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Thermo-Responsive Polymer Nanoparticles CoDeliver RSV F Trimers with a TLR-7/8 Adjuvant Joseph R. Francica, Geoffrey M. Lynn, Richard Laga, Michael Gordon Joyce, Tracy J. Ruckwardt, Kaitlyn M. Moribito, Man Chen, Rajoshi Chaudhuri, Baoshan Zhang, Mallika Sastry, Kiyoon Ko, Misook Choe, Michal Pechar, Ivelin S. Georgiev, Lisa A. Kueltzo, Leonard W. Seymour, John R. Mascola, Peter D. Kwong, Barney S Graham, and Robert A. Seder Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00370 • Publication Date (Web): 01 Sep 2016 Downloaded from http://pubs.acs.org on September 3, 2016
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Bioconjugate Chemistry
Thermo-Responsive Polymer Nanoparticles Co-Deliver RSV F Trimers with a TLR-7/8 Adjuvant
Joseph R. Francica1, Geoffrey M. Lynn1, Richard Laga2, M. Gordon Joyce1, Tracy J. Ruckwardt1, Kaitlyn M. Moribito1, Man Chen1, Rajoshi Chaudhuri3, Baoshan Zhang1, Mallika Sastry1, Kiyoon Ko1, Misook Choe1, Michal Pechar2, Ivelin S. Georgiev1, Lisa A. Kueltzo3, Leonard W. Seymour4, John R. Mascola1, Peter D. Kwong1, Barney S. Graham1, and Robert A. Seder1*
1
Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
2
Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic
3 Vaccine Production Program, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Gaithersburg, MD 20878, USA 4
Department of Oncology, University of Oxford, Oxford, UK
* Corresponding Author Cellular Immunology Section Vaccine Research Center National Institute of Allergy and Infectious Disease National Institutes of Health 40 Convent Drive, MSC 3025, Building 40, Room 3512 Bethesda, MD 20892 E-mail address:
[email protected] ACS Paragon Plus Environment
Bioconjugate Chemistry
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Abstract
Structure-based vaccine design has been used to develop immunogens that display conserved neutralization sites on pathogens such as HIV-1, respiratory syncytial virus (RSV) and influenza. Improving the immunogenicity of these designed immunogens with adjuvants will require formulations that do not alter protein antigenicity. Here we show that nanoparticle-forming thermo-responsive polymers (TRP) allow for co-delivery of RSV fusion (F) protein trimers with Toll-like receptor 7 and 8 agonists (TLR7/8a) to enhance protective immunity. While primary amine conjugation of TLR-7/8a to F trimers severely disrupted the recognition of critical neutralizing epitopes, F trimers site-selectively coupled to TRP nanoparticles retained appropriate antigenicity and elicited high titers of prefusion-specific, TH1 isotype anti-RSV F antibodies following vaccination. Moreover, coupling F trimers to TRP delivering TLR-7/8a resulted in ~3-fold higher binding and neutralizing antibody titers than soluble F trimers admixed with TLR-7/8a, and conferred protection from intranasal RSV challenge. Overall, these data show that TRP nanoparticles may provide a broadly applicable platform for eliciting neutralizing antibodies to structuredependent epitopes on RSV, influenza, HIV-1, or other pathogens.
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Introduction
Structural vaccinology has been used to improve the design of protein immunogens for vaccination 1,2
against a number of infectious diseases . Integral to this process is the presentation of conserved neutralizing epitopes, shielding of non-neutralizing sites, and the prevention of conformational changes that can present immunodominant non-neutralizing epitopes. This immunofocusing reduces “off-target” 3
immune responses and may enhance responses to protective neutralizing epitopes . Structure-based 4-6
7
vaccine design has been applied to the HIV-1 envelope (Env) , influenza hemagglutinin (HA) , Epstein8
3
Barr virus gp350 and respiratory syncytial virus fusion (RSV F) proteins. However, such subunit 9
vaccines alone are often less immunogenic than intact or live-attenuated pathogens and so must be formulated with an exogenous adjuvant to improve the magnitude and quality of humoral and cellular immunity. Even multivalently arrayed subunit vaccines that cross-link B cell receptors are often further improved by adjuvants to increase the magnitude of antibody titers
7,8,10
, highlighting the need to optimize
both the antigen and the adjuvant to maximize adaptive immune responses.
