Polymer–Protein Core–Shell Nanoparticles for Enhanced Antigen

Letter pubs.acs.org/macroletters

Polymer−Protein Core−Shell Nanoparticles for Enhanced Antigen Immunogenicity Xiaolei Zhang,†,‡ Xia Zhao,§ Jittima Amie Luckanagul,†,∥ Jing Yan,† Yuzhe Nie,†,⊥ L. Andrew Lee,† and Qian Wang*,† †

Department of Chemistry and Biochemistry, University of South Carolina, Columbia, South Carolina 29208, United States MicroSep Biological Science Co. Ltd., Wuxi, Jiangsu 214400, People’s Republic of China § State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, People’s Republic of China ∥ Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmaceutical Sciences, Chulalongkorn University, 254 Phayathai Road, Bangkok, 10330, Thailand ⊥ Department of Life Science, Northeast Forestry University, Harbin, Heilongjiang 150040, People’s Republic of China ‡

S Supporting Information *

ABSTRACT: Nanoengineered vaccine platforms can be modeled after viruses and other pathogens with highly organized and repetitive structures that trigger the host immune system. Here we demonstrated a pyridine-grafted poly(ε-caprolactone)-based polymer−protein core−shell nanoparticles (PPCS-NPs) platform can effectively trigger the host immune system and lead to significantly higher antibody titers.

N

assembled with the polymers containing pyridine groups (Scheme 1).42−49 The self-assembly is primarily driven by

anoparticles (NPs) as candidates for delivering antigens hold great promise as vaccines, because of large surface area to volume ratio for immobilizing multiple therapeutic agents at high densities to efficiently elicit host immune system.1−8 Many reports demonstrate the effective use of viruslike particles (VLPs) as scaffolds to display pathogenic haptens or antigens with repetitive arrays, especially glycan-based haptens, for the purpose of augmenting immunogenicity.9−20 However, there are still significant challenges to universally apply VLPs as prophylactic vaccines.21 Alternatively, self-assembling amphiphilic polymers have also been demonstrated as platform for vaccine development. Their flexibility and processability enable the polymers to form a variety of polymer-based carriers, including dextran particles,22 poly(lactic acid)23 and poly(ortho-esters)24 composites, polyanhydrides matrices,25 poly(acrylamide) hydrogel,26 poly(βamino ester)s delivery system,27 polyacrylate conjugates,28−31 poly(alkyl cyanoacrylate) nanospheres,32 polyanhydride copolymers microspheres,33 and poly(lactide-co-glycolide acid) based nanoparticles.34−38 These delivery systems have certain advantages over traditional vaccines,39 but applications as prophylactic vaccines have been limited by the complexity of preparation and potential antigen denaturation.5,40,41 Our previous studies demonstrated a versatile strategy to fabricate nanometer-sized polymer−protein core−shell nanoparticles (PPCS-NPs) without affecting the protein’s native functionalities. Using the self-assembly method, a broad number of proteins with different properties have been © XXXX American Chemical Society

Scheme 1. Schematic Illustration of the Assembly of Polymer−Protein Core−Shell Nanoparticles

functionalized polymers that are unstable in aqueous solutions. In aqueous solution, the pyridine modified polymer aggregates and crashes out of solution. However, in the presence of proteins, the proteins act as amphiphiles to stabilize the polymeric core by multiple interactions including hydrogen bonding between proteins and pyridine-grafted side chain. In this study, we self-assembled polymers and antigenic proteins to form PPCS-NPs, to display repetitive arrays of antigens, while eliminating the need for complex genetic engineering of the scaffold to incorporate foreign antigens. Based on the prior Received: January 24, 2017 Accepted: March 30, 2017

