key: cord-0839317-tx6ucusk
authors: Farfán-Castro, Susan; García Soto, Mariano J.; Comas-García, Mauricio; Arévalo-Villalobos, Jaime I.; Palestino, Gabriela; González-Ortega, Omar; Rosales-Mendoza, Sergio
title: Synthesis and immunogenicity assessment of a gold nanoparticle conjugate for the delivery of a peptide from SARS-CoV-2
date: 2021-03-02
journal: Nanomedicine
DOI: 10.1016/j.nano.2021.102372
sha: b42ffebfba164f3d5c7424d7c2653a29641b8841
doc_id: 839317
cord_uid: tx6ucusk

The development of vaccines is a crucial response against the COVID-19 pandemic and innovative nanovaccines could increase the potential to address this remarkable challenge. In the present study a B cell epitope (S(461–493)) from the spike protein of SARS-CoV-2 was selected and its immunogenicity validated in sheep. This synthetic peptide was coupled to gold nanoparticles (AuNP) functionalized with SH-PEG-NH(2) via glutaraldehyde-mediated coupling to obtain the AuNP-S(461–493) candidate, which showed in s.c.-immunized mice a superior immunogenicity (IgG responses) when compared to soluble S(461–493); and lead to increased expression of relevant cytokines in splenocytes cultures. Interestingly, the response triggered by AuNP-S(461–493) was similar in magnitude to that induced using a conventional strong adjuvant (Freund's adjuvant). This study provides a platform for the development of AuNP-based nanovaccines targeting specific SARS-CoV-2 epitopes.

The new coronavirus, SARS-CoV-2, affecting human health is leading to devastating effects in terms of morbidity, mortality, and economic impact [1] . In response to this emergency, the development of vaccines has been rapidly implemented and thus far at least ten candidates have reached phase 3 clinical trials and about forty are under phase I/II evaluations [2, 3] . Most of the candidates target the spike protein (S), which is a critical target to achieve virus neutralization through antibodies by preventing its entry into the host cell [4, 5] . The protein S has also been associated with the induction of cellular responses of relevance for clearance of infected cells [6] . Most of the vaccine candidates are formulated with the full-length S protein and based on viral vectors or inactivated viruses. Such formulations, although highly immunogenic, could be associated to side effects such as high reactogenicity and the induction of suboptimal, if not unsafe, immune responses [2, 4, 5, 7] The rational design of vaccines should be rapidly implemented in the case of SARS-CoV-2 to produce the next generation vaccines as an alternative path for the induction of optimal immune responses. The possible critical points in this regard are the induction of robust neutralizing humoral responses that avoid the induction of antibodies that act as antibody-dependent enhancement inducers [8, 9] . An approach to achieve such goals relies in the use of epitope-based vaccines formulated with synthetic peptides, which allow focusing the response on the particular epitopes that induce neutralizing antibodies [10] . Therefore, epitope-based vaccines are proposed as fine vaccine formulations capable of achieving the optimal immune responses [4, 9] . However, the use of such simplified antigenic formulations compared to whole virus-based vaccines imposes the challenge of antigen carriers have been described. For instance, it is known that AuNP are efficiently captured by dendritic cells and this process is improved if they are covered with polyethylene glycol at a reasonable graft density [14] ; cellular internalization occurs is mediated by given by phagocytosis, macropinocytosis and receptor-mediated endocytosis in dendritic cells and is largely dependent on the physicochemical properties of AuNP, determined by the size and surface modification of the particles [15, 16] . Once internalized, the peptides are loaded onto MHC molecules and expressed on the cell surface to be presented to lymphocytes [17] .

It is known that AuNP-antigen conjugates increase the antigen presentation process [18] by promoting effective maturation of dendritic cells, proliferation of Th and NK cells and increased secretion of cytokines IL-4, IFNg, IL-12 and IL-10 is achieved [16, 19] .

Interestingly, a recent review has covered the outlook on the application of nanomaterials for the development of vaccines against SARSCoV-2, highlighting the potential of some previously reported vaccines against other viruses, based in metallic particles, including AuNP [20] .In fact, previous efforts have been reported on the development of AuNPbased vaccine against coronavirus, but optimization in the formulation is required given the limited neutralization potential and some concerns derived from the use of full-length S protein, which might induced an suboptimal humoral response [21] . Thus far the preclinical evaluation of several AuNP-based vaccines has been reported with promising findings [20] [21] [22] [23] . Therefore, AuNP-based vaccines augur a promising path for the development of rationally designed subunit vaccines. Herein, a peptide derived from the SARS-CoV-2 S protein sequence was successfully coupled to AuNP and the bioconjugate obtained was characterized and used to assess its immunogenic potential in mice. obtained by chemical synthesis. In addition, a cysteine was included at the N-terminus of the peptide to facilitate its eventual attachment to carriers via the thiol group. The final sequence of the S 461-493 peptide was: CLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQ, synthesized by Synpeptide Inc., China.

The S 461-493 peptide was conjugated to the keyhole limpet hemocyanin (KLH; Geno Technology Inc., St. Louis, MO) carrier using ethyl-3- [3-dimethylaminopropyl] carbodiimide (EDC). The reaction was performed according to the instructions from the manufacturer (Thermo Fisher Scientific, Waltham, MA) with some modifications comprising 500 µg of peptide and 1 mg of KLH, which reacted with EDC previously dissolved in DMSO. The reaction conditions and purification of the conjugate were performed according to a previous report [24] .

The protocols involving test animals were performed according to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH, Bethesda, MD) and protocols approved by the Institutional Research Ethics Committee (CEID-2020-07R1). Blood samples were obtained by jugular puncture, the recovered sera were stored at −40 °C until further use.

The levels of anti-S 461-493 antibodies were determined from the sheep sera samples by ELISA, which comprised coating, overnight at 4 °C, ninety-six-well polystyrene plates with conjugated anti- For the synthesis of AuNP, all glassware was cleaned with aqua regia and rinsed thoroughly with deionized water [25] . Stock solutions of 1 % HAuCl 4 and 1 % Na 3 C 6 H 5 O 7

were prepared with Milli-Q water one day before their use. A reaction mixture containing 0.25 % HAuCl 4 , 0.25 % Na 3 C 6 H 5 O 7 , and water was prepared by adding each component in sequence and under mixing. This mixture was incubated for 5 min and added rapidly to a flask with water already boiling for a final concentration of 0.01 % HAuCl 4 and 0.01 % Na 3 C 6 H 5 O 7 . Reflux occurred for 30 min. The as-prepared AuNP were stored at 4 °C.

For the functionalization of AuNP, the amount of HS-PEG-NH 2 was calculated considering a grafting density of one molecule covering 2 nm 2 [26] . Per 1 mL of as-prepared AuNP, 5 μL For the conjugation of AuNP-PEG-NH 2 , the same grafting density as described above was considered. First, glutaraldehyde (GTA) was added 100 times in excess, the solution mixed AuNP and 30 % glycerol deposited in a 0.75 % agarose gel [27] . The gel was prepared with Tris-acetate-EDTA (TAE) 1× buffer, casted and immersed horizontally in a chamber with TA 1× buffer at pH 7, and run for 30 min at 100 V.

The size and morphology of the AuNP before and after conjugation was determined using a JEM-JEOL-2100 Transmission Electron Microscope at 200 kV. The AuNP suspension was diluted until the first dilution became colorless. Afterwards, 5 µL of sample were deposited on a nickel formvar/carbon coated grid (Ted Pella Inc) for 1 min, washed with Milli-Q water, and left to dry for 24 h in a desiccator. The conjugated AuNP were analyzed using the aforementioned method and also by negatively staining them with 1 % uranyl acetate. The size distribution was calculated using at least 100 measurements of the average diameter. The diameter of each particle was estimated from the geometric mean of two orthogonal measurements.

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The cytotoxicity of the different conjugates was evaluated using trypan blue staining and the resazurin assay. HEK-293T cells were grown in DMEM (Corning Inc., Corning, NY) supplemented with ampicillin/streptomycin (Thermo Fisher Scientific), and 10 % heatinactivated fetal bovine serum (Gibco®) at 37 C and 5 % CO 2 using T75 flasks (Corning) until reaching confluence. One day before the cytotoxicity evaluation, 1 × 10 5 cells were seeded by triplicate in a 12-well culture plate. Each experiment was done with two different cell passages. As controls each plate had cells treated with the vehicle alone or H 2 O 2 (40 mM). The cells were subsequently exposed to different concentrations of AuNP In parallel, the resazurin-based cell viability was estimated. For this purpose, the cells treated during 48 h with the respective AuNP concentrations were exposed to 30 µg/mL resazurin for 3 h and fluorescence (560 nm/590 nm) was recorded in a FlexStation II scanning fluorimeter (Molecular Devices LLC, San Jose, CA).

Test mice (BALB/c strain, 8-10 weeks old) groups were established randomly (n = 5). The Statistical analysis was performed with Statistica® 12.7 (TIBCO Software Inc., Palo Alto, CA).

Two test mice treated with the AuNP-S 461-493 vaccine were sacrificed and the isolation of splenocyte was performed under standard methods. The spleen was isolated and placed into a cell strainer fitted on a 50 mL tube. The organ was pressed with a 1 mL syringe plunger while adding and 5 ml of culture medium (RPMI/10 %FBS/1 % pen-strep) to release the cells. The cells suspension was spun at 1500 rpm for 5 min and resuspended in RPMI/10 % FBS/1 % pen-strep. Cell viability was estimated with the trypan blue method and if this parameter was above 95 % the cells were used to set the stimulation protocol. into AuNP-PEG-GTA, we also tested one-and two-step conditions in PB and PBS 1× [32] .

For the functionalization of AuNP with HS-PEG-NH 2 and their activation with GTA, 0.01 % Tween 20 was useful, as its exclusion in either step resulted in losing particles as they adhered on the container. GTA in solution produces cyclic, dimeric, and/or polymeric reactive species that will crosslink all the reactants available [33] . 

Test mice groups were subjected to a sub-cutaneous (s.c.) immunization scheme to explore the capability of the AuNP carriers to enhance the immunogenicity of the S 461-493

peptide Herein a prototype of a nanovaccine based on AuNP was generated as an effort to expand the possibilities for the development of rationally designed vaccines against SARS-CoV-2. It is well-known that nanosized materials, organic or inorganic, can lead to improved immunogenicity of antigens, which is especially important when the vaccine is formulated with peptides that are typically poor immunogens. In particular, AuNP are considered biocompatible, noncytotoxic, and nonimmunogenic entities, which make them highly attractive for biomedical applications that include vaccine development [37] . AuNP can act as carriers that exert immunodulator effects [38] [39] [40] .

We focused in the S 461-493 sequence; whose immunogenicity was first validated in sheep given the lack of experimental validation of protective epitopes from the S protein. the allergic inflammatory responses [20] . Therefore, even though AuNP are carriers that enhance the immune response, further approaches must direct the response to the immune relevant epitopes. Among the approaches to achieve this goal, the use of synthetic peptides covering those key epitopes is attractive to achieve robust, protective, and safe immune responses [20] . The perspectives for this study contemplate the evaluation of the neutralizing potential of the induced immune response, expanding the number of target epitopes included in the formulation, and characterizing the mucosal immune response induced.

As long as this pandemic advances, the development of new immunization approaches against SARS-CoV-2 should be continued to favor the development of the next generation vaccines that could sum to the vaccines that are expected to get approved in the midterm,

increasing the potential to address the remarkable challenge of achieving global vaccination against this highly relevant emerging pathogen. In conclusion, this study opens the path for the formulation of epitope-based vaccines against SARS-CoV-2, which will be of high relevance in the development of the next generation vaccines against this and other emerging pathogens. cell viability. In contrast, the addition of H 2 O 2 resulted in a cell viability of less than 10 %.

The asterisk indicates statistically significant differences versus the control. 

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This work was supported by Consejo Nacional de Ciencia y Tecnología (CONACyT, grant 311879 to SRM, and grant A1-S-31287 to GP), Consejo Potosino de Ciencia y Tecnología (COPOCyT, grant FIIS-FS04- 19) , and Programa para el Desarrollo Profesional Docente