Predicting the Assembly of the Transmembrane Domains of Viral Channel Forming Proteins and Peptide Drug Screening Using a Docking Approach.

A de novo assembly algorithm is provided to propose the assembly of bitopic transmembrane domains (TMDs) of membrane proteins. The algorithm is probed using, in particular, viral channel forming proteins (VCPs) such as M2 of influenza A virus, E protein of severe acute respiratory syndrome corona virus (SARS-CoV), 6K of Chikungunya virus (CHIKV), SH of human respiratory syncytial virus (hRSV), and Vpu of human immunodeficiency virus type 2 (HIV-2). The generation of the structures is based on screening a 7-dimensional space. Assembly of the TMDs can be achieved either by simultaneously docking the individual TMDs or via a sequential docking. Scoring based on estimated binding energies (EBEs) of the oligomeric structures is obtained by the tilt to decipher the handedness of the bundles. The bundles match especially well for all-atom models of M2 referring to an experimentally reported tetrameric bundle. Docking of helical poly-peptides to experimental structures of M2 and E protein identifies improving EBEs for positively charged (K,R,H) and aromatic amino acids (F,Y,W). Data are improved when using polypeptides for which the coordinates of the amino acids are adapted to the Cα coordinates of the respective experimentally derived structures of the TMDs of the target proteins.

In silico structural inhibition of ACE-2 binding site of SARS-CoV-2 and SARS-CoV-2 omicron spike protein by lectin antiviral dyad system to treat COVID-19.

Spike glycoprotein of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) binds angiotensin-converting enzyme-2 (ACE-2) receptors via its receptor-binding domain (RBD) and mediates virus-to-host cell fusion. Recently emerged omicron variant of SARS-CoV-2 possesses around 30 mutations in spike protein where N501Y tremendously increases viral infectivity and transmission. Lectins interact with glycoproteins and mediate innate immunity displaying antiviral, antibacterial, and anticarcinogenic properties. In this study, we analyzed the potential of lectin, and lectin-antibody (spike-specific) complex to inhibit the ACE-2 binding site of wild and N501Y mutated spike protein by utilizing in silico molecular docking and simulation approach. Docking of lectin at reported ACE-2 binding spike-RBD residues displayed the ZDock scores of 1907 for wild and 1750 for N501Y mutated spike-RBD. Binding of lectin with antibody to form proposed dyad complex gave ZDock score of 1174 revealing stable binding. Docking of dyad complex with wild and N501Y mutated spike-RBD, at lectin and antibody individually, showed high efficiency binding hence, effective structural inhibition of spike-RBD. MD simulation of 100 ns of each complex proved high stability of complexes with RMSD values ranging from 0.2 to 1.5 nm. Consistent interactions of lead ACE-2 binding spike residues with lectin during simulation disclosed efficient structural inhibition by lectin against formation of spike RBD-ACE-2 complex. Hence, lectins along with their ability to induce innate immunity against spike glycoprotein can structurally inhibit the spike-RBD when given as lectin-antibody dyad system and thus can be developed into a dual effect treatment against COVID-19. Moreover, the high binding specificity of this system with spike-RBD can be exploited for development of diagnostic and drug-delivery systems.

Crystal structure of the Rubella virus protease reveals a unique papain-like protease fold.

Rubella, a viral disease characterized by a red skin rash, is well controlled because of an effective vaccine, but outbreaks are still occurring in the absence of available antiviral treatments. The Rubella virus (RUBV) papain-like protease (RubPro) is crucial for RUBV replication, cleaving the nonstructural polyprotein p200 into two multifunctional proteins, p150 and p90. This protease could represent a potential drug target, but structural and mechanistic details important for the inhibition of this enzyme are unclear. Here, we report a novel crystal structure of RubPro at a resolution of 1.64 Å. The RubPro adopts a unique papain-like protease fold, with a similar catalytic core to that of proteases from Severe acute respiratory syndrome coronavirus 2 and foot-and-mouth disease virus while having a distinctive N-terminal fingers domain. RubPro has well-conserved sequence motifs that are also found in its newly discovered Rubivirus relatives. In addition, we show that the RubPro construct has protease activity in trans against a construct of RUBV protease-helicase and fluorogenic peptides. A protease-helicase construct, exogenously expressed in Escherichia coli, was also cleaved at the p150-p90 cleavage junction, demonstrating protease activity of the protease-helicase protein. We also demonstrate that RubPro possesses deubiquitylation activity, suggesting a potential role of RubPro in modulating the host's innate immune responses. We anticipate that these structural and functional insights of RubPro will advance our current understanding of its function and help facilitate more structure-based research into the RUBV replication machinery, in hopes of developing antiviral therapeutics against RUBV.