442

DOI: 10.1021/acsmacrolett.7b00049 ACS Macro Lett. 2017, 6, 442−446

Letter

ACS Macro Letters

μg of protein and three-dose regimen of OVA or PyPHEMAOVA were administered at days 0, 14, and 28. The mice sera were collected and the OVA specific IgG titers in the mice sera were measured by a standard enzyme-linked immunosorbent assay (ELISA). As shown in Figure 2, the antibody titers in the

studies, we hypothesize that PPCS-NPs assembled with antigenic proteins will be effective at eliciting strong immune response compared to antigenic protein alone. Furthermore, the stability and simplicity of synthesizing PPCS-NPs with a wide range of purified proteins are attractive features for the development of a novel vaccine platform. Many evidences suggest that antigens presented in such highly repetitive densities facilitate a stronger immune response,50−54 and subsequent studies will explore whether PPCS-NPs elicit pattern recognition receptors or other receptors. In this study, the primary focus is whether a stronger immunogenic response toward the antigens is observed when displayed as PPCS-NPs without toxic effects. For the initial proof of concept study, pyridine-grafted poly(hydroxyethyl methacrylate) (PyPHEMA) was assembled with ovalbumin (OVA) using the one-step PPCS-NPs preparation method.48 The assembly was initiated by adding the solution of PyPHEMA dissolved in dimethylformamide (DMF) dropwise into the solution of OVA in 10 mM phosphate saline buffer (PBS, pH 7.4) with stirring. Several rounds of dialysis were conducted to remove the organic solvent prior to downstream analysis. Dynamic light scattering (DLS) measurement and transmission electron microscopy (TEM) showed the particles with a size range of 100−200 nm in diameter (Figure 1a,d). Field emission scanning electron

Figure 2. Total specific anti-OVA IgG titers in BALB/c mice following three subcutaneous immunizations. Asterisks over bars indicated degree of significance, where **p < 0.01; ***p < 0.001; ns = not significant.

mice group immunized with PyPHEMA-OVA did not significantly differ from the control group immunized with free OVA on day 14. However, by day 28, the group administered with PyPHEMA-OVA exhibited 3-fold higher titers against OVA. By day 35, the sera from the same group exhibited a 2-fold increase in antibody titers over the group inoculated with OVA alone. Day 35 titer levels for mice inoculated with OVA alone were only similar to the titers detected on day 28 for PyPHEMA-OVA inoculated mice. Histological analysis of spleen and liver showed significant increase in spleen weight and liver toxicities likely due to high levels of endotoxin in OVA antigen or the poor biodegradability of PyPHEMA polymers (data not shown). The toxicity of the polymers was addressed by using FDA-approved poly(εcaprolactone) (PCL), as PCL is well characterized for its biodegradable and biocompatible properties.55,56 PCL was synthesized and conjugated with pyridinyl units (Scheme 2). In

Figure 1. Physicochemical characterization of PyPHEMA-OVA PPCSNPs: (a) TEM analysis; (b, c) FESEM analysis; (d) Size distribution; and (e) Zeta potential from dynamic light scattering (DLS) measurement. (f) CD spectra comparison between PPCS-NPs and free protein.

Scheme 2. Synthesis of Pyridine-grafted-poly(εcaprolactone)

microscopy (FESEM) showed their uniform spherical structure with a smooth surface morphology (Figure 1b,c). By comparing with free OVA, the PyPHEMA-OVA PPCS-NPs exhibited lower zeta potential (Figure 1e), which indicated the event resulting from polymer−protein assembly. The assembly was subjected to centrifugation at 9000 rcf for 10 min, and the supernatant was collected and very low concentration of free proteins could be detected. Circular dichroism (CD) analysis showed that PyPHEMA-OVA displayed two minima at 211 and 224 nm, respectively, which were consistent with the CD spectrum of native OVA (Figure 1f). This observation suggested the proteins on nanoparticle corona still maintained their original folded conformations upon assembly. With these PyPHEMA-OVA PPCS-NPs in hand, BALB/c mice were inoculated with PyPHEMA-OVA NPs by subcutaneous injection. The mice were segregated into two groups, where the control group was inoculated with OVA alone, and another group with PyPHEMA-OVA. Each dose contained 10

addition, endotoxin-free antigens, dengue virus serotype-2 envelope protein (DV2EP)57 and Plasmodium falciparum malaria circumsporozoite protein (CSP),58 the crucial structural proteins for dengue and malaria, were used as the vaccine candidates, which were assembled with PyPCL. The assembly processes were similar as discussed in the previous sections to afford core−shell particles, PyPCL-DV2EP and PyPCL-CSP, accordingly. DLS measurement indicated the 443

