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U. S. Patent

Jan. 10, 2017

Sheet 41 of 53

US 9, 539, 210 B2

FIG. 22A

VI10YEN В cells

popLN brachLN

FIG. 22B

WT В cells

popLN brachLN

	₉

	‘ 1
	♦ ’
	. 1


9? W	' 2


„. •- « ъ л , ^A


*	I ■ . -r 2


	-



¹ ♦ 5.


'7'	'*•


gO

98РЭ

	. 45
... , *

Cd69

[2	’• 3

о......	......... 0
*	^82

o.......	....... ~ т
ЗДЯ\|	и. ^; ⁴ 9

U. S. Patent Jan. 10, 2017

Sheet 42 of 53

US 9, 539, 210 B2

VSV-IND

VI10YEN В cells
B220

VSV-IND VI10YEN В cells C57BL/6 В cells

0 min

30 min

VSV-IND

polyclonal В cells

'VSV-IND polyclonal

''B cells VSV-IND

polyclonal В cells

VSV-IND

VI10YEN В cells

VSV

VI10YEN _vsv

В cells

■ I.

FIG. 23

U. S. Patent

Jan. 10, 2017

Sheet 43 of 53

US 9, 539, 210 B2

Targeted NP accumulate on SCS-Mph

Non-Targeted

i 11~~mu|~~ iniinq iiiiniij iii~~unq ^l]~~

0 10² 10³ 10⁴ 10⁵

Control nanoparticles

L- medulla

1 h post injection

SCS-Mph (CD169)

Control nanoparticles Medulla (Lyve-1)

anti-CD169

0 10² 10³ 10⁴ 10⁵

Targeted nanoparticles

anti-CD169

В cells

1 h post injection

SCS-Mph (CD169)

Targeted nanoparticles

В cells (B220)

250K

200K

Й150К

100K

50K

Targeted nanoparticles

0 10² 10³ 10⁴ 10⁵

medulla

24h post injection

SCS-Mph (CD169)

Targeted
nanoparticles
Medulla (Lyve-1)

anti-CD169

FIG. 24A

U. S. Patent

Jan. 10, 2017

Sheet 44 of 53

US 9, 539, 210 B2

Fc-NP target SCS-Mph and follicular dendritic cells

24h post injection

Fc-targeted NPS; Control NPS; Bcells

FIG. 24B

Alternative SCS-MPh targeting strategy: Macrophage-expressed CX3CR1

Double-knockin mouse LN:

CX3CR (SCS-Mph)

CCR2 (medulla/cortex)

FIG. 25

U. S. Patent

Jan. 10, 2017

Sheet 45 of 53

US 9, 539, 210 B2

< APC-A>: CRFc

SCS-Mph and dendritic cells express CX3CR1

1 x 10⁵

10000 1000

0 100 1000 10000 1x10⁵

< FITC-A>: CX3CR1 GFP

SCS-Mph (CRFc⁺)

(CRFc'CD11c⁺)

FIG. 26

Amine modified latex nanoparticies accumulate on SCS-Mph

Carboxy

microspheres

anti-CD169

amine'

Amine

CD 169 microspheres

amine

anti-CD169

-carboxy

FIG. 27

U. S. Patent Jan. 10, 2017 Sheet 46 of 53 US 9, 539, 210 B2

Antigen-bearing targeted nanoparticles are highly immunogenic

VSV

VSV-G

> A

Soluble VSV-G (10x)

NP-VSV-G

Immunize

<
cn
c

'■ 3
TO
N

з ф

90000-1
80000-
70000-
60000-
50000-
40000-
30000-

20000¹10000'

Immunize DO, boost D55, obtain sera after 10 weeks

Sera tested for 50% neutralization of plaque-forming capacity

□			«0
		A A
					*0
		—1—		" *T^-“	f^T
> 0		E	0 u.		E	0 LL
>	(Л	TO	Q.	C/)	TO	" O
: >	>	c	Z	>	c	TO
Z)	Ф _Q			CL Z		и
		>			>
		(Л			V)
	tn	>			>	>
		Ф			Q.	CL
		JO			Z	Z

О w

U. S. Patent Jan. 10, 2017 Sheet 47 of 53 US 9, 539, 210 B2

FIG. 28A

Vaccination with antigen-bearing targeted nanoparticies protects against lethal infection with VSV