High level SARS-CoV-2 nucleocapsid refolding using mild condition for inclusion bodies solubilization: Application of high pressure at pH 9.0.

SARS-CoV-2 Nucleocapsid (N) is the most abundant viral protein expressed in host samples and is an important antigen for diagnosis. N is a 45 kDa protein that does not present disulfide bonds. Intending to avoid non-specific binding of SARS-CoV-2 N to antibodies from patients who previously had different coronaviruses, a 35 kDa fragment of N was expressed without a conserved motif in E. coli as inclusion bodies (N122-419-IB). Culture media and IB washing conditions were chosen to obtain N122-419-IB with high yield (370 mg/L bacterial culture) and protein purity (90%). High pressure solubilizes protein aggregates by weakening hydrophobic and ionic interactions and alkaline pH promotes solubilization by electrostatic repulsion. The association of pH 9.0 and 2.4 kbar promoted efficient solubilization of N122-419-IB without loss of native-like tertiary structure that N presents in IB. N122-419 was refolded with a yield of 85% (326 mg/L culture) and 95% purity. The refolding process takes only 2 hours and the protein is ready for use after pH adjustment, avoiding the necessity of dialysis or purification. Antibody binding of COVID-19-positive patients sera to N122-419 was confirmed by Western blotting. ELISA using N122-419 is effective in distinguishing between sera presenting antibodies against SARS-CoV-2 from those who do not. To the best of our knowledge, the proposed condition for IB solubilization is one of the mildest described. It is possible that the refolding process can be extended to a wide range of proteins with high yields and purity, even those that are sensible to very alkaline pH.

The influence of new SARS-CoV-2 variant Omicron (B.1.1.529) on vaccine efficacy, its correlation to Delta variants: A computational approach.

The newly discovered COVID variant B.1.1.529 in Botswana has more than 30 mutations in spike and many other in non-spike proteins, far more than any other SARS-CoV-2 variant accepted as a variant of concern by the WHO and officially named Omicron, and has sparked concern among scientists and the general public. Our findings provide insights into structural modification caused by the mutations in the Omicrons receptor-binding domain and look into the effects on interaction with the hosts neutralizing antibodies CR3022, B38, CB6, P2B-2F6, and REGN, as well as ACE2R using an in silico approach. Computational analysis revealed that the Omicron variant has a higher binding affinity for the human ACE2 receptor than the wild and Delta (AY.1 and AY.2 strains), but lower than the Delta AY.3 strain. MD simulation and docking analysis suggest that the omicron and Delta AY.3 were found to have relatively unstable RBD structures and hampered interactions with antibodies more than wild and Delta (AY.1 and AY.2), which may lead to relatively more pathogenicity and antibody escape. In addition, we observed lower binding affinity of Omicron for human monoclonal antibodies (CR3022, B38, CB6, and P2B2F6) when compared to wild and Delta (AY.1 & AY.2). However, the binding affinity of Omicron RBD variants for CR3022, B38, and P2B2F6 antibodies is lower as compared to Delta AY.3, which might promote immune evasion and reinfection and needs further experimental investigation.

Structural Basis for Human Receptor Recognition by SARS-CoV-2 Omicron Variant BA.1.

The highly contagious and fast-spreading omicron variant of SARS-CoV-2 infects the respiratory tracts efficiently. The receptor-binding domain (RBD) of the omicron spike protein recognizes human angiotensin-converting enzyme 2 (ACE2) as its receptor and plays a critical role in the tissue tropism of SARS-CoV-2. Here, we showed that the omicron RBD (strain BA.1) binds to ACE2 more strongly than does the prototypic RBD from the original Wuhan strain. We also measured how individual omicron mutations affect ACE2 binding. We further determined the crystal structure of the omicron RBD (engineered to facilitate crystallization) complexed with ACE2 at 2.6 Å. The structure shows that omicron mutations caused significant structural rearrangements of two mutational hot spots at the RBD/ACE2 interface