DOI: 10.1021/acsmacrolett.7b00049 ACS Macro Lett. 2017, 6, 442−446

Letter

ACS Macro Letters

dendritic cells, and can enter the lymphatic system at the distal sites of the lymph vessels.52,54 In this study, the mass ratio of polymer and protein was fixed, which yielded PPCS-NPs with a size range of 100−200 nm in diameter, it was in the size range that required for efficiently processing by antigen presenting cell (APCs).52,54 A second possibility is the prolonged circulation of protein antigens, similar to that of alum and other adjuvants.65,66 Previous study using IL-1R showed that PPCS-NPs exhibited longer residence time in tissues.67 And last, the PPCS-NPs may display the antigens in a highly repetitive manner, which may trigger the host pattern recognition receptors. In a parallel study, we have used green fluorescent protein-tags proteins to test the assembly process and found the proteins indeed distributed as the corona of the final core−shell particles. In addition, there was little exchange between the surface proteins with solution proteins upon the formation of the final assemblies. However, when we tried to use small-angle X-ray diffraction to investigate the protein arrangement, no clear diffraction pattern has been observed. While the exact mechanism for the PPCS-NPs triggering a strong immunogenic response is not yet clear, the surface stabilized antigens on the polymeric core appear to be potent for presenting immunogens. However, it will be very interesting to further investigate if the immune response is indeed trigger by the whole particles or the released proteins. To demonstrate the safety of PPCS-NPs, the physiological indices were examined after the animals were euthanized. No significant body and spleen weight change were observed in comparison to protein-injected mice (Figure S2b,c,e,f). The blood cell counts in the mice were shown to be in the normal range for both antigen alone and PPCS-NPs inoculated groups (Figure S3a,d). Additionally, there were no obvious lesions or damage to liver, kidney and spleen after tissues hematoxylin and eosin staining (Figure 4). In summary, we have synthesized PyPCL and investigated its assembly with antigens to PPCS-NPs. Subcutaneous immunization of the PPCS-NPs to mice showed an increased antibody titer as well as the subtypes IgG2a and IgG1, which suggests a preferential elicitation of Th1 and Th2-like immune responses.39,59 Such immune-enhancement effect could be attributed to the ideal structure that promises an efficient contact between antigens and immune system. PyPCL was shown to be a safety polymeric material, which permits the potential in vivo applications of these PPCS-NPs system. The nontoxic PyPCL-based PPCS-NPs should have broad applic-

diameters of both PyPCL-DV2EP and PyPCL-CSP nanoparticles were about 100−150 nm, and the results were consistent with particles observed by TEM (Figure S1c,f). PyPCL-CSP and PyPCL-DV2EP PPCS-NPs were then subcutaneously injected into mice, respectively. For the CSP group, the immunizations were given at days 0, 11, and 26, and the DV2EP group was inoculated at days 0, 14, and 28. Consistent with the previous OVA antigen immunizations, the specific IgG titers for CSP (Figure 3a) and DV2EP (Figure 3b)

Figure 3. Total specific anti-CSP (a) and anti-DV2EP (b) IgG titers. Asterisks over bars indicated degree of significance, where **p < 0.01; ***p < 0.001.

in PPCS-NPs immunized mice were significantly higher than the control groups. Moreover, immunization with PPCS-NPs resulted in an enhancement of IgG1 and IgG2a in both CSP and DV2EP groups (Table 1). Following the first immunization on day 0, the mice inoculated with PPCS-NPs display an order of magnitude higher level of IgG1 titers than mice inoculated with protein antigen alone. This trend continues to the end of the study with nearly 2 orders of magnitude higher levels of IgG1 levels for mice inoculated with PPCS-NPs (Figure S2a,b). Of the six mice that were inoculated with protein antigen alone, only one exhibited IgG1 or IgG2a titers above the threshold levels, whereas the PPCS-NPs formed with PyPCL and antigens induced a significantly higher titer level across all six mice (Table 1). Only the two groups inoculated with antigens assembled as PPCS-NPs show higher levels of IgG2a, whereas the two groups inoculated with antigen protein alone has lower levels of IgG2a titers. The increased IgG2a titers are indicative of a Th1-like response,39,59−61 which are required for both prophylactic and therapeutic vaccines.62−64 The ability of PPCS-NPs in effectively stimulating the immune system could be attributed to multiple factors associated with PPCS-NPs. First, entities that are smaller than 100−200 nm can be taken up by both macrophages and

