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. Author manuscript; available in PMC: 2016 Dec 20.
Published in final edited form as: Nat Microbiol. 2016 Jun 20;1(8):16080. doi: 10.1038/nmicrobiol.2016.80

Figure 1.

Figure 1

Lentiviral screening approach. Alpacas are immunized with the desired antigen mix (here: inactivated influenza A virus, IAV, and vesicular stomatitis virus, VSV). After repeated immunizations, we draw a blood sample, purify lymphocytes, extract mRNA, reversely transcribe RNA to cDNA, amplify VHH coding sequences and clone them into a lentiviral vector. Alternatively, VHH coding sequences can be subcloned from an existing VHH library. 293T cells are transfected with the lentiviral library as well as packaging vectors, and lentivirus is harvested from the supernatant 2 days later. We transduce the cell line of interest (here: A549 cells) and induce VHH expression with doxycycline. Cells are then subjected to a selection assay that allows identification of cells expressing the desired VHHs (here: survival of a lethal infection with IAV or VSV). Finally, we prepared genomic DNA from selected (surviving) cells, amplified the VHH sequences by PCR and determined the VHH sequence encoded.