1. Table E1: Virus titres used in mouse and ALI experiments
VP/100 ml
TU/100 ml
Relative
titre
rSIV.F/HN-CMV-EGFPLux-IC
5E10
1E8 1
rSIV.F/HN-EF1a-EGFPLux-IC
1.8E10
6.1E7
0.616
rSIV.F/HN-hCEF-EGFPLux-IC
5.7E10
1.6E8
1.61
rSIV.F/HN-CMV-EGFPLux-ID
3.6E10
1.2E8
1.15
rSIV.F/HN-hCEF-EGFPLux- ID
7.8E10
2.8E8
2.8
For each vector configuration the maximum feasible volume (100 ml) was used
leading to variation in titres applied. For calculation of gene expression the titre was
Ratioed relative to that of rSIV.F/HN-CMV-EGFPLux-IC (=1).
VP=virus particles, TU= Transduction Units, IC=Integrase competent,
ID=integrase deficient

2. Table E2: Primary and secondary antibodies used for EGFP detection and cell type
characterisation
Cell type
Primary antibody
(dilution used)
Supplier of primary antibody
Secondary antibody
Supplier of secondary antibody
Not applicable
Chicken anti-GFP
(1:100)
Abcam plc Cambridge, UK
Goat anti-Chicken
Alexa Fluor 488
(1:200)
Life Technology Eugene, OR, USA
Rabbit anti-GFP
1/100
Invitrogen
Rockford, IL, USA
Goat anti-Rabbit Alexa Fluor 488 (1:200)
Ciliated airway epithelial cells
Mouse anti β-Tubulin (1:100)
Chemicon Int, Temecula, Ca
Goat anti-Mouse
Alexa Fluor 594
Clara cells
Rabbit anti-Uteroglobin (1:100)
Goat anti-Rabbit Alexa Fluor 594 (1:200)
Goblet cells
Mouse anti-Mucin 5Ac[45M1]
(1: 100)
Basal cells
Rabbit anti-Cytokeratin 5 (1:100)
Type 1 pneumocytes
Hamster anti-Podoplanin
Santa Cruz Biotechnology, Inc
Dallas, Texas, USA
Goat anti-Hamster
Life Technology Eugene, OR ,USA
Type 2 pneumocytes
Rabbit anti-Surfactant protein C
Millipore, Temecula, CA
Pulmonary macrophages
Rat anti-Mouse F4/80
(1:50)
Serotec, Kidlington, UK
Goat anti-Rat

3. Table E3: Compilation of mouse lung, mouse nose and human ALI samples subject to IS analysis
rSIV.F/HN transgene1
Sampling site
No of samples
Input DNA per sample (ng)
Total no of reads2
High stringency reads3
Medium stringency reads4
VCN/cell
High stringency IS5
Medium stringency IS6
hCEF-EGFPLux
mouse lung 4
405995
11475
25006 82
104
hCEF-soCFTR2 2
21273 49 63 3
mouse nose
54922
566
847 12 10
98373
131
137
hALI
49806 48 50
1Genotype of rSIV.F/HN vector used in initial transduction
2Raw Ion Torrent sequencing reads
3High stringency reads remaining after stripping primer, linker, vector and genomic repeat sequences
4Medium stringency reads remaining after stripping primer, linker, vector and genomic repeat sequences
5Unique high stringency IS remaining after quality control filtering
6Unique medium stringency IS remaining after quality control filtering
VCN: Vector Copy Number

4. Figure E1 PBS mice SIV l a i rSIV.F/HN (RC) v u s t n c r e P6 12 18 24 50 60 70 80 90
100
PBS mice
SIV
rSIV.F/HN (RC)
Month after treatment P e r c n t s u v i a l
Figure E1

5. A
IS ± 10 kb
IS ± 100 kb C
Figure E2

6. A. C. B. D. Figure E3 Untreated mice Untreated miceS F 1 : 4 8 6 3 2 50
100
150 % G P p o s i t v e c l ( r a d u m f n ) A. C. S F 1 : 4 8 6 3 2 50
100
150 % G P p o s i t v e c l ( r a d u m f n )
Untreated mice
Untreated mice S F 1 : 4 8 6 3 2 50
100
150 % G P p o s i t v e c l ( r a d u m f n ) S F 1 : 4 8 6 3 2 50
100
150 % G P p o s i t v e c l ( r a d u m f n ) B. D.
rSIV.F/HN transduced mice
rSIV.F/HN transduced mice
Figure E3