Until recently, aluminum salts (“alum”) or oil and water emulsions were the only approved adjuvants available to enhance antibody responses to protein subunit vaccines. More recently, Toll-like receptor 11
agonists (TLRa) have been used as vaccine adjuvants . Monophosphoryl lipid A (MPL), a TLR4a, has been approved for human use against human papillomavirus (HPV) when administered with alum and 12
shows increased antibody responses compared to alum alone . Similarly, subunit vaccines containing 13
MPL enhance immunogenicity and protection against malaria
14
and hepatitis B . The safe and successful
use of MPL has provided the necessary proof of concept for advancing the development of other TLRa that may further improve immunity and protection based on stimulation through alternative innate pathways.
Based on unique patterns of TLR expression across different immune cell subsets, TLRa can be rationally selected to induce qualitatively distinct immune responses that would be most protective against 15
a given pathogen . Accordingly, imidazoquinoline compounds have been identified that signal through
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TLR7 to directly activate plasmacytoid dendritic cells (DCs) stimulate monocytes and other DC subsets
16,19
16,17
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18
and B cells , and through TLR8 to
, leading to robust humoral and cellular immunity.
However, small molecule TLRa, e.g. TLR-7/8a, rapidly diffuse away from the site of injection, reducing their ability to prime immune cells and potentially causing systemic side effects
20,21
. To better focus their
activity in lymph nodes draining the sites of immunization while reducing systemic distribution, TLRa may be formulated with a delivery vehicle that prevents systemic dissemination 22
immune cells. Insoluble aluminum salts , oil-in-water emulsions
23
22
and enhances uptake by
and liposomal vesicles
24
among others
have been used for this purpose. Additionally, physically linking TLRa with protein immunogens to ensure their co-delivery to antigen presenting cells (APC) has been shown to be beneficial for priming T cell 25-30
immunity
. We and others have shown that direct chemical conjugation of TLR-7/8a to immunogens is 31-33
an effective co-delivery approach for inducing T cell immunity
. However, the chemical modification of
31
proteins to link TLR-7/8a may result in a loss of antigenicity and aggregation
32
of the immunogen, which
limits this approach for antibody-based vaccines.
To address these challenges, we recently showed that synthetic, biocompatible polymer chains are an effective vehicle for improving the pharmacokinetic properties of TLR-7/8a and coupling them with protein 20
immunogens to ensure co-delivery . Accordingly, our previous work characterized thermo-responsive polymers (TRP) comprised of single, amphiphilic, di-block co-polymer chains that are soluble at room temperature but can be induced to self-assemble into nanoparticles above a chemically programmable transition temperature. Coupling TLR-7/8a and protein immunogens to TRP nanoparticles improved APC 20
uptake, leading to higher magnitude and persistence of protective cellular immune responses .
The goal of this study was to investigate the utility of the TRP platform for co-delivering a structurally stabilized B cell immunogen with TLR-7/8a in a formulation that enhances immunogenicity while preserving critical neutralizing epitopes. To this end, the trimeric fusion (F) protein from RSV was selected as a model immunogen since the structural integrity of this immunogen is critical to the generation of protective neutralizing antibody responses
3,34-36
. RSV virions have an array of F trimers on the viral 37
membrane and a single-stranded RNA genome that can signal through TLR7 and TLR8 . To mimic
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these properties of RSV, we investigated the effects of immunogen multivalency and co-delivery with TLR-7/8a on anti-RSV immunity. However, natural RSV infection does not confer protective immunity, in part because F trimers can exist on the virion surface in the postfusion conformation, acting as decoys to 36
distract the immune response from neutralizing epitopes found only in the prefusion conformation . Thus, the ability of the TRP platform to preserve the F trimer prefusion conformation and focus antibody responses against specific neutralizing sites was assessed. Moreover, as a previous RSV vaccine that induced TH2-biased responses enhanced RSV illness in children
38-40
, the ability of TLR-7/8a to promote
TH1 skewed responses was also determined. Here we provide the first evidence that a structurally stabilized immunogen can be site-selectively coupled with TLR-7/8a to polymer nanoparticles, preserving antigenicity and eliciting protective TH1 immunity.