Table 1. Free Protein and PPCS-NPs Induced IgG1 and IgG2a: (a, b) for CSP and PyPCL-CSP, and (c, d) for DV2EP and PyPCL-DV2EP (a) day day day day

day day day day

0 11 26 40

0 14 28 42

(b)

CSP only

PyPCL-CSP

IgG1 threshold

CSP only

0/6 1/6 0/6 0/6

0/6 6/6 6/6 6/6 (c)

>200 >1000 >100000

0/6 0/6 0/6 0/6

0/6 0/6 6/6 6/6 (d)

DV2EP only

PyPCL-DV2EP

IgG1 threshold

DV2EP only

PyPCL-DV2EP

IgG2a threshold

0/6 0/6 0/6 0/6

0/6 0/6 5/6 6/6

>1000 >100000

0/6 0/6 0/6 2/6

0/6 0/6 6/6 6/6

>10 >100

444

PyPCL-CSP

IgG2a threshold

>20 >1000

DOI: 10.1021/acsmacrolett.7b00049 ACS Macro Lett. 2017, 6, 442−446

Letter

ACS Macro Letters

(10) Miermont, A.; Barnhill, H.; Strable, E.; Lu, X.; Wall, K. A.; Wang, Q.; Finn, M. G.; Huang, X. Chem. - Eur. J. 2008, 14, 4939. (11) Raja, K. S.; Wang, Q.; Gonzalez, M. J.; Manchester, M.; Johnson, J. E.; Finn, M. G. Biomacromolecules 2003, 4, 472. (12) Rae, C. S.; Khor, I. W.; Wang, Q.; Destito, G.; Gonzalez, M. J.; Singh, P.; Thomas, D. M.; Estrada, M. N.; Powell, E.; Finn, M. G.; Manchester, M. Virology 2005, 343, 224. (13) Steinmetz, N. F.; Hong, V.; Spoerke, E. D.; Lu, P.; Breitenkamp, K.; Finn, M. G.; Manchester, M. J. Am. Chem. Soc. 2009, 131, 17093. (14) Astronomo, R. D.; Kaltgrad, E.; Udit, A. K.; Wang, S.-K.; Doores, K. J.; Huang, C.-Y.; Pantophlet, R.; Paulson, J. C.; Wong, C.H.; Finn, M. G.; Burton, D. R. Chem. Biol. 2010, 17, 357. (15) Pokorski, J. K.; Breitenkamp, K.; Liepold, L. O.; Qazi, S.; Finn, M. G. J. Am. Chem. Soc. 2011, 133, 9242. (16) Kaltgrad, E.; Sen Gupta, S.; Punna, S.; Huang, C.-Y.; Chang, A.; Wong, C.-H.; Finn, M. G.; Blixt, O. ChemBioChem 2007, 8, 1455. (17) Brennan, F. R.; Bellaby, T.; Helliwell, S. M.; Jones, T. D.; Kamstrup, S.; Dalsgaard, K.; Flock, J. I.; Hamilton, W. D. J. Virol. 1999, 73, 930. (18) Gilleland, H. E.; Gilleland, L. B.; Staczek, J.; Harty, R. N.; García-Sastre, A.; Palese, P.; Brennan, F. R.; Hamilton, W. D.; Bendahmane, M.; Beachy, R. N. FEMS Immunol. Med. Microbiol. 2000, 27, 291. (19) McInerney, T. L.; Brennan, F. R.; Jones, T. D.; Dimmock, N. J. Vaccine 1999, 17, 1359. (20) Porta, C.; Spall, V. E.; Loveland, J.; Johnson, J. E.; Barker, P. J.; Lomonossoff, G. P. Virology 1994, 202, 949. (21) Smith, D. M.; Simon, J. K.; Baker, J. R. Nat. Rev. Immunol. 2013, 13, 592. (22) Bachelder, E. M.; Beaudette, T. T.; Broaders, K. E.; Dashe, J.; Fréchet, J. M. J. J. Am. Chem. Soc. 2008, 130, 10494. (23) Yolles, S.; Leafe, T. D.; Meyer, F. J. J. Pharm. Sci. 1975, 64, 115. (24) Heller, J. Ann. N. Y. Acad. Sci. 1985, 446, 51. (25) Rosen, H. B.; Chang, J.; Wnek, G. E.; Linhardt, R. J.; Langer, R. Biomaterials 1983, 4, 131. (26) Delgado, M.; Lee, K. J.; Altobell, L.; Spanka, C.; Wentworth, P.; Janda, K. D. J. Am. Chem. Soc. 2002, 124, 4946. (27) Anderson, D. G.; Lynn, D. M.; Langer, R. Angew. Chem., Int. Ed. 2003, 42, 3153. (28) Liu, T.