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European Biopharmaceutical Review

Protein on the Fly

Developers of a protein-based vaccine for placental malaria are turning to insect cell systems as a possible low-cost manufacturing solution, which could meet the needs of philanthropically-funded vaccination programmes in developing countries.

The most deadly form of malaria is caused by the parasite Plasmodium falciparum, which is transmitted to humans by infected mosquitoes. The multiple different disease forms are influenced by the expression of antigens on the surface of the infected erythrocyte that enable the parasite to sequester to the vascular lining. The binding is mediated by a family of antigens called P falciparum erythrocyte membrane protein 1 (PfEMP1) encoded by var genes, of which there are about 60 in each genome. Protective antibodies block the binding between the infected red blood cells and receptors on vascular endothelium.

Women, who during childhood developed immunity against malaria, are susceptible to malaria during first pregnancies. Parasites accumulate in the placenta, where a combination of altered blood flow and expression of chondroitin sulphate A (CSA) provides a new niche for parasites to sequester (1). Constituting a major health problem in areas south of the Sahara, malaria in pregnant women manifests as death, anaemia in the mother, impaired fetal development, low birth weight or spontaneous abortion. Fortunately, women can acquire immunity against placental malaria (PM). The average birth weight is significantly higher among second and third time childbirth than at first childbirth. This relatively fast development of immunity has raised the hope that a vaccine to protect against placental malaria can be developed.

Discovery of a Possible Vaccine Target

The Centre for Medical Parasitology (CMP) at the University of Copenhagen (UCPH) identified in 2003 the malaria protein which is responsible for parasite binding to CSA in the placenta. It was initially shown that only one gene, named VAR2CSA, was highly expressed in only CSA selected parasites (2). Expression studies have since shown that parasites isolated from infected placenta expresses VAR2CSA in high quantities, and that this gene family is uniquely conserved (3).

Only women who have had placental malaria have antibodies to the VAR2CSA protein and the levels of antibodies are acquired as a function of the parity. Furthermore, women who have acquired VAR2CSA specific antibodies give birth on average to children that are 500g heavier than women without these protective antibodies (4). Both in vitro CSA-selected parasites and parasites directly from the placenta express VAR2CSA that contain cross-reactive B-cell epitopes on the surface of the infected erythrocyte. Furthermore, it has been shown that VAR2CSA-knockout parasites cannot bind CSA. This data argues strongly for the possibility of using VAR2CSA as a vaccine against PM. The aim of a vaccine is to induce antibodies that can hinder parasite adhesion in the placenta and mediate circulation, followed by destruction, of infected red blood cells in the spleen. VAR2CSA is a complex 350kDa protein with seven large domains. Many expression systems have been explored to express the complex subparts of VAR2CSA with limited success; either no expression was obtained or the produced protein did not induce functional antibodies, indicating a wrong fold of the recombinant protein. Using baculovirus transfected insect cells as well as Drosophila S2 cells, several parts of VAR2CSA have recently been produced. These protein variants were effective at inducing antibodies in animal models which inhibit parasite binding to the placental receptor VAR2CSA in vitro (5). A promising alternative approach is being followed by the Institut de Recherche pour le Développement (IRD), where a VAR2CSA DNA vaccine is under development. The next steps for the protein-based vaccine are to address manufacturability of the VAR2CSA antigen in insect cells and commence the toxicological test in animals.

The Drosophila S2 Expression System

Drosophila S2 insect cell expression is less well known than the extensively used Spodoptera or Trichoplusia ni (Hi-5) insect cell based Baculovirus expression system (BEVS). Nevertheless it has been used in research for almost 40 years. The cell line was derived from late stage Drosophila melanogaster (fruit fly) embryos by Schneider in the early 1970s, who named the cell line Drosophila Schneider line 2 (synonyms: S2, SL2, D mel 2); see Figure 1 (page 26) for an image of the cells. The system has been widely applied to fundamental research, where the availability of the whole genome sequence of D melanogaster and the S2 cells’ susceptibility to RNA interference methods have enabled genome wide RNAi screening and whole genome expression analysis techniques to be used to great effect (6-9). S2 cells have proved to be highly effective for the production of proteins from a great variety of protein classes (10), such as viral proteins, toxins, membrane proteins and enzymes (see Table 1, page 26).

Insect Cells for Clinical Material Manufacture

Over the last decade, insect cell systems have progressed from protein expression for basic research to product manufacture for the clinic and market. BEVS has been used to produce two recently marketed drugs, namely Cervarix, GSK’s HPV vaccine, and the protein antigen component of Dendreon’s Provenge, as well as Protein Sciences’ FluBløk which is nearing the market. These success stories have paved the way for regulatory approval of insect cell based systems. S2 cells are the only other insect cell system currently used in the clinic. To date, it has been applied to the manufacture of three subunit vaccines for clinical trials, namely Merck Sharp & Dohme's penta-valent Dengue virus vaccine, Hawaii Biotech’s West Nile virus vaccine, and Pharmexa’s (now Affitech) Her-2 positive breast cancer vaccine. An overview of insect produced vaccine programmes and products can be seen in Table 2.

