Effect of Several HIV Antigens Simultaneously Loaded with G2-NN16

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Effect of several HIV-antigens simultaneously loaded with G2-NN16 carbosilane dendrimer in the cell uptake and functionality of human dendritic cells Daniel Sepúlveda-Crespo, Enrique Vacas-Córdoba, Valeria Márquez-Miranda, Ingrid Araya-Duran, Rafael Gómez, F. Javier de la Mata, Fernando Danilo Gonzalez-Nilo, and M. Ángeles Muñoz-Fernández Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00623 • Publication Date (Web): 15 Nov 2016 Downloaded from http://pubs.acs.org on November 25, 2016

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Bioconjugate Chemistry

Effect of several HIV-antigens simultaneously loaded with G2-NN16 carbosilane dendrimer in the cell uptake and functionality of human dendritic cells

Daniel Sepúlveda-Crespo†,Ψ, Enrique Vacas-Córdoba†,Ψ, Valeria Márquez-Miranda‡,§, Ingrid Araya-Durán‡,§, Rafael Gómezǁ, Francisco Javier de la Mataǁ, Fernando Danilo GonzálezNilo‡,§,┴, Mª Ángeles Muñoz-Fernández*,†,Φ †

Laboratorio InmunoBiología Molecular, Hospital General Universitario Gregorio Marañón (HGUGM), Madrid, Spain; Instituto de Investigación Sanitaria Gregorio Marañón (IiSGM), Spanish HIV-HGM BioBank, Madrid, Spain; Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Madrid, Spain

‡

Universidad Andres Bello, Facultad de Biología, Center for Bioinformatics and Integrative Biology (CBIB), Av. República 239, Santiago, Chile

§

Fundación Fraunhofer Chile Research, Las Condes, Chile

ǁ

Departamento Química Orgánica y Química Inorgánica, Universidad de Alcalá Henares, Spain. CIBER-BBN, Madrid, Spain. ┴

Centro Interdisciplinario de Neurociencia de Valparaíso, Facultad de Ciencias, Universidad de Valparaíso, Valparaíso, Chile.

Φ

Plataforma de Laboratorio, HGUGM, Madrid, Spain; IiSGM, Madrid, Spain. CIBER-BBN, Madrid, Spain.

Corresponding author: Mª Ángeles Muñoz-Fernández Laboratorio InmunoBiología Molecular Hospital General Universitario Gregorio Marañón, CIBER-BBN. IiSGM C/Dr. Esquerdo 46, 28007 Madrid, Spain; Telephone number: +34 915 868 565. E-mail: [email protected] Ψ

Both authors contributed equally to this work

- Conflicts of Interest: The authors do not have commercial or other associations that might pose a conflict of interest.

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TABLE OF CONTENTS


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ABSTRACT Dendrimers are highly branched, star-shaped and nano-sized polymers that have been proposed as new carriers for specific HIV-1 peptides. Dendritic cells (DCs) are the most potent antigen-presenting cells which play a major role in development of cell-mediated immunotherapy due to the generation and regulation of adaptive immune responses against HIV-1. This article reports on the associated behavior of two or three HIV-derived-peptides simultaneously (p24/gp160 or p24/gp160/NEF) with cationic carbosilane dendrimer G2NN16. We have found that: (i) immature DCs (iDCs) and mature (mDCs) did not capture efficiently HIV-peptides regarding the uptake level when cells were treated with G2NN16/peptide complex alone; (ii) the ability of DCs to migrate was not depending on the peptides presence; and (iii) by using molecular dynamic simulation, a mixture of peptides decreased the cell uptake of the other peptides, particularly, NEF hinders the binding of more peptides, especially obstructing the binding of gp160 to G2-NN16. The results suggest that G2-NN16 cannot be considered as an alternative carrier for delivering two or more HIVderived peptides to DCs.