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Results
Direct conjugation of TLR-7/8a to RSV F protein disrupts antigenicity. Prior studies by us and others have shown that direct chemical conjugation of TLRa to protein antigens is an effective co-delivery approach 30,32,41,42
for improving T cell immunogenicity
. To advance this approach for inducing antibody responses,
the stabilized prefusion RSV F trimer (DS-Cav1) was first analyzed for accessible primary amines to directly couple TLR-7/8a molecules (Fig. 1a). In total, 32 residues on the surface of each protomer are available for amine coupling composing ~9% of the molecular surface area of the RSV F trimer (Fig. 1b). However, 18/32 residues with primary amines are located within or adjacent to previously described RSV F sites of neutralization, suggesting that coupling TLR-7/8a to primary amines could physically block B cell receptor (BCR) recognition of neutralizing epitopes on RSV F trimers. To address this directly, primary amine chemistry was used to conjugate an imidazoquinoline-based TLR-7/8a (2BXy)
43-45
to the
surface of RSV F trimers using 10 or 20 molar equivalents of the TLR-7/8a to F protein (F lo TLR-7/8a and F hi TLR-7/8a, respectively, Fig. 1c; supplementary Fig. 1).
The ability to generate protective antibody responses against RSV requires that neutralizing epitopes on the RSV F protein immunogen are accessible to binding by B cells. One means of assessing how a vaccine formulation influences the accessibility of sites of neutralization is to measure the binding of monoclonal antibodies (mAbs). Therefore, biolayer interferometry (BLI) was used to perform an antigenic assessment of the F-TLR-7/8a conjugates, using mAbs against defined neutralizing epitope sites (Fig. 1dg). BLI demonstrated that direct coupling of TLR-7/8a to F trimers resulted in a concentration-dependent decrease in maximum binding to the following mAbs: AM14 (site V, prefusion-specific), D25 (site ∅, prefusion-specific), MPE8 (site III, prefusion-specific) and Motavizumab (site II, prefusion and postfusion). Similarly, both apparent kon and koff rates of these mAbs were detrimentally affected by the TLR-7/8a conjugation (Fig. 1h). These data show that direct coupling of TLR-7/8a using primary amine chemistry blocks antibody binding to critical sites of neutralization on the stabilized prefusion RSV F protein.
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b
RSV F
Residue Distance to antigenic site
2BXy (TLR-7/8a)
NH 2
c
N
N
PEG-TLR-7/8a
N
HN
NH 2
Gln 26 Lys 42 Lys 65
Site II AM14 site Site IV
Lys 68 Lys 75 Lys 77 Lys 80 Lys 85
Site I
90o
Lys 87 Lys 156 Lys 166 Lys 168 Lys 176
site Ø site Ø 15.4 Å (site Ø) 8.7 Å (site Ø)
HN PEG4-DBCO O
NHS-PEG4-Azide
NH 2
HN
2BXy-PEG 4-DBCO
PEG4-Azide
100 mM HEPES, pH 8.5
H N
100 mM HEPES, pH 8.5
PE G-T LR -7 /8a
RSV F NH 2
d
HN
e
AM14
1.5
PEG4-Azide
NH PEG-TLR-7/8a
O
D25
1.5
5.3 Å (site Ø) 12.1 Å (site II) 2.3 Å (site Ø) 9.6 Å (AM14) 4.6 Å (AM14) 7.5 Å (site Ø) 3.7 Å (site II)
Lys 191 Lys 196
5.2 Å (site Ø) site Ø
Lys 201 Lys 209
site Ø site Ø
Lys 226
4.1 Å (site Ø)
Lys 271 Lys 272
site II
Lys 293 Lys 327
Reactive amines
13.1 Å (AM14) 15.7 Å (site I)
Response (nm)
a
site II 11.3 Å (AM14) 13.9 Å (site I) site I
Lys 390 Lys 419 Lys 421
13.7 Å (site I) 4.0 Å (site IV)
Lys 427 Lys 433
site IV and AM14 site Site IV
Lys 445 Lys 461
AM14 site 4.2 Å (AM14)
Lys 465
3.5 Å (AM14)
Lys 470 Lys 508
16.8 Å (site IV) 25.1 Å (site IV)
1.0
1.0
0.5
0.5
0
0 400
600
800
1000
1200
Time (s)
f
400
1.0
0.5
0.5
0
1000
1200
F (unconjugated ) F lo TLR7/8a F hi TLR7/8a
0 400
600
800
1000
1200
400
Time (s)
h
800
Motavizumab
1.5
1.0
600
Time (s)
g
MPE8
1.5
Response (nm)
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Ligand AM14 IgG
D25 IgG
MPE8 IgG
Motavizumab IgG
600
800
1000
1200
Time (s)
k on (1/Ms)
k off (1/s)
K D (app.)(M)
F (unconjugated)
2.04E+05
