AbDEN

The Problem

Dengue fever is a mosquito-borne viral disease found in tropical countries like India. It affects and kills thousands each year, and with climate change, the numbers are only expected to rise[1]. However, no cure for the disease exists. The treatment is majorly symptom control[2] and vector control is the only preventative step taken. The WHO has classified Dengue as a Neglected Tropical Disease (NTD)[3]. A vaccine exists, but it has its own problems arising from a phenomenon called antibody-dependent enhancement (ADE)[4][5][6], which is also the reason that no monoclonal antibody therapy for dengue is on the market.

In 2020, a large proportion of dengue cases in India and other places were misdiagnosed as COVID-19[7][8].

Our own city, Pune, faced a major outbreak of Dengue recently. Most of our team has been infected at least once. We decided that the creation of a true therapeutic against the disease was long overdue.

Antibody-Dependent Enhancement

The dengue virus has four major serotypes, and most antibodies generated by our immune response target only the serotype that has infected the body in the first (or primary) infection.

Thus, when someone gets infected with dengue for the second time, as a first line of defence, the immune response generates antibodies against the former serotype to target the newer one. These antibodies are unable to neutralise the virus, and act as Trojan horses to the immune system. When they signal the myeloid cells to consume the immune complex, the virus escapes from the vacuole inside the immune cells and starts to multiply – leading to increase in viremia and severe outcomes like Dengue shock syndrome and dengue haemorrhagic fever[9].

Fig: A visual depiction of the difference in viral fate upon internalisation during a primary and a secondary infection. The neutralising antibodies produced during a primary infection ensure viral destruction whereas the non-neutralising antibodies in a secondary infection allow for an alternative path for cell infection leading to an increased viremia.

This phenomenon has led to failures of vaccine development and efforts at treatment with monoclonal antibodies. Antibodies that are not cross-neutralising across serotypes, or present in sub-neutralising concentrations, can elicit ADE, leading to the patient developing severe dengue on second infection with a different serotype.

The Solution: Antibody Engineering

Antibodies are a special class of proteins that have the perfect balance of surface geometry and surface chemistry. This makes them immensely versatile, able to specifically target and bind almost any protein or biomolecule.

They are also incredibly modular - most of the antibody remains identical to others of its kind, antigen-specificity is determined only by a few amino acid loops in two domains of the protein. The remaining domains of a natural antibody are used for communication with the broader immune system.

Recombinant DNA technology has made it possible to manipulate these domains to create different forms of antibodies and antibody fragments, engineered for various purposes[10]. After a thorough review of existing literature and conversations with many experts, we identified the best approach to create an ideal therapeutic for Dengue - an antibody fragment called an scFv (single chain Fragment variable). It would not possess effector functions, so it would be able to bind to and neutralise the virus without causing ADE.

Fig: A visual description of the assembly of our NeoFv. The scFv was derived from the variable domains of a full-length C-10 IgG antibody, where the variable domains were held together with a flexible linker peptide. An FcRn binding peptide was then attached between the linker peptide, to facilitate FcRn binding, to create the NeoFv.

NeoFvs

A single chain fragment variable, or an scFv, is an antibody fragment created by joining the variable regions of the heavy and light chains of an antibody using a short, flexible peptide linker.

They are quite small, so they tend to be cleared from the body fairly quickly. This was a problem because it would create issues down the line with high dosages, which could possibly make the therapeutic quite expensive and more likely to be toxic to the patient. To address this, we engineered our scFv to bind to the receptor in the body that typically governs and extends the half-life of native full-length antibodies(FcRn, or neonatal Fc receptors). We added an FcRn-binding peptide to the scFv, that has been demonstrated in-vitro to extend the half-life of the molecule. We decided to call this assembly a NeoFv[11].

Since scFvs do not have effector functions, they cannot cause ADE[12]. They will simply act as neutralising agents by binding to the virus and preventing it from undergoing the structural changes required for pathogenicity. The virus-fragment complexes will get degraded by nonspecific proteolytic pathways in the bloodstream.

The Path to the Solution: Synthetic Biology

We needed a platform for the quick generation of our recombinant antibody formats, that would fold properly and work as intended. Eukaryotic cells are the canonical medium for antibody production, but are difficult and expensive to manipulate and grow. A prokaryotic system would be ideal, but they cannot produce disulfide bonds outside their periplasm, which makes overexpression of a large, complex, multimeric protein challenging.

Enter SHuffle: a special strain of E. coli engineered to be able to form disulfide bonds in its cytoplasm. The strain has mutated reductive pathways (trx-, gor-) so it has an oxidative cytoplasm;alternate reductive pathways (ahPc*) have been introduced so that the knockout mutations are not lethal. Additionally, the strain expresses disulfide-bond isomerase (DsbC) cytoplasmically, to help form disulfide bonds in the cytoplasm[13].