-Y.; Hussein, W. M.; Giddam, A. K.; Jia, Z.; Reiman, J. M.; Zaman, M.; McMillan, N. A. J.; Good, M. F.; Monteiro, M. J.; Toth, I.; Skwarczynski, M. J. Med. Chem. 2015, 58, 888. (29) Skwarczynski, M.; Zaman, M.; Urbani, C. N.; Lin, I.-C.; Jia, Z.; Batzloff, M. R.; Good, M. F.; Monteiro, M. J.; Toth, I. Angew. Chem., Int. Ed. 2010, 49, 5742. (30) Zaman, M.; Skwarczynski, M.; Malcolm, J. M.; Urbani, C. N.; Jia, Z.; Batzloff, M. R.; Good, M. F.; Monteiro, M. J.; Toth, I. Nanomedicine 2011, 7, 168. (31) Ahmad Fuaad, A. A. H.; Jia, Z.; Zaman, M.; Hartas, J.; Ziora, Z. M.; Lin, I.-C.; Moyle, P. M.; Batzloff, M. R.; Good, M. F.; Monteiro, M. J.; Skwarczynski, M.; Toth, I. Nanomedicine (London, U. K.) 2014, 9, 35. (32) Damgé, C.; Vranckx, H.; Balschmidt, P.; Couvreur, P. J. Pharm. Sci. 1997, 86, 1403. (33) Mathiowitz, E.; Jacob, J. S.; Jong, Y. S.; Carino, G. P.; Chickering, D. E.; Chaturvedi, P.; Santos, C. A.; Vijayaraghavan, K.; Montgomery, S.; Bassett, M.; Morrell, C. Nature 1997, 386, 410. (34) Lim, Y. T.; Noh, Y.-W.; Han, J. H.; Cai, Q.-Y.; Yoon, K.-H.; Chung, B. H. Small 2008, 4, 1640. (35) Kishimoto, T. K.; Ferrari, J. D.; LaMothe, R. A.; Kolte, P. N.; Griset, A. P.; O’Neil, C.; Chan, V.; Browning, E.; Chalishazar, A.; Kuhlman, W.; Fu, F.-N.; Viseux, N.; Altreuter, D. H.; Johnston, L.; Maldonado, R. A. Nat. Nanotechnol. 2016, 11, 890. (36) Siefert, A. L.; Caplan, M. J.; Fahmy, T. M. Biomaterials 2016, 97, 85. (37) Zhu, G.; Mallery, S. R.; Schwendeman, S. P. Nat. Biotechnol. 2000, 18, 52.

Figure 4. Histological analysis of the effect of PyPCL-based PPCSNPs administration on the spleen, kidney, and liver at 50× and 100× magnifications.

ability to vaccine development where the antigen is a weak immunogen and should be suitable for all candidate antigens.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.7b00049. Experimental details, DLS data, TEM images, mice IgG1 and IgG2a titers, and animals’ physiological indices (PDF).

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Qian Wang: 0000-0002-2149-384X Funding

The Research was partially supported by US NSF CHE1307319. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank University of South Carolina and Molecular Pathology/Special Procedures/Chemistry Laboratory Director Delecia R. LaFrance for assistance with the histological analysis.