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Dendrimers are hyperbranched, star-shaped and nano-sized polymers consisting of tree-like arms or branches constructed through the sequential addition of branching units from an initiator.1-5 Dendrimers have emerged as promising candidates for many applications in biology and nanomedicine due to monodispersity and multivalency. The presence of several end-groups in dendrimers provides a versatile functionalization much stronger than monovalent interactions to inhibit undesired biological interactions, to promote desired cellular responses and/or to control recognition at the surface.6-9 Therefore, the multivalency of dendrimers is one of their most important properties recognized, along with their welldefined structure and ease functionalization, for drug/gene delivery and vaccine technology.1013

Moreover, the correct choice of terminal groups is a critical task for designing new

dendrimers as nanocarriers of molecules. Despite the inherent potency of current combination antiretroviral therapy (cART) to suppress virus or to control viral load and to achieve the immune recovery in human immunodeficiency virus (HIV)-patients, cART does not eradicate the viral reservoirs when cART is discontinued. Therefore, there are concerns about the emergence of multidrug-resistant mutants, risk of side effects, daily administration for lifetime and high daily costs. In this sense, therapeutic HIV-vaccines have been proposed as the best alternative to eradicate the long-term virus. Several types of strategies, such as inactivated virus, live attenuated virus, recombinant viral vectors and HIV-1 virus-like particles have shown limited efficacy for the development of HIV-1 vaccines.14 A growing effort has been focused on the development of therapeutic HIV-1 vaccines based on dendritic cells (DCs) because DCs-based vaccines can play a key role in HIV-1 infection inducing a strong CD4+ T cell response, a sustained and effective HIV-specific CD8+ response and suppressing HIV-1 replication. Although the results of DCs-based vaccines in clinical HIV-1 trials are promising, the correct choice of


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HIV-1 antigens and the lack of persistence of antigen loaded in DCs remain the main problems to overcome.14 In this sense, several glycodendrimers and polycationic dendrimers have been employed as non-viral vectors of HIV-derived peptides for DCs-based immunotherapy, such as polyamidoamine (PAMAM) dendrimers, polypropyleneimine (PPI) dendrimers, poly-ʟ-lysine (PLL) dendrimers, polyglycerol (PG) dendrimers, phosphorus-containing dendrimers and carbosilane dendrimers.15-20 In previous studies, various HIV-derived peptides employed as antigens (NEF, gp160 and p24), were individually associated with water soluble carbosilane dendrimer G2-NN16 (see Supporting Information for details from dendrimer) to form peptide-dendrimer complexes in order to be captured by human monocytes-derived DCs for efficient immune responses.17,18 These peptide-dendrimer complexes showed to be a successful technique because G2-NN16 enhanced peptides loading efficiency into DCs. Moreover, molecular modeling represents a valuable tool to understand phenomena beyond of the view of classical experiments. In this sense, our group has developed several methods to study the complexation between dendrimers and drugs.21,22 Specially, our group has employed molecular dynamics method to design and evaluate new dendrimers as carrier of nucleic acids, aiming at providing an interpretation for experimental evidences.23-25 In this study, we proposed to evaluate the G2-NN16 ability to transport two or more HIVderived peptides into DCs. First, we evaluated whether G2-NN16 in association with two or more peptides increased the uptake of these peptides into DCs in comparison to peptides alone or peptides associated with G2-NN16 separately. Moreover, we showed whether DCs treated with G2-NN16 associated with two or more peptides are functional with respect to migration and justified the obtained results by using computational simulations (for detailed experimental data, see Supporting Information). In this sense, we checked whether G2-



