Preclinical Immune Response and Safety Evaluation of the Protein Subunit Vaccine Nanocovax for COVID-19

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global health concern. The development of vaccines with high immunogenicity and safety is crucial for controlling the global COVID-19 pandemic and preventing further illness and fatalities. Here, we report the development of a SARS-CoV-2 vaccine candidate, Nanocovax, based on recombinant protein production of the extracellular (soluble) portion of the spike (S) protein of SARS-CoV-2. The results showed that Nanocovax induced high levels of S protein-specific IgG and neutralizing antibodies in three animal models: BALB/c mouse, Syrian hamster, and a non-human primate (Macaca leonina). In addition, a viral challenge study using the hamster model showed that Nanocovax protected the upper respiratory tract from SARS-CoV-2 infection. Nanocovax did not induce any adverse effects in mice (Mus musculus var. albino) and rats (Rattus norvegicus). These preclinical results indicate that Nanocovax is safe and effective.

• Aside from a truncated N-Terminus and a heterogeneous C-terminus, the complete sequence was verified for the SARS-CoV-2 spike protein.
• The mass spectrometric data suggests a truncation of N-terminal serine (compared to the theoretical N-terminus of the SARS-CoV-2 spike protein without the signal sequence as published for UnitProtKB entry P0DTC2) and complete pyroglutamate formation of the N-terminal glutamine.
• The C-terminus shows a high heterogeneity with a C-terminal peptide with truncation of AA 1215-1222 as the most abundant variant. The following sequence was used for data analysis:

N-Deglycosylation
The samples were N-deglycosylated with PNGase F at enzyme specific conditions.

Reduction and alkylation
The samples were reduced and denatured with DTT in the presence of urea or GuHCl.
The samples were then alkylated with IAA.

Enzymatic digestion
The samples were enzymatically digested with the following enzymes at enzyme specific conditions to achieve a high sequence coverage: • Trypsin • Chymotrypsin • AspN

Sample measurement with LC-ESI-MS
The samples were acidified in approx. 0.5% TFA and separated on a HPLC-system (Agilent 1100) using a reversed phase column (AdvanceBio Peptide Map 2.1 x 100 mm, 2.7 µm, Agilent). Eluents were 0.1% FA in water and 0.1% FA in acetonitrile. The mass spectrometric analysis was performed with a Compact QTOF mass spectrometer (Bruker Daltonik).

Data analysis
The recorded LC-ESI-MS and -MS/MS spectra were processed, annotated and searched against a customized sequence database using Mascot (Matrix Science).
The following modifications were taken into consideration: • Carbamidomethyl (C) • Deamidation (NQ) • Oxidation (M) • N-terminal pyroglutamate formation (Q) Modified peptides were identified by their exact mass and retention time and quantified by their mass spectrometric signal intensity.

Sequence verification
The LC-ESI-MS measurements were performed successfully. An exemplary fragment mass spectrum of the peptide SFIEDLLFNKVTLADAGFIK (AA 816-835) is shown in Figure 1.

Figure 1: Exemplary fragment mass spectrum of peptide AGGVLVASHLQSFLEVSYR (AA 816-835) of sample recombinant SARS-Cov-2-Spike Protein (PN006-2021-001). Annotated are b-and y-ion series of the fragmentation pattern.
Aside from a truncated N-Terminus and a heterogeneous C-terminus, complete sequence coverage was achieved for the SARS-CoV-2 spike protein. The following modifications were observed: • N-terminal pyroglutamate

• Several oxidations and deamidations
The full sequence data obtained with LC-ESI-MS measurements is shown in Figure 2.
The sequence was matched to the sequence of the SARS-CoV-2 spike protein. The sequence region AA 708-718 (SNNSIAIPTNF) was verified manually (data not shown).

N-and C-terminal modifications
The  Table 1. The peptide masses used for analysis of N-and C-terminal modifications are summarized in Table 2 in the appendix.

Summary
Aim of the project was the peptide mapping of a recombinant SARS-CoV-2 spike protein by LC-ESI-MS using different digestion strategies. Focus of the peptide mapping was: • Sequence verification • Analysis of N-and C-terminal modifications The LC-ESI-MS measurements were performed successfully. The following observations were made: • Aside from a truncated N-Terminus and a heterogeneous C-terminus, the complete sequence was verified for the SARS-CoV-2 spike protein.
• The mass spectrometric data suggests a truncation of N-terminal serine (compared to the theoretical N-terminus of the SARS-CoV-2 spike protein without the signal sequence as published for UnitProtKB entry P0DTC2) and complete pyroglutamate formation of the N-terminal glutamine.
• The C-terminus shows a high heterogeneity with a C-terminal peptide with truncation of AA 1215-1222 as the most abundant variant.