRA hybridomas were cloned and rescreened for gamete/zygote surface reactivity, TRA and antigen reactivity. Antigen reactivity was assessed by immunoblot, surface reactivity with gametes/zygotes that do not express Pfs230 (Pfs230 minus IFA), and mass spectroscopy (MS/MS).

Figure 2

Monoclonal (mAb) sexual stage parasite reactivity. Immunoblots of purified gametocyte (gc) or gamete/zygote (gm) Triton X-100 extract without β-mercaptoethanol or gamete/zygote Triton-X-100 extract with (r) and without (nr) β-mercaptoethanol were probed with the indicated mAb or as positive controls, anti-recombinant MBP.Pfs230.C (α230) (A) or MBP.Pfs48/45 (α48/45) serum (B). Previously reported mAb specific for Pfs230 (1B3) (A) or Pfs25 (4B7) (C) are also included as controls. Immunoblots are grouped according to the mAb pattern: A) Pfs230-like, B) Pfs48/45-like, C) Pfs25-like, and D) No reactivity. 5-Bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT) were used to visualize the bands.

Figure 3

Monoclonal (mAb) gamete/zygote surface reactivity. Purified wild-type strain NF54 (wt) or Pfs230Δ_452–3135 (Pfs230Δ) gametes/zygotes were incubated with the indicated mAb, washed, and then stained with Alexa Fluor 488-labeled anti-mouse IgG and membrane potential dye DiIC₁ (5). Fluorescence was monitored using flow cytometry and fluorescence microscopy. For each antibody and gamete/zygote population, the DiIC1(5) and Alexa Fluor 488 fluorescence of each cell is plotted and representative fluorescent and bright field images are shown.

Figure 4

Comparison of the profiles of monoclonals (mAbs) generated by each of the immunogens. mAbs that were derived from mice immunized with gamete/zygote membranes are in the light blue boxes and those from mice immunized with material immunoprecipitated by TRA mAbs are in the dark blue boxes. Within each box mAbs have been divided first based on their immunoblot reactivity and TRA mAbs are underlined. The immunoblot-negative mAbs are further separated based on their lack of reactivity with Pfs230Δ_357–3135 gametes/zygotes (Pfs230 Assoc). The two immunoblot-negative mAbs that still reacted with Pfs230Δ_357–3135 gametes/zygotes, 11A6 and 6C6, were then separated based on TRA and mass spectrometry data.

Figure 5

Monoclonals (mAbs) do not react with recombinant Pfs230. A) Schematic of the structure of Pfs230. Glutamate-rich repeats are indicated by vvv and 6-cys domains are depicted by boxes numbered 1 to 14 with the actual number of cysteines in each domain indicated in the box. The Pfs230 regions included in the recombinant proteins tested for reactivity are shown, the non-structured domain, Glu⁴⁴⁴-Val⁵⁹² without (EcPfs230ns, □) and with domains 1 and 2, Glu⁴⁴⁴-Asn⁹¹⁵ (EcPfs230/nsd1d2, ■) produced in E. coli (Ec) and domain 1, Ser⁵⁴²-Gly⁷³⁶ (Pfs230D1M, ■) produced in Pichia pastoris. (B) The 3 recombinant proteins were tested for reactivity with the indicated mAb using ELISA. Red boxes indicate mAbs with transmission-reducing activity, including the positive control 4F12. Alkaline phosphatase-labeled anti-mouse secondary antibody and p-nitrophenyl disodium phosphate substrate were used for detection.

Figure 6

Monoclonal (mAb) F6.30C5.11A6 does not react with recombinant Pfs47. A) Schematic of the three Pfs47 6-cys domains included in the 7 E. coli-produced recombinant proteins tested for antibody recognition using ELISA. B) The reactivity of the 6 antibodies, JH11 (▪), mAb1 (▪), mAb5 (▪), IB2 (▪), mIgG (▪), 11A6 (▪) against the indicated recombinant Pfs47 proteins (1–7) is plotted. Alkaline phosphatase-labeled anti-mouse secondary antibody and p-nitrophenyl disodium phosphate substrate were used for detection.

Figure 7

Recombinant Pfs25 Reactivity. An immunoblot of purified recombinant Pfs25 (rPfs25, aa 22–193) produced in Pichia pastoris pretreated with (r) or without (nr) 5% β-mercaptoethanol was probed with the indicated monoclonal.