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NN16 as carriers of two or more HIV-derived peptides could be used as tool as cellular vaccine or adjuvant for HIV-1 immunotherapy. We analyzed whether DCs enhanced the uptake of two or more peptides simultaneously (p24/gp160 or p24/gp160/NEF) more efficiently when complexed with G2-NN16 compared with non-complexed peptides or G2-NN16/peptide complex alone. Detection of capturing peptides in DCs was performed by flow cytometry using fluorescent peptides. The optimum charge ratio of G2-NN16/peptide system was a molar ratio 1:3 according to previous studies.17,18 We washed the loaded DCs with an acid buffer to detach all peptides fixed at the DCs surface and to make sure that the observed signal came from internalized HIV-1 peptides.26 TAMRA-labeled gp160 or p24, or FITC-labeled NEF were complexed with G2NN16 at a ratio 3:1 using the maximum non-toxic concentration of dendrimer (2.5µM) associated with diverse peptides. The findings are in agreement with results previously reported18 due to dendriplexes (G2-NN16/peptide) increased cellular uptake of peptides in iDCs and mDCs at 2h and 24h compared with non-complexed peptides (Figure 1). However, G2-NN16/p24/gp160 dendriplex decreased the peptides uptake at 2h post-treatment in iDCs (1.3% p24; 1.5% gp160) compared with G2-NN16/peptide complex alone (26.7% p24 for G2-NN16/p24 or 13.7% gp160 for G2-NN16/gp160 dendriplexes) (Figure 1A). Practically the same tendency was observed at 24h post-treatment (19.6% p24; 1.1% gp160) compared with G2-NN16/peptide complex alone (45.5% p24 for G2-NN16/p24 or 1.1% gp160 for G2NN16/gp160 dendriplexes) (Figure 1B). G2-NN16/p24/gp160 dendriplex also decreased uptake of peptides into mDCs compared with G2-NN16/peptide complex alone at 2h (2.3% p24; 1.0% gp160) and 24h (2.0% p24; 0.7% gp160) post-treatment (Figure 1C-D). On the other hand, G2-NN16/p24/gp160/NEF dendriplex showed to decline peptide loading at 2h (1.9% p24; 2.7% gp160) and 24h (30.1% p24; 0.5% gp160) post-treatment in iDCs compared to the peptides associated alone with G2-NN16 (Figure 1A-B). G2[6] ACS Paragon Plus Environment

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NN16/p24/gp160/NEF dendriplex slightly decreased the peptides uptake at 2h post-treatment (0.3% p24; 0.3% gp160) and at 24h (0.3% p24; 0.2% gp160) post-treatment in mDCs compared with G2-NN16/peptide complex alone (Figure 1C-D).

Figure 1. HIV-derived peptides uptake by dendritic cells. Capture levels by iDCs and mDCs after (A, C) 2h or (B, D) 24h of treatment with untreated control (NT), HIV-derived peptides (NEF, gp160 and/or p24) or G2-NN16/HIV-derived peptides complexes at 1:3, 1:3:3 or 1:3:3:3 molar ratios. Data are represented as mean ± SD of two independent donors. *: p < 0.05; **: p < 0.01 vs. untreated control (NT).

The migratory capacity of mDCs is a key property in a DCs-based immunotherapy because when mDCs are intradermally injected, mDCs need to migrate to the secondary lymphoid organs where they can stimulate T cells and to activate immune system. Therefore, we analyzed whether migration of mDCs could be altered in the presence of two or more peptides (p24/gp160 or p24/gp160/NEF) simultaneously complexed with G2-NN16. As we expected, iDCs hardly migrate towards CCL19 and CCL21, whereas mDCs migrated efficiently in



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transwell chemotaxis assays regardless whether mDCs captured G2-NN16, or G2NN16/p24/gp160, G2-NN16/p24/gp160/NEF dendriplexes (Figure 2). mDCs treated with G2-NN16/p24/gp160 or G2-NN16/p24/gp160/NEF dendriplexes showed a increased ability to migrate forward CCL19, whereas they showed a slightly decreased ability to migrate forward CCL21 than the initial non-migrated populations, although these differences were not significant (Figure 2).

Figure

2.