Fig: A visual representation of the various pathways in the cytoplasm of SHuffle B E.coli that ensure that the cytoplasm is made oxidative, which along with the overexpression of DsbC facilitates the correct folding of full-length antibodies within the cytoplasm at higher yields.

SHuffle is a modular, easily genetically manipulable platform for the production of antibodies, antibody fragments in various formats, and other complex proteins, in higher yields compared to periplasmic expression in other strains[14]. We present a plug-and-play system for the generation of therapeutic antibodies.

Goals

  • To identify and design the right antibodies that target all dengue serotypes.
  • To synthesize antibodies and fragments in SHuffle E. coli
  • To assay these antibodies and prove they bind to the target and don’t cause ADE.
  • Scale up the production of antibodies in higher yields.
  • Educate and raise awareness about Dengue and neglected tropical diseases.
To read more on how we will achieve these goals, read the Design page.

References

  1. Campbell LP, Luther C, Moo-Llanes D, Ramsey JM, Danis-Lozano R, Peterson AT. Climate change influences on global distributions of dengue and chikungunya virus vectors. Philos Trans R Soc Lond B Biol Sci. 2015 Apr 5;370(1665):20140135. DOI
    PMID: 25688023; PMCID: PMC4342968.
  2. Symptom control for the treatment of dengue
  3. Neglected tropical diseases
  4. Dengue vaccine
  5. Dengvaxia controversy
  6. Antibody dependant enhancement
  7. Kulkarni R. Antibody-Dependent Enhancement of Viral Infections. Dynamics of Immune Activation in Viral Diseases. 2019 Nov 5:9–41. DOI
    PMCID: PMC7119964.
  8. Phadke R, Mohan A, Çavdaroğlu S, Dapke K, Costa ACDS, Riaz MMA, Hashim HT, Essar MY, Ahmad S. Dengue amidst COVID-19 in India: The mystery of plummeting cases. J Med Virol. 2021 Jul;93(7):4120-4121. DOI
    Epub 2021 Apr 6. PMID: 33788285; PMCID: PMC8250513.
  9. Yan G, Lee CK, Lam LTM, Yan B, Chua YX, Lim AYN, Phang KF, Kew GS, Teng H, Ngai CH, Lin L, Foo RM, Pada S, Ng LC, Tambyah PA. Covert COVID-19 and false-positive dengue serology in Singapore. Lancet Infect Dis. 2020 May;20(5):536. DOI
    Epub 2020 Mar 4. PMID: 32145189 PMCID: PMC7128937.
  10. Bates A, Power CA. David vs. Goliath: The Structure, Function, and Clinical Prospects of Antibody Fragments. Antibodies (Basel). 2019 Apr 9;8(2):28. DOI
    PMID: 31544834; PMCID: PMC6640713.
  11. Fink AL, Williams KL, Harris E, Alvine TD, Henderson T, Schiltz J, Nilles ML, Bradley DS. Dengue virus specific IgY provides protection following lethal dengue virus challenge and is neutralizing in the absence of inducing antibody dependent enhancement. PLoS Negl Trop Dis. 2017 Jul 7;11(7) :e0005721. DOI
    PMID: 28686617; PMCID: PMC5517069.
  12. Kelly, V. W., & Sirk, S. J. (2022). SHORT FCRN-binding peptides enable salvage and transcytosis of scFv antibody fragments. ACS Chemical Biology, 17(2), 404–413. DOI
  13. Balsitis SJ, Williams KL, Lachica R, Flores D, Kyle JL, Mehlhop E, Johnson S, Diamond MS, Beatty PR, Harris E. Lethal antibody enhancement of dengue disease in mice is prevented by Fc modification. LoS Pathog. 2010 Feb 12;6(2):e1000790. DOI
    PMID: 20168989; PMCID: PMC2820409.
  14. Ju MS, Jung ST. Aglycosylated full-length IgG antibodies: steps toward next-generation immunotherapeutics. Curr Opin Biotechnol. 2014 Dec;30:128-39. DOI
    Epub 2014 Jul 16. PMID: 25035939.
  15. Lobstein, J., Emrich, C.A., Jeans, C. et al. SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm. Microb Cell Fact 11, 753 (2012). DOI
  16. Robinson MP, Ke N, Lobstein J, Peterson C, Szkodny A, Mansell TJ, Tuckey C, Riggs PD, Colussi PA, Noren CJ, Taron CH, DeLisa MP, Berkmen M. Efficient expression of full-length antibodies in the cytoplasm of engineered bacteria. Nat Commun. 2015 Aug 27;6:8072. DOI
    PMID: 26311203; PMCID: PMC4560801.