% survival

100-ь

75-

50-

Ф —B- NP-Fc-VSV-G

—M— NP-Fc-VSV-G + alum

£ ]

VSV-G + alum

D150: immunized challenged with a lethal dose of VSV

FIG. 28B

U. S. Patent Jan. 10, 2017 Sheet 48 of 53 US 9, 539, 210 B2

CD4 T cell response to OVA-NPs mixed with CpG adjuvant (TLR9 agonist)

PLA-OVA

exp 23 NP-OVA_#5 draining. fcs... CFSE⁺

OVA

exp 23 NP-OVA_#4 draining. fcs... CFSE⁺

PLGA-OVA

exp 23 NP-OVA_#6 draining. fcs... CFSE⁺

popLN

ф20
О
*10

	96. 1

0....................................

о Ю²10³ 10⁴ 10⁵

< FITC-A>: CFSE

	99. 2,

	100.
	L

spleen

0Ю²10³ 10⁴ 10⁵

< FITC-A>: CFSE

exp 23 NP-OVA_#4 non draining. fcs.. , CFSE⁺ exp 23 NP-OVA_#5 non draining. fcs.. , CFSE⁺

0 Ю²10³ 10⁴ 10⁵

< FITC-A>: CFSE

exp 23 NP-OVA_#6 non draining. fcs... CFSE⁺

brachial LN

	63. 9
	И	L

	77. 6 ₍
	L1

< FITC-A>: CFSE

	88. 5,
	Ljl

0 Ю²10³ 10⁴ 10⁵ < FITC-A>: CFSE

exp 23 NP-OVA_#4 spleen. fcs... CFSE⁺

О Ю²10³ 10⁴ 10⁵

< FITC-A>: CFSE

о Ю²1 0³ 10⁴ 10⁵ < FITC-A>: CFSE

exp 23 NP-OVA_#6 spleen. fcs... CFSE⁺

		99. 8
	Г	L

0 Ю²10³ 10⁴ 10⁵

< FITC-A>: CFSE

U. S. Patent Jan. 10, 2017 Sheet 49 of 53 US 9, 539, 210 B2

FIG. 29A

CD4 T cell response to OVA-NPs mixed with CpG adjuvant (TLR9 agonist)

OVA PLA-OVA

exp 23 NP-OVA_#1 draining. fcs.. , CFSE⁺ exp 23 NP-OVAJ2 draining. fcs.. . CFSE⁺

Draining LN

< FITC-A>: CFSE

w " 3 o

300 i


f

° 010² 10³ 10⁴ 10⁵

< FITC-A>: CFSE

exp 23 NP-0VA_#1 non draining. fcs... CFSE⁺ exp 23 NP-0VA_#2 non draining. fcs... CFSE⁺

PLGA-OVA

exp 23 NP-0VAJ3 draining. fcs... CFSE⁺

< FITC-A>: CFSE

exp 23 NP-OVA_#3 non draining. fcs... CFSE⁺

" 2 15

		⁹⁹>
		......... '. ни,.

jig " 3

Non-draining О

«V

О Ю²10³ 10⁴ 10⁵

CF4F

exp 23 NP-0VAJ1 spleen. fcs... CFSE⁺

spleen

J2 " 3 О %

24. 1

0Ю²10³ 10⁴ 10⁵

< FITC-A>: CFSE

exp 23 NP-0VA_#2 spleen. fcs... CFSE⁺

0Ю²10³ 10⁴ 10⁵ < FITC-A>: CFSE

exp 23 NP-OVA #3 spleen. fcs... CFSE⁺

1,.....

0 10² 10³ 10⁴ 1 0⁵

< FITC-A>: CFSE

u»

®
О
%

66. 6

	⁹⁷,

0Ю²10³ 10⁴ 10⁵

< FITC-A>: CFSE

U. S. Patent Jan. 10, 2017 Sheet 50 of 53 US 9, 539, 210 B2

FIG. 29B

PEG-PLA

CD8 T cell activation

NP-CL097[OVA]

FIG. 29C

U. S. Patent Jan. 10, 2017 Sheet 51 of 53 US 9, 539, 210 B2

Nicotine conjugation strategy

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