Chemotaxis ability of mature dendritic cells. Percentage of total number of mDCs that crossed the transwell membrane towards the basal part of the transwell. Untreated mDCs were loaded with HIVderived peptides alone, G2-NN16 or G2-NN16/HIV-derived peptides (at 1:3, 1:3:3 or 1:3:3:3 molar ratios) for 4h and were placed in the top part of transwells (4.5µm pore size). DCs-medium alone (NT), CCL19 or CCL21 chemokines were added in the basal part of the transwell. After 4h of migration, migrating cells detected in the basal part were counted by fluorescence-activated cell sorting (FACS) during 60s. Data are represented as mean ± SD of two independent experiments. Not significant: p > 0.05 vs. untreated mDCs (mDC).

To address the 1:1 interaction between G2-NN16 and peptides, molecular dynamics (MD) simulations and molecular mechanics/generalized Born surface area (MM-GBSA) method were carried out for each peptide. From these simulations, radial distribution function (RDF), as a way to evaluate the location of the different peptides, was calculated a function of the [8] ACS Paragon Plus Environment

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center-of-mass (COM) of G2-NN16. NEF was identified as the peptide that reaches a better interaction in the inner cavities of G2-NN16 (Figure 3C and 3F), followed by gp160 (Figure 3B and 3E), as judging by the peaks inside the distribution of G2-NN16. Meanwhile, p24 tends to interact better in the surface of G2-NN16 rather than in the cavities (Figure 3A and 3D).



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Figure 3. Analysis of G2-NN16/peptide complex at 1:1 molar ratio. (A-C) Radial distribution function (RDF) of each peptide as a function of the center-of-mass of G2-NN16. Snapshots of the last frame of molecular dynamic (MD) simulation for (D) G2-NN16/p24, (E) G2-NN16/gp160 and (F) G2-NN16/NEF complexes. Mean number of contacts established between G2-NN16 and each aminoacid of (G) p24 peptide, (H) gp160 peptide, (I) NEF peptide, during the last 10ns of MD trajectory. (J) Solvent coverage for each peptide in presence of G2-NN16 obtained as a percentage from surface solvent accessible surface area values (SASA) along the trajectory and normalized by SASA of each peptide. (K) Binding energy of each G2-NN16/peptide complex, obtained from molecular mechanics/generalized Born surface area (MM-GBSA) method

To further elucidate whether composition of the different peptides p24, gp160 and NEF are eliciting any influence in the interaction with the dendrimer, the mean number of contacts that each amino acid (aa) can establish with G2-NN16 at 4Å was calculated during the last 10ns of MD simulation (i.e. when the complex becomes stability). This analysis revealed the key role of aromatic aa in the stabilization of peptides in the cavities of G2-NN16. Therefore, p24 peptide seems to anchor G2-NN16 through its only aromatic residue (Trp), while negative charged residues, such as Glu and Asp, orientate towards the solvent, adopting a less number of contacts with G2-NN16 (Figure 3G). In contrast, gp160 does not have aromatic groups, except for Tyr residues that are also somewhat polar. Instead, the negative residues in its terminal ends displayed a poor interaction with G2-NN16 (Figure 3H). In the case of NEF, the high content of aromatic residues (2 Phe, 1 Trp), aimed by other aliphatic hydrophobic residues (2 Leu) are mediating a greater number of contacts compared to other types of aa (Figure 3I), which explains NEF distribution obtained by RDF analysis in Figure 3C. Additionally, to quantify the degree of coverage of the different peptides in complex with the dendrimer, solvent accessible surface area (SASA) for each peptide was calculated during the whole MD simulations, normalized by SASA of each peptide in solution in absence of dendrimer. SASA information revealed that all the peptides reach a similar level of coverage from the solvent (about 30%) in presence of G2-NN16 in a 1:1 ratio (Figure 3J). [10] ACS Paragon Plus Environment

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Interestingly, gp160 showed a faster interaction, being already covered at 10ns of MD simulation. This effect can be explained by the electrostatic interaction yielded by the negative charged residues. Hydrophobic anchoring of the peptides gp160 and NEF appear elicited in a slower way. In terms of binding energy, MM-GBSA method represents in a straightforward manner both electrostatic and van der Waals terms, together with associating the non-polar contributions to the solvent accesibility (i.e. SASA). Therefore, MM-GBSA results showed that G2-NN16 has a more favorable interaction (i.e. more negative ∆Gbind) with NEF, which as described above might be associated to strong interaction of the hydrophobic groups with the cavities of G2NN16. It must be noted that even when gp160 has more aa than p24, both displayed similar

∆Gbind values, suggesting once again that one single aa, such as Trp of p24, could have a relevant role in the G2-NN16/peptide interaction (Figure 3K). Dynamics of the peptides interaction with the dendrimer during the MD simulation were inspected through G2-NN16/peptide COM distance (Figure 4A). As seen before, gp160 was covered by G2-NN16 faster than the other peptides in a 1:1 ratio system, which could be due to the electrostatic interactions and a more negative nature (-4). The same behavior was seen in the 1:3:3:3 system, where G2-NN16/gp160(1) interaction was established before the others. The second peptide that interacts with G2-NN16 was p24(2). Having the peptide a formal charge equal to -4, interaction with G2-NN16 is also aided by p24 negative residues and later promoted by hydrophobic interactions. Meanwhile, in a competition with electrostatic interaction promoted by p24 and gp160, a more hydrophobic NEF(1) seems less prone to interact at first. But once one p24 and one gp160 form a complex with G2-NN16, by establishing contacts with their quaternary amines and becoming more neutral the overall complex, the possibilities of NEF(1) to interact with G2-NN16 increase. However, the number of contacts of NEF(1) in this 1:3:3:3 system is lesser than in the 1:1 system, because [11] ACS Paragon Plus Environment


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of the steric impedance that displaces NEF towards a more peripheral position regarding to G2-NN16. In this 1:3:3:3 system, NEF Try residue still has a key role. Up to this point, G2NN16 becomes saturated; its charges appeared be totally screened by the peptides, and then only hydrophobic interactions become relevant. In this manner, just one small p24(1) can interact with G2-NN16 surface, mainly mediated by its only hydrophobic residue (Try). Finally, NEF(2) appeared stabilized by hydrophobic interactions in the outer shell of this aggregate composed by G2-NN16 and the peptides. The final distribution of the peptides regarding to G2-NN16 is represented in the RDF of the different molecular species (Figure 4B) and in the final snapshot (Figure 4E). In this kind of aggregate, it must be noted that only three peptides conserved similar levels of coverage that in 1:1 system (SASA, Figure 4C). Even if more peptides would interact with the aggregate, they are not well protected by G2NN16 and eventually can be degraded inside the cells. NEF(1) hinders the binding of more peptides, especially obstructing the binding of a second gp160 peptide. NEF(1) in this case is not obstructing the binding of p24(1). This evidence can explain why mixtures of peptides including NEF are decreasing the cell uptake of the other peptides, as experimental assays showed in this study, being more dramatic the decreasing of gp160 uptake. Notably, from Figure 4C appears clear that only three peptides conserved a coverage level greater than 20%: p24(2), gp160(1) and p24(1), being the coverage much lesser in the case of NEF(1) and NEF(2). Importantly, as more peptides bind G2-NN16, the coverage and number of contacts for p24 is progressively losing (Figure 4D). This evidence also supports the experimental findings, in terms of ideal G2-NN16/peptide ratio was determined as 1:3.


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Figure 4. Analysis of G2-NN16/peptide complex at 1:3:3:3 molar ratio. (A) G2-NN16/peptide center-of-mass (COM) distance, calculated along the molecular dynamic (MD) trajectory only for peptides that actually were in contact with G2-NN16. (B) Radial distribution function (RDF) of each peptide as a function of COM of G2-NN16. (C) Solvent coverage for each peptide in presence of G2NN16 obtained as a percentage from surface solvent accessible surface area (SASA) values along the trajectory and normalized by SASA of each peptide. (D) Mean number of contacts established between G2-NN16 and each aminoacid of dendrimer closest peptides: p24 (1), p24 (2), gp160 (1) and NEF (1) peptides, obtained from the last 10ns of MD trajectory. (E) Snapshots of the last frame of MD simulation. Only the dendrimer closest peptides are shown.

Summarizing, our results show that G2-NN16/p24/gp160 or G2-NN16/p24/gp160/NEF dendriplexes did not generate better HIV-peptide uptake both in iDCs and mDCs at short and long times regarding the uptake level obtained when cells were treated with G2-NN16/peptide complex alone. However, the uptake of two or more peptides simultaneously did not affect their migratory ability and did not depend on the peptide presence. NEF(1) hinders the [13] ACS Paragon Plus Environment


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binding of more peptides, especially obstructing the binding of a second gp160 peptide. NEF(1) in this case is not obstructing the binding of a second p24 peptide(1). This evidence can explain why mixtures of peptides including NEF are decreasing the cell uptake of the other peptides, as experimental assays showed in this study, being more dramatic the decreasing of gp160 uptake. Eventually, in a system with only p24 peptides, G2-NN16 can encapsulate three of these peptides, differently that in a solution with a mixture of different peptides, where only two p24 peptides are captured, which explains the cell uptake decreases in such conditions. More p24 peptides were encapsulated and protected by G2-NN16 in presence of other peptides, which can explain that their cell uptake do not decay at the level of gp160. The binding energy of G2-NN16/peptides is not directly correlated with the degree of coverage of the peptide elicited by G2-NN16. SASA parameter as a function of MD simulation time seems a more predictive parameter for the degree of protection. Therefore, our results show that G2-NN16 associated with two or more HIV-derived peptides could not used as a vaccine because it does not offer the potential and correct ability to deliver peptides as non-viral vectors.

ACKNOWLEDGMENTS This work has been (partially) funded by the RD12/0017/0037, project as part of the project as part of the Acción Estratégica en Salud, Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica 2008-2011 and cofinanced by Instituto de Salud Carlos III (Subdirección General de Evaluación) and Fondo Europeo de Desarrollo Regional (FEDER), RETIC PT13/0010/0028, Fondo de Investigacion Sanitaria (FIS) (grant number PI13/02016), CTQ2014-54004-P (MIMECO), Comunidad de Madrid (grant numbers S2010/BMD-2351 and S-2010/BMD-2332], CYTED 214RT0482. CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008-2011, IniciativaIngenio 2010, the Consolider [14] ACS Paragon Plus Environment

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Program, and CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. This work was supported partially by a Marie Curie International Research Staff Exchange Scheme Fellowship within the 7th European Community Framework

Program, project No. PIRSES-GA-2012-316730

NANOGENE, co-financed by the Polish Ministry of Science and Higher Education (grant No. W21/7PR/ 2013). V.M.M. thanks CONICYT for a Ph.D. Scholarship (21120785) and CONICYT + PAI/ “Concurso Nacional Tesis de Doctorado en la Empresa” 2014 (781413007). D.G.N. thanks for support of Fraunhofer Chile Research, Innova-Chile CORFO (FCR-CSB 09CEII-6991). The Centro Interdisciplinario de Neurociencia de Valparaíso (CINV) is a Millennium Institute supported by the Millennium Scientific Initiative of the Ministerio de Economía, Fomento y Turismo. SUPPORTING INFORMATION The Supporting Information is available free of charge on the ACS Publications website at DOI:



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Figure 1 1930x1216mm (96 x 96 DPI)

ACS Paragon Plus Environment

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Figure 2 1040x620mm (96 x 96 DPI)



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Figure 3 951x1293mm (96 x 96 DPI)


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Figure 4 200x149mm (150 x 150 DPI)


Effect of Several HIV Antigens Simultaneously Loaded with G2-NN16

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