Engineering chimeric antibodies aimed for passive mucosal
immunization against HRSV Shruti Bakshi1,2, Paloma Juarez1,2, Jorge Palaci1,2, Vikram Virdi1,2, Bert Schepens3,4, Xavier Saelens3,4 and Ann Depicker1,2
1Department of Plant System Biology, VIB, Ghent, Belgium; 2Department of Plant Biotechnology and Bioinformatics, Ghent University, Belgium; 3Medical Biotechnology
Center, VIB, Ghent, Belgium; 4Department of Biomedical Molecular Biology, Ghent University, Belgium
Human Respiratory Syncytial Virus
Human Respiratory Syncytial Virus (HRSV) is the leading cause of acute lower respiratory tract infection in infants and frequently causes severe disease in the elderly. There is no licensed HRSV vaccine. As an alternative, the prophylactic treatment with an HRSV fusion protein targeting monoclonal antibody (mAb) is often recommended for infants that are at risk for developing HRSV infection. However, the high cost that is associated with the current mammalian cell-based mAb manufacturing systems hampers the broad implementation of this therapy. Also, it is possible that IgA type antibodies against HRSV may contribute even better to protection. Therefore, we aim to compare the effectiveness of IgG with secretory IgA based passive immunization against HRSV. In this context, we choose for the transient expression platform in plants that permits rapid small scale production of IgG and IgA based antibody versions [1].
Future Perspectives:
• Protein quantification to compare efficiency of our chimeric antibodies with commercially available antibodies.
• Fc effector function: In vivo (mouse models) comparisons will be done to test the effect of Fc tails. E4 antibodies (binding, but not neutralizing) will be useful for this purpose.
• Production of the most efficient neutralizing antibody in Arabidopsis seeds and challenge experiments in animal models.
Human IgG1
Fc
Mouse IgG2a
Fc
Mouse IgA
Fc
Mouse dIgA
Fc
Mouse sIgA
Fc
VHHC4
VHHD3
VHHE4
VHHV2
Monomeric Dimeric Secretory
Our Approach: We have genetically fused three different single domain antibodies [1] that are specific for the HRSV fusion protein F to the fragment crystallizable part (Fc) of different murine and human monomeric IgA and IgG antibodies. In order to obtain all different permutations we took advantage of the GoldenBraid2.0 [2] cloning system.
Since secretory IgA are the predominant antibodies in mucosal surfaces, they might be more effective than monomeric IgA and IgG in virus neutralization; therefore, IgA
based antibodies will also be tested in their dimeric and secretory forms.
A total of 15 different versions of chimeric antibodies against HRSV and 5 negative controls have been engineered and produced in Nicotiana benthamiana via Agrobacterium tumefaciens – mediated transient expression.
Synagis (50 µg/ml)
(Positive control)
VHH C4 + HuIgG1
VHH V2 + HuIgG1
(negative control)
Dilution Factor
Plaque reduction assay to assess the neutralization potency of RSV specific chimeric antibodies
Conclusions
• E4 nanobodies with the highest binding activity levels do
not neutralize RSV infection.
• A high binding activity of a given Nanobody® does not
translate to a high neutralizing activity.
a) Vacuum infiltration
b) Manual Infiltration
All the RSV specific antibodies showed binding activity for Fusion protein.
By means of combinatorial approaches using novel
synthetic biology tools, it is possible to transiently
express 20 different chimeric antibodies against RSV
in N. benthamiana,.
An
tI-F
pro
tein
Negative
control
1. Schepens, B., et al. (2011). "Nanobodies(R) specific for respiratory syncytial virus fusion protein protect against infection by inhibition of fusion." J Infect Dis 204(11): 1692-1701.
2. Sarrion-Perdigones, A., et al. (2011). "GoldenBraid: an iterative cloning system for standardized assembly of reusable genetic modules." PLoS ONE 6(7): e21622.
Monomeric Dimeric +
Monomeric
Secretory + Dimeric +
Monomeric
A B
C D E
Expression of the chimeric antibodies by agro-infiltration
in N. benthamiana plants
A B
C D E
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
10 100 1000 10000
OD
@ 4
92
nm
Dilution Factor
VHH + Human IgG1
C4-huIgG1
D3-HuIgG1
E4-HuIgG1
V2-HuIgG1
0
0,05
0,1
0,15
0,2
0,25
0,3
0,35
0,4
0,45
10 100 1000 10000
OD
@ 4
92
nm
Dilution Factor
VHH + Murine IgG2a
C4-MuIgG2a
D3-MuIgG2a
E4-MuIgG2a
V2-MuIgG2a
0
0,05
0,1
0,15
0,2
0,25
0,3
0,35
10 100 1000 10000
OD
@ 4
92
nm
Dilution Factor
VHH + Murine IgA (Monomeric)
C4-MuIgA
D3-MuIgA
E4-MuIgA
V2-MuIgA
0
0,05
0,1
0,15
0,2
0,25
0,3
0,35
10 100 1000 10000
OD
@ 4
92
nm
Dilution Factor
VHH + Murine IgA (Dimeric)
C4-MuIgA
D3-MuIgA
E4-MuIgA
V2-MuIgA
0
0,05
0,1
0,15
0,2
0,25
0,3
0,35
10 100 1000 10000
OD
@ 4
92
nm
Dilution Factor
VHH + Murine IgA (Secretory)
VHH C4
VHH D3
VHH E4
VHH V2
0
10
20
30
40
50
60
Nu
mb
er
of
Vir
us
Pla
qu
es
Dilution Factor
VHH-E4 chimeric antibody
E4-huIgG1
E4-muIgG2a
E4-muIgA
E4-muIgA (dimeric)
E4-muIgA (secretory)
Synagis (50 µg/ml)
PBS
0
10
20
30
40
50
60
Nu
mb
er
of
Vir
us
Pla
qu
es
Dilution Factor
VHH-D3 chimeric antibody
D3-huIgG1
D3-muIgG2a
D3-muIgA
D3-muIgA (dimeric)
D3-muIgA (secretory)
Synagis (50 µg/ml)
PBS
0
10
20
30
40
50
60
Nu
mb
er
of
Vir
us
Pla
qu
es
Dilution Factor
VHH-C4 chimeric antibody
C4-huIgG1
C4-muIgG2a
C4-muIgA
C4-muIgA (dimeric)
C4-muIgA (secretory)
Synagis (50 µg/ml)
PBS
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