7. IgG IgA Figure E4 % G F P p o s i t v e c l ( r a d n b y ) % G F P p1 : 2 4 8 6 3 20 40 60 80
100 % G F P p o s i t v e c l ( r a d n b y ) 1 : 2 4 8 6 3 20 40 60 80
100 % G F P p o s i t v e c l ( r a d n b y )
IgG
IgA
Figure E4

8. A. B. Figure E5 Serum BALF human human O D 4 5 n m 100 ml IP 400 ml IN
0.0
0.5
1.0
1.5
2.0
Serum O D 4 5 n m
100 ml IP
human
400 ml IN A.
0.0
0.5
1.0
1.5
2.0
2.5
BALF O D 4 5 n m B.
100 ml IP
human
400 ml IN
Figure E5

9. A. B. C. Figure E6 ) i l C ° s ( b u t a o r f p m g e n T h c1 2 3 4 -2 -1 T e m p r a t u ( C ) c h n g f o b s l i
Untreated
SiV only
SiV+IVIg IP
SiV+IVIg IN A.
Days after IVIg administration B. UT IP IN 18 20 22 24 26 28 30 B o d y w e i g h t ( )
Day 5
Day 28 C.
Median in bodyweight, KW no diff
Mean and SEM in temp
Figure E6

10. A. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 n o i t ) p y m a d u e / s n s o u c o m e r / t g a ( W
SeV
SeV
SIV
Weeks after transduction B. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Weeks after transduction F o d c n s u m p t i ( g / e a y )
SeV
SIV
Figure E7

11. C. D. Figure E7 cont’d SeV SIV 10 20 30 40 50 60 70 80 90 100 -4 -2 210 20 30 40 50 60 70 80 90
100 -4 -2 2 4
Days after transduction C h a n g e i b o d y t m p r u ( D l f s )
SeV
SIV
Figure E7 cont’d

12. E. Serum BALF Figure E7 cont’d 4 3 m n 5 2 D O 1 UT SeV 1E7 SIV 1E61 2 3 4 O D 5 n m UT
SeV
1E7
SIV
1E6
Serum
BALF
****
****
****
$$$$
$$$
****
+SIV
+SIV
Anova and post hoc Tuckeys comparing all groups
Figure E7 cont’d

13. 2.0
1.5 m n 5 4
1.0 D O
0.5
0.0
Adults
Children
Figure E8 A

14. B. C. D. E. Figure E8 % G P p o s i t v e c l ( r a n d u m f ) % G P1 : 8 6 3 2 4 5 S F 50
100
150 % G P p o s i t v e c l ( r a n d u m f ) 1 : 8 6 3 2 4 5 S F 50
100
150 % G P p o s i t v e c l ( r a d u m f n ) D. E. 1 : 8 6 3 2 4 5 S F 50
100
150 % G P p o s i t v e c l ( r a d u m f n ) 1 : 8 6 3 2 4 5 S F 50
100
150 % G P p o s i t v e c l ( r a n d u m f )
Adults SA3 IgG pos
Child SC8 IgG neg
Figure E8

15. F. **** Figure E8 cont’d ) d r % f 5 s v e c a h p m n o t u ( l i D
F/HN
VSVG 4 8 16 32 64
128
256
512
1024 D i l u t o n h a c e v s 5 % r d ( m p f )
****
Figure E8 cont’d

16. A. Figure E9 m n 5 4 D O IgG IgA Adults Children 0.0 0.5 1.0 1.5 2.0
2.5
3.0 O D 4 5 n m
cut off point IgG
cut off point IgA
IgG
IgA
Figure E9

17. B. % G F P p o s i t v e c l ( r a n d B A L f ) rSIV.F/HN rSIV.VSV-G
150
100 50
Ab pos
Ab neg
Ab pos
Ab neg
rSIV.F/HN
rSIV.VSV-G
Figure E9 cont’d

18. **** Figure E10 ) i d e m l / U R ( x u L G 1000000 100000 10000 1000UT NC 1 2 3 4 1 2 3 4
Exp 1
Exp 2
Figure E10