■

REFERENCES

(1) Reddy, S. T.; Swartz, M. A.; Hubbell, J. A. Trends Immunol. 2006, 27, 573. (2) Peek, L. J.; Middaugh, C. R.; Berkland, C. Adv. Drug Delivery Rev. 2008, 60, 915. (3) Sharma, S.; Mukkur, T. K. S.; Benson, H. A. E.; Chen, Y. J. Pharm. Sci. 2009, 98, 812. (4) Zhao, Z.; Leong, K. W. J. Pharm. Sci. 1996, 85, 1261. (5) Cho, N.-H.; Cheong, T.-C.; Min, J. H.; Wu, J. H.; Lee, S. J.; Kim, D.; Yang, J.-S.; Kim, S.; Kim, Y. K.; Seong, S.-Y. Nat. Nanotechnol. 2011, 6, 675. (6) Sun, C.; Lee, J. S. H.; Zhang, M. Adv. Drug Delivery Rev. 2008, 60, 1252. (7) Klippstein, R.; Pozo, D. Nanomedicine 2010, 6, 523. (8) Singh, A.; Agarwal, R.; Diaz-Ruiz, C. A.; Willett, N. J.; Wang, P.; Lee, L. A.; Wang, Q.; Guldberg, R. E.; García, A. J. Adv. Healthcare Mater. 2014, 3, 1562. (9) Kaltgrad, E.; O’Reilly, M. K.; Liao, L.; Han, S.; Paulson, J. C.; Finn, M. G. J. Am. Chem. Soc. 2008, 130, 4578. 445

DOI: 10.1021/acsmacrolett.7b00049 ACS Macro Lett. 2017, 6, 442−446

Letter

ACS Macro Letters

K.; Lu, X.; Erdman, D. D.; Spearman, P.; Moore, M. L. Nat. Commun. 2016, 7, 12838. (67) Agarwal, R.; Volkmer, T. M.; Wang, P.; Lee, L. A.; Wang, Q.; Garcı ́a, A. J. Biomed. Mater. Res., Part A 2016, 104A, 595−599.