Considerations for Development of a VAR2CSA Manufacturing Process

The geographic distribution of malaria and the philanthropic funding sources involved require the vaccine production to be as cost-efficient as possible. Both BEVS and Drosophila S2 based systems have been shown to produce the VAR2CSA antigen in an immunologically relevant form (in other words correctly folded). However, the S2 system has unique advantages for low-cost production compared to BEVS as it is a stable cell line based, non-viral and non-lytic system. This allows for a wide variety of upstream processing options compared to the obligatory batch process approach of the high-yielding, but lytic BEVS. The most established of these potential processes are fed-batch (leading to higher yields) and perfusion (improved yield, smaller footprint installations, with much reduced building costs). Fed-batch processes generally have significantly higher yields (two to more than tenfold) compared to simple batch processes. Implementing perfusion was shown by a recent study to save up to 42 per cent in capital investment, although only three per cent in cost-of-goods, compared to fed-batch (11). Further processing options are available with recent advances in concentrated fed-batch and concentrated perfusion techniques, which generally employ the ATFTM cell retention system. Here it was shown that, compared to a fed-batch process, savings of 25 per cent and 45 per cent can be attained by using concentrated fed-batch or concentrated perfusion processes (12). S2 cells are well suited to perfusion and concentrated perfusion cultures as they do not aggregate (a frequent issue in mammalian perfusion cultures), even at high densities (140 million cells per mL). A S2 cell culture perfusion process was used for the production of the Phase 1 and 2 Her-2 breast cancer vaccine trials mentioned above, as well as for the late pre-clinical stage RANK Ligand project. Excellent yield and product consistency were achieved for all five perfusion runs required up to the Phase 2 trial (see Figure 2). This demonstrates the viability of a perfusion-based approach for the production of cost-sensitive protein based vaccines.

Clinical Development of a Placental Malaria Vaccine

The insect cell produced VAR2CSA vaccine is currently in the development stage, with initial process development in progress and a limited panel of lead variants being screened for expression yield, protein characteristics and immunological profile in small-scale cultures. Upstream process development goals are focused on developing a fed-batch or concentrated fed-batch process for Phase 1, with further development for Phase 2 focused on a concentrated perfusion process. The goal is to have tox material produced by Q4 2013 and the first Phase 1 clinical trial initiated by Q3 2014.

Placental malaria is a disease with a high cost to society and represents an urgent medical need. Early development with the S2 system has shown promising results in vaccine efficacy and considerable potential for delivering a low-cost vaccine production process. Preparations for a Phase 1 clinical trial are underway.


  1. Fried M and Duffy PE, Adherence of Plasmodium falciparum to chondroitin sulfate A in the human placenta, Science 7,272 (5,267): pp1,502-1,504, 1996
  2. Salanti A et al, Selective upregulation of a single distinctly structured var gene in chondroitin sulphate A-adhering Plasmodium falciparum involved in pregnancy-associated malaria, Molecular Microbiology 49(1): pp179-91, 2003
  3. Tuikue NG et al, High level of VAR2CSA transcription by Plasmodium falciparum isolated from the placenta, Journal of Infectious Disease 192(2): pp331-5, 2005
  4. Salanti A et al, Evidence for the involvement of VAR2CSA in pregnancyassociated malaria, Journal of Experimental Medicine 200(9): pp1,197-1,203, 2004
  5. Dahlback M et al, The chondroitin sulfate A-binding site of the VAR2CSA protein involves multiple N-terminal domains, Journal of Biological Chemistry 286(18): pp15,908-15,917, 2011
  6. Adams MD et al, The genome sequence of drosophila melanogaster, Science 287: pp2,185-2,195, 2000
  7. Ashburner M et al, Drosophila melanogaster: a case study of a model genomic sequence and its consequences, Genome Research 15(12): pp1,661-1,667, 2005
  8. Neumüller RA et al, Where gene discovery turns into systems biology: genome-scale RNAi screens in Drosophila, Wiley Interdisciplinary Reviews Systems Biology and Medicine 3(4): pp471-478, 2011
  9. D’Ambrosio MV et al, A whole genome RNAi screen of Drosophila S2 cell spreading performed using automated computational image analysis, Journal of Cell Biology 191(3): pp471-8, 2010
  10. Schetz JA et al, Protein expression in the Drosophila schneider 2 cell system, Current Protocols in Neuroscience 4(16), 2004
  11. Lim AC et al, A computer-aided approach to compare the production economics of fed-batch and perfusion culture under uncertainty, Biotechnology and Bioengineering 93(4): pp687-697, 2006
  12. Lim J et al, An economic comparison of three cell culture techniques, BioPharm International 24(2): pp54-60, 2011

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Wian de Jongh is Vice President, Product Development and Co-Founder of ExpreS2ion Biotechnologies. Wian has a BSc and MSc in Chemical Engineering from the University of Stellenbosch, South Africa, and a PhD in Biotechnology from the Technical University of Denmark. Wian has been instrumental in developing a Drosophila S2 protein expression system, and has six years of experience in applying the system to process development and clinical material manufacture. Email:

Ali Salanti is an Associate Professor at the University of Copenhagen. He has an MA in Molecular Biology and a PhD in Medicine from the Faculty of Health at the University of Copenhagen, and leads a group of 25 scientists working on malaria vaccine development, with a particular focus on malaria during pregnancy. His group was the first to describe the VAR2CSA antigen, which is now the main focus of a malaria vaccine for pregnant women. Email:

Wian de Jongh
Ali Salanti
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