A target-agnostic approach to identify candidate vaccine antigens expressed at different stages of the P. falciparum life cycle using monoclonal antibodies from African volunteers experimentally infected with live sporozoites

1. Overview of the project or Platform developed

The research proposal entitled “A target-agnostic approach to identify candidate vaccine antigens expressed at different stages of the P. falciparum life cycle using monoclonal antibodies from African volunteers  experimentally infected with live sporozoites” brings together three Swiss research groups with complementary and unique expertise. The groups have developed new tools and approaches that effectively circumvent major bottlenecks that have limited in the past the analysis of the human antibody response to P. falciparum. The aim of the proposal is to isolate human monoclonal antibodies that target functional and conserved antigens expressed on all different stages of the parasite life cycle. The team will use memory B cells from experimentally malaria exposed volunteers and novel screening approaches that take advantage of purified sporozoites as well as P. falciparum mutants that overexpress var antigens and generate large number of gametocytes in vitro. This project will deliver a large panel of well-characterized human monoclonal antibodies that block P. falciparum infectivity and pathology, which will provide new insights into the human protective response and help prioritize antigens for inclusion in malaria vaccines.

The research project has the following specific objectives:

1. To isolate from memory B cells of immune donors a panel of monoclonal antibodies that bind to the surface of sporozoites, merozoites, infected erythrocytes and gametocytes.
2. To identify conserved functional antigens in sporozoites, merozoites, infected erythrocytes and gametocytes using different approaches.
3. To use this information to identify vaccine candidates and to dissect mechanisms of protection and pathogenesis under experimental malaria vaccination followed by CHMI.

2. Research work achieved during the funding period

MTA and consortium agreement (All participants)

An MTA and agreement has been finalized between Sanaria, PP, IHI, SwissTPH and IRB to ensure the transfer of sporozoites and recombinant CSP from Sanaria to IRB as screening tool for detection of vaccination-induced antibodies targeting the pre-erythrocytic development.

Patient samples for the isolation of memory B cells (Daubenberger)

In total, 40 volunteers were successfully inoculated five times with two different doses of live attenuated, purified sporozoites of P. falciparum (strain NF54) during the BSPZV1 trial. Three weeks after the vaccination period, all volunteers were challenged intravenously with live, non-attenuated sporozoites of P. falciparum (strain NF54). All controls included here became positive for asexual blood stage antigens after the challenge proving that the sporozoite dose and route was sufficient to infect 100 % of non-vaccinated volunteers. Several volunteers withstood the challenge proving that the vaccination confers protection. From all volunteers, peripheral blood mononuclear cells were obtained by the PhD student Isabelle Zenklusen during her stay in Bagamoyo, stored in liquid nitrogen and transferred to Basel, Switzerland from Tanzania. After un-blinding of the clinical trial, a clear vaccine dose dependency of the protection level against homologous challenge three weeks after last vaccination was observed. In the group of volunteers vaccinated with the lower dose, 1/20 showed sterile protection, while in the higher dose 4/20 volunteers withstood the homologous challenge. We transferred PBMC from these vaccinees to IRB for establishment of human monoclonal antibodies targeting the sporozoite stage.

Tool development for the functional dissection and validation of vaccine candidates (Voss)

We generated a novel transgenic parasite line allowing us to induce sexual conversion and gametocyte differentiation in a synchronous manner at a rate exceeding 50% (M. Filarsky, unpublished). This line was used to screen the sera from the 40 volunteers experimentally infected with live P. falciparum sporozoites (CHMI trial) for the presence of antibodies against the surface of trophozoite- and gametocyte-infected red blood cells (see below). In the meantime, we developed our transgenic gametocyte hyper-producer line further in two major ways using CRISPR/Cas9-mediated gene editing approaches. First, we cloned several versions of the conditional expression cassette required for the induction of sexual conversion and integrated these into the parasite genome. We are currently evaluating the efficiency of sexual conversion for these lines and are hopeful to soon have identified a stable mutant cell line that can achieve sexual conversion rates of 80-100%. Second, in these parasites we deleted various genes encoding proteins predicted to be exported to the RBC cytosol/membrane specifically in gametocytes. In parallel, we also modified these loci such that the encoded proteins are expressed as C-terminal GFP, TdTomato or 3xHA fusions. This powerful tool – high-level induction of sexual conversion combined with fast and precise CRISPR/Cas9-mediated gene deletions/modifications – opens up completely new possibilities to study gene function in gametocytes, gametes and sporozoites at unprecedented speed, quality and throughput, and will be particularly useful for the functional dissection and validation of vaccine candidates.

Serum antibodies against antigens expressed on sporozoites, merozoites, erythrocytes and gametocytes (Daubenberger, Voss)

All sera showed specific reactivity with both trophozoites and gametocytes in indirect immunofluorescence assays (IFA) using permeabilised cells, delivering distinct staining patterns throughout the parasite as well as host cell cytosol and membrane. All sera also reacted with the surface of live trophozoite-infected RBCs as expected. However, none of these sera recognized antigens on the surface of gametocyte-infected RBCs (tested for strain NF54 and clone 3D7; tested by IFA on live cells and by flow cytometry). Noteworthy, antigens associated with the surface of gametes (exposed after gametocytes egressed from the RBC) were readily detected by these sera. This suggests either that, in contrast to trophozoites and schizonts, gametocytes do not expose (conserved) antigens on the surface of the infected RBC, or that the sera tested here don’t contain antibodies against such antigens. Based on these negative results we didn’t pursue the planned flow cytometry-based isolation of human monoclonal antibodies against gametocyte surface antigens.

Human monoclonal antibodies against sporozoites (Lanzavecchia)

PBMC from two protected volunteers were used to isolate eight IgG antibodies to P. falciparum sporozoites. The supernatants of immortalized memory B cells were screened for their capacity to stain the surface of intact sporozoites (obtained from Sanaria). This approach was designed to capture all the possible specificities. Eight IgG antibodies that stained intact sporozoites were isolated and further characterized using recombinant proteins and peptides. All were found to bind to the NANP repeats in the CSP protein. Three of them also bound to N terminal peptide and one to the C terminal peptide. The monoclonals are currently tested by Isabelle Zenklusen in the laboratory of Stefan Kappe at CIDR, Seattle, for functional analysis. Interestingly, 50 % (4/8) of the antibodies showed in vitro strong sporozoite invasion and traversal inhibition using a human liver cell line and live non-attenuated sporozoites. Several antibodies (both, inhibitory and non-inhibitory) are currently tested in humanized mouse models for their in vivo functional relevance in relation to sporozoite and liver stage development inhibition. Results are expected within the next month.

Human monoclonal antibodies against malaria infected erythrocytes (Lanzavecchia)

In order to set up the screening assays and the methods for antigen identification, we started to work on a set of samples that we previously obtained from Dr. Peter Bull working at the Kenya Medical Research Institute (KEMRI) in Kilifi, Kenya. By immortalizing memory B cells from Kenyan samples and screening with the new flow-based assay we isolated a large panel of human monoclonal antibodies that stain different proportions of IE and show different patterns of reactivity with field isolates of Pf. These broad spectrum antibodies were found to be produced by a new mechanism of DNA transposition whereby a fragment encoding the extracellular domain of LAIR-1 (a collagen binding inhibitory receptor encoded on chromosome 19) is inserted between the V and DJ. We found that the binding to IE was exclusively mediated by the mutated LAIR-1 domain and identified the target antigens as members of the RIFIN family. We also found that LAIR-1 containing antibodies agglutinate and promote phagocytosis of IE. We have recently extended the analysis of broadly reactive antibodies to the Tanzanian cohort (Daubenberger) in Bagamoyo and the Malian cohort (collaboration with Peter Crompton, NIAID) and isolated several additional LAIR-1 containing clones. Interestingly in some of these clones the LAIR-1 DNA is inserted in the switch region leading to the expression of the LAIR-1 domain in the elbow between the VH and the CH1 domains. We also estimated that approximately 5% of individuals living in malaria endemic regions make LAIR-1 containing antibodies. These results will be the subject of a new publication.

3. Main research results, their relevance and impact

LAIR-1 containing antibodies to IE: These findings illustrate, with a biologically relevant example, a novel mechanism of antibody diversification by interchromosomal DNA transposition and demonstrate the existence of conserved epitopes that may be suitable candidates for the development of a malaria vaccine.

Sporozoite-specific antibodies from vaccinated African donors: From these exciting outcomes, it is for the first time within reach to define target structures recognized by sporozoite inhibitory and non-inhibitory antibodies induced by whole attenuated sporozoite vaccination in African Volunteers residing in malaria epidemic countries. This information will be essential for potentially defining biomarkers of protection of the whole sporozoite vaccine approach. Sanaria has obtained recently the FDA Fast Track Designation (http://www.fda.gov/ForPatients/Approvals/Fast/ucm405399.htm) (http://www.sanaria.com/pdf/Fast%20Track%20Press%20Release%2022SEP2016.pdf) which details that identification of biomarkers of protection will support review and licensure process of this vaccine. This FDA Fast Track Designation has never been obtained by a malaria vaccine before.

4. Future research activities

The team aims to identify additional surface structures of sporozoites that might be recognized by the antibodies of sporozoite vaccinated volunteers. If identified, human monoclonal antibodies will be established and tested similarly to the CSP specific antibodies currently under functional analysis.

In a follow up trial conducted in 2016, we have obtained 100 % protection in a group of volunteers vaccinated three times with a much higher dose of sporozoites. PBMC of these volunteers have been collected and stored in liquid nitrogen and are available for cellular and humoral immune response studies.

5. Personnel involved and collaborations

IRB: Joshua Tan, Luana Perlini, Kathrin Pieper, PhD; Sonia Barbieri, PhD; Silvia Preite, PhD; Chiara Silacci:

STI: Isabelle Zenklusen; Armin Passecker; Enja Kipfer; Nicole Bertschi

Conclusion
This work illustrates the success of using the new human monoclonal antibody technology on samples obtained from well-defined cohorts in clinical trials where malaria is still prevalent.

We have successfully isolated human monoclonal antibodies against the IE and sporozoite stages of the P. falciparum life cycle. The antibodies against the IE gained broad reactivity against different parasite isolates through the insertion of a DNA fragment of the inhibitory receptor LAIR1. This illustrates a new mechanism of antibody diversification which has been observed in substantial fraction of individuals from malaria-endemic areas. The anti-sporozoite antibodies are able to potently inhibit the invasion of sporozoites into a liver cell line and are currently being tested in an in vivo mouse model.

6. Reference and publications

Tan J, Pieper K, Piccoli L, Abdi A, Foglierini M, Geiger R, Tully CM, Jarrossay D, Ndungu FM, Wambua J, Bejon P, Fregni CS, Fernandez-Rodriguez B, Barbieri S, Bianchi S, Marsh K, Thathy V, Corti D, Sallusto F, Bull P, Lanzavecchia A. 2016. A LAIR1 insertion generates broadly reactive antibodies against malaria variant antigens. Nature 529: 105-9 

Lanzavecchia A, Fruhwirth A, Perez L, Corti D. 2016. Antibody-guided vaccine design: identification of protective epitopes. Curr Opin Immunol 41: 62-7