(38) McKeever, U.; Barman, S.; Hao, T.; Chambers, P.; Song, S.; Lunsford, L.; Hsu, Y.-Y.; Roy, K.; Hedley, M. L. Vaccine 2002, 20, 1524. (39) Sahdev, P.; Ochyl, L. J.; Moon, J. Pharm. Res. 2014, 31, 2563. (40) Tan, Y.; Huang, L. J. Drug Target. 2002, 10, 153. (41) Rae, C. S.; Khor, I. W.; Wang, Q.; Destito, G.; Gonzalez, M. J.; Singh, P.; Thomas, D. M.; Estrada, M. N.; Powell, E.; Finn, M. G.; Manchester, M. Virology 2005, 343, 224. (42) Russell, J. T.; Lin, Y.; Böker, A.; Su, L.; Carl, P.; Zettl, H.; He, J.; Sill, K.; Tangirala, R.; Emrick, T.; Littrell, K.; Thiyagarajan, P.; Cookson, D.; Fery, A.; Wang, Q.; Russell, T. P. Angew. Chem., Int. Ed. 2005, 44, 2420. (43) Li, T.; Niu, Z.; Emrick, T.; Russell, T. P.; Wang, Q. Small 2008, 4, 1624. (44) Li, T.; Wu, L.; Suthiwangcharoen, N.; Bruckman, M. A.; Cash, D.; Hudson, J. S.; Ghoshroy, S.; Wang, Q. Chem. Commun. 2009, 20, 2869. (45) Li, T.; Ye, B.; Niu, Z.; Thompson, P.; Seifert, S.; Lee, B.; Wang, Q. Chem. Mater. 2009, 21, 1046. (46) Suthiwangcharoen, N.; Li, T.; Li, K.; Thompson, P.; You, S.; Wang, Q. Nano Res. 2011, 4, 483. (47) Wu, L.; Li, T.; Blom, D.; Zhao, J.; Ghoshroy, S.; Wang, Q. Microsc. Res. Tech. 2011, 74, 636. (48) Suthiwangcharoen, N.; Li, T.; Wu, L.; Reno, H. B.; Thompson, P.; Wang, Q. Biomacromolecules 2014, 15, 948. (49) Lu, L.; Yuan, L.; Yan, J.; Tang, C.; Wang, Q. Biomacromolecules 2016, 17, 2321. (50) Zinkernagel, R. M.; Hengartner, H. Science 2001, 293, 251. (51) Bachmann, M. F.; Rohrer, U. H.; Kündig, T. M.; Bürki, K.; Hengartner, H.; Zinkernagel, R. M. Science 1993, 262, 1448. (52) Bachmann, M. F.; Jennings, G. T. Nat. Rev. Immunol. 2010, 10, 787. (53) Liu, W.; Peng, Z.; Liu, Z.; Lu, Y.; Ding, J.; Chen, Y.-H. Vaccine 2004, 23, 366. (54) Gamvrellis, A.; Leong, D.; Hanley, J. C.; Xiang, S. D.; Mottram, P.; Plebanski, M. Immunol. Cell Biol. 2004, 82, 506. (55) Zong, C.; Wang, M.; Yang, F.; Chen, G.; Chen, J.; Tang, Z.; Liu, Q.; Gao, C.; Ma, L.; Wang, J. J. Tissue Eng. Regener. Med. 2015, n/a. (56) Wang, J.; Wang, J.; Lu, P.; Cai, Y.; Wang, Y.; Hong, L.; Ren, H.; Heng, B. C.; Liu, H.; Zhou, J.; Ouyang, H. Biomaterials 2015, 62, 76. (57) Méndez, J. A.; Usme-Ciro, J. A.; Domingo, C.; Rey, G. J.; Sánchez, J. A.; Tenorio, A.; Gallego-Gomez, J. C. Virol. J. 2012, 9, 64. (58) Singh, A. P.; Buscaglia, C. A.; Wang, Q.; Levay, A.; Nussenzweig, D. R.; Walker, J. R.; Winzeler, E. A.; Fujii, H.; Fontoura, B. M. A.; Nussenzweig, V. Cell 2007, 131, 492. (59) Kanchan, V.; Panda, A. K. Biomaterials 2007, 28, 5344. (60) Mosmann, T. R.; Coffman, R. L. Immunol. Today 1987, 8, 223. (61) Snapper, C. M.; Paul, W. E. Science 1987, 236, 944. (62) Pawar, D.; Mangal, S.; Goswami, R.; Jaganathan, K. S. Eur. J. Pharm. Biopharm. 2013, 85, 550. (63) Wibowo, N.; Chuan, Y. P.; Seth, A.; Cordoba, Y.; Lua, L. H. L.; Middelberg, A. P. Vaccine 2014, 32, 3664. (64) Bachmann, M. F.; Hengartner, H.; Zinkernagel, R. M. Med. Microbiol. Immunol. 1994, 183, 315. (65) Vaccari, M.; Gordon, S. N.; Fourati, S.; Schifanella, L.; Liyanage, N. P. M.; Cameron, M.; Keele, B. F.; Shen, X.; Tomaras, G. D.; Billings, E.; Rao, M.; Chung, A. W.; Dowell, K. G.; Bailey-Kellogg, C.; Brown, E. P.; Ackerman, M. E.; Vargas-Inchaustegui, D. A.; Whitney, S.; Doster, M. N.; Binello, N.; Pegu, P.; Montefiori, D. C.; Foulds, K.; Quinn, D. S.; Donaldson, M.; Liang, F.; Loré, K.; Roederer, M.; Koup, R. A.; McDermott, A.; Ma, Z.-M.; Miller, C. J.; Phan, T. B.; Forthal, D. N.; Blackburn, M.; Caccuri, F.; Bissa, M.; Ferrari, G.; Kalyanaraman, V.; Ferrari, M. G.; Thompson, D.; Robert-Guroff, M.; Ratto-Kim, S.; Kim, J. H.; Michael, N. L.; Phogat, S.; Barnett, S. W.; Tartaglia, J.; Venzon, D.; Stablein, D. M.; Alter, G.; Sekaly, R.-P.; Franchini, G. Nat. Med. 2016, 22, 762. (66) Lee, S.; Nguyen, M. T.; Currier, M. G.; Jenkins, J. B.; Strobert, E. A.; Kajon, A. E.; Madan-Lala, R.; Bochkov, Y. A.; Gern, J. E.; Roy, 446

DOI: 10.1021/acsmacrolett.7b00049 ACS Macro Lett. 2017, 6, 442−446