Antivenoms Using Phage Display AntibodiesApis Mellifera is a honeybee that has a massive defensive reaction.This reaction involves secretion of Melittin and PLA2 venoms via stinging.
Thistoxic venoms can lead to hemolysis, edema, and eventually death. A. Melliferabee is diffused heavily in the United States. Indeed, this bee is widespread inthe Americas and has caused more than 249 deaths in Brazil since 2000. Thistype of envenomation is usually treated using an immune serum that is capableof minimizing the effect of the poison. Though, the sera generated by researchhave proven to trigger undesirable reactions. Due to the fact that there is alack of medicament that efficiently targets the toxics of the bee, a lab at theUniversity of São Paulo decided to compact a treatment using phage display.Thus, antibody fragment will be generated to handle the toxic effect of the bee.
To achieve this goal, there are several technologies to obtainantibodies. The most powerful one is the phage display, which generatesantibodies that attach to the antigen targets with relatively high affinity. Infact, Phage display is a technique which allows to detect protein-proteininteraction and produces exogenous polypeptides on the surface of a phageparticle; it was introduced by Georgy Smith in 1985. One of the main componentscommonly used in phage display is Filamentous bacteriophage (M13), a bacteriaphage virus that only infects bacteria. It has a single-stranded circular DNA andis enclosed by different viral coat proteins. The end of the M13 particle iscapped with 5 copies of PIII coat protein. PIII is typically exploited as afusion partner for the displayed antibody. In addition, the use of M13 hasseveral advantages.
For instance, the phage coat proteins in it can be modifiedto exhibit fused particles without decreasing its infectivity. Additionally, M13is also stable under a broad range of PH and temperature. The phage display method starts by building a phage library by theproduction of gene fragments. This library is generated with variant sequencesof single-chain fragment variable (ScFv), which is sub-cloned to M13 phages’circular DNA in a way that ScFv upstreams the PIII protein sequence usingoverlap extension PCR method.
In this case, the ScFv-s is displayed on the PIIIof the phage. These ScFv protein antibodies consist of two variable regions:heavy and light chains. Furthermore, it has been found by previous literaturethat the displayed ScFv antibodies can induce the binding desire to thetargeted antigen and are genetically more stable due to their small size.
So in this experiment, we are adding genetic materials that producea phenotypic phage library on the surface of the coat PIII. To use this phagelibrary a process called panning is done by incubating the library with antigensof interest in this case, Melittin and PLA2. The phage library with the ScFvantibodies will bind to the target, and the unbounded segment will be washed/removed.The bound phage will be eluted with an acidic condition. After that, theantibody solution is neutralized with a base.
Then the eluted phage isamplified by infecting an E. coli. The step of panning must be repeatedmultiple times for successful phage display. To recover the eluted antibodies,the infected bacteria are cultured and stored at -20C temperature. Furtheranalysis and DNA sequencing are obtained on the phage antibodies todifferentiate between the antibodies of the two toxins, Melittin and PLA2,using ELISA. Finally, production of purified and soluble ScFv antibodies hasbeen made and full-length human antibodies were generated. The specific clones for Melittin and PLA2were named Afribumab 1 and Afribumab 2. This Phage display is called phagedisplay antibodies (because antibodies are displayed outside of the phage onthe surface of PIII) with positive selection (because we want phage that bindsto the desired antigen, which is the Melittin, and PLA2 venoms in this case).
The Antivenoms of Apis Mellifera’s Melittin and PLA2 (Afribumab 1and Afribumab 2) were tested in vitro and in vivo assays to evaluate theirabilities to curb the bee’s toxins. The results were fascinating in vitro.Afribumab 1 targets the melittin and decreased the hemolysis significantly.Afribumab 2, targets PLA2, inhabited the hemolysis; however, with a less rationthan Afribumab 1.
The combination of two antibodies was also effective in thereduction of hemolysis. In vivo, on the other hand, Afribumab 1 and 2 were ableto reduce the edema in the paws of mice injected with A. Mellifera’s venom. Inaddition, the new antibodies, together, were able to increase the survivalprobability by 50 percent in the infected mice population.
It is important tomention that in this study researchers did not focus on the amount of theantibody dose needed for the best result; but rather, it was stated that a largeramount of antibodies could result in a greater reduction of venomousness.Although Phage display is a powerful method for displaying proteinantibodies with anticipated biological properties, it has possible limitations.Phage display antibodies may miss the targeted antigens due to the loss of DNAduring phage library creation. Another issue is the difficulty behindextracting venom antigens. Therefore, it is important to use purified toxins inorder to have effective results while presenting the baits to the antibodylibraries. On the other hand, phage display has major advantages for obtainingantibodies.
For instance, it rapidly identifies phages to their target due tothe phage libraries. Also, it is not essential to immunize animals, especiallyhumans, for antibodies production.In conclusion, I believe phage display is an easy and quick way toproduce antibodies, which can be very useful in vaccine and drug design orrapid therapy. Phage display has other application, such as detectingprotein-protein interaction and epitope determination. It has also shown anincredible growth in the last three decades due to its flexibility. Thus, some futureaims of Phage display can include improving the affinity of the developedantibodies since they could be relatively short-lived in vivo. In addition,combining Phage display with high productivity DNA sequencing methods, likeIllumina dye sequencing, could be a sturdy technique to understand the created antibodies’responses in vivo.