Table1_Synthesis and evaluation of gold nanoparticles conjugated with five antigenic peptides derived from the spike protein of SARS-CoV-2 for vaccine development.pdf

Introduction: The development of innovative anti-COVID-19 vaccines is a need to ensure the population’s immunity worldwide, with broad protection against variants of concern and low cost as the main goals. Gold nanocarriers are potential entities that could aid in the development of innovative vaccines having thermal stability, high immunogenicity, and safety as the main attributes. Moreover, this approach could lead to adjuvant-free formulations, which will reduce the costs of vaccines.Methods: In this study, five peptides (P1, P2, P3, P4, and P5) corresponding to linear epitopes of the SARS-CoV-2 spike (S) protein were chemisorbed on gold nanoparticles (AuNP) of 20 nm, prefunctionalized with heterobifunctional polyethylene glycol, by using glutaraldehyde as crosslinker to generate nanovaccine prototypes.Results and discussion: The surface modification was confirmed by DLS with an increase of 31.7 ± 1.8 nm in the hydrodynamic diameter and an average ζ potential of −8.3 ± 2.2 mV in PBS (as excipient). The coupling concentration achieved was 23.7 ± 7.1 μg of peptide per mg AuNP. These AuNP-based conjugates showed no inherent toxicity in assays performed with HEK293T cells, in which a 100–1,000 μg/mL concentration range only led to a temporary decrease of up to 30% in cell viability after 48 h of treatment with restoration by 72 h. The immunogenicity of the conjugates produced was assessed in test mice subjected to three subcutaneous doses at 2-week intervals. Significant levels of IgM against each target peptide were observed at an early stage of the immunization scheme in all groups, reaching maximum levels after the second dose, whereas the IgG response increased after the third dose. The AuNP-P2, AuNP-P3, and AuNP-P5 conjugates induced the highest levels of IgG antibodies, lasting for at least 2 months after the last boost, with a predominance of the IgG1 subclass. Although the magnitude of the response induced by the gold conjugates was comparable to that with alum as adjuvant, these nanoconjugates induced a longer response. Our data support the use of AuNP as carriers in innovative vaccines against SARS-CoV-2.</p

Immunolocalization of the S3Pvac peptides in <i>E. granulosus</i> and <i>E. multilocularis</i>.

<p>Sections of larval specimens reveal a strong binding to the tegument (<b>T</b>) as well as in the parenchyma (<b>P</b>) of <i>E. multilocularis</i> and in tegument of <i>E. granulosus</i>. (<b>A</b>) Control labeled with rabbit pre immune serum. (<b>B</b>) Positive control labeled with polyclonal sera from rabbit immunized with a total extract of <i>Taenia crassiceps</i> ORF cysticerci.</p

Multiple sequence alignment GK1 peptides.

<p>The DNA and amino acid sequences of GK1 in several species of the Taeniidae family were aligned. GK1 alignments of amino acids (A) and cDNA (B) are shown in the upper part of the figure; the region encoding the actual peptides is marked with a double-headed arrow on the top of the alignment. On every alignment, boxes corresponding to the regions subjected to dN/dS-ratio analysis are marked accordingly. Models of the 3D structure of the GK1 peptide are shown in panel C. The model in red corresponds to <i>T. solium</i>, <i>T. saginata</i> and <i>E. granulosus</i>' GK1 and in purple to <i>T. crassiceps</i> ORF and WFU and <i>E. multilocularis</i>. A very good fitting between the two structures is shown as expected from the strong similarity of the amino acid sequence.</p

Sequences used to model the 3D structure of KETc1, GK1 and KETc12.

1<p>GK1 and KETc1 3D models include more than just the peptide sequence because the HMMSTR-server requires sequences of at least 20 amino acids length. Upper case marks the sequences of peptides while lower caps mark the preceding sequence to them.</p>2<p>There is no “complete” sequence for any of the strains of <i>T. crassiceps.</i> However, considering the strong similarity between the <i>Taenia</i> species we decided to construct a hypothetical sequence for each strain assuming that the preceding regions to KETc1 in <i>T. crassiceps</i> spp. are identical to those preceding the peptide in the other <i>Taenia</i> species.</p

Immunolocalization of the S3Pvac peptides in larval specimens of the four Taeniidae families.

<p>Metacestode cryostat parasite sections were incubated with a rabbit pre-immune serum (A) or with the three different rabbit antibodies (anti-GK1, anti-KETc1, anti-KETc12) and developed with biotinylated goat anti-rabbit IgG plus streptavidin peroxidase conjugate and counterstained with hematoxilin. The arrows indicate the areas in the metacestode where the peptides were localized. 10X (A and C), 40X (B and D). Tegument (<b>T</b>), parenchyma (<b>P</b>), tegument of the spiral canal (<b>TSC</b>), parenchymal folds (<b>PF</b>), microthrix (<b>MT</b>), parenchyma (<b>P</b>). The three peptides are present in metacestode stage of four cestodes analyzed.</p

Primers used for PCR amplification of the KETc1 and GK-1 (KETc7) homologous sequences in <i>T. crassiceps</i> ORF, <i>T. crassiceps</i> WFU, <i>T. solium</i>, <i>T. saginata</i>, <i>E. multilocularis</i>, and <i>E. granulosus.</i>

<p>Primers used for PCR amplification of the KETc1 and GK-1 (KETc7) homologous sequences in <i>T. crassiceps</i> ORF, <i>T. crassiceps</i> WFU, <i>T. solium</i>, <i>T. saginata</i>, <i>E. multilocularis</i>, and <i>E. granulosus.</i></p

S3Pvac peptides were detected in the tegument of <i>Taenia</i> tapeworms and <i>T. solium</i> eggs.

<p>No reaction was detected in control sections, for which naive rabbit serum was used in place of the specific polyclonal rabbit serum. <b>S.</b>- Sucker; <b>Sc.</b> Scolex, <b>MP.</b>- Medullar parenchyma, <b>N.</b>- Neck; <b>DC.</b>- Distal cytoplasm region; <b>PE.</b>- perinuclear cytoplasm region. <b>O.</b>- oncospheres.</p

Multiple sequence alignment of KETc1 peptides.

<p>The DNA and amino acid sequences of KETc1 of several species of the Taeniidae family were aligned. KETc1 alignments of amino acids (A) and cDNA (B) are shown in the upper part of the figure. On every alignment, boxes corresponding to the regions subjected to dN/dS-ratio analysis are marked accordingly. Panel C and D shows the 3D models of the complete and truncated version of KETc1. Panel D depicts the models corresponding to <i>T. solium</i> (red), <i>T. crassiceps</i> ORF and WFU (pink), <i>T. saginata</i> (light green) and <i>Echinococcus</i> group (dark green). Structures are aligned with very good general agreement. Panel D shows the alignment of the truncated KETc1 peptide where the yellow model corresponds to <i>T. solium</i> and <i>T. saginata</i>; blue corresponds to <i>T. crassiceps</i> ORF and WFU and brown to the <i>Echinococcus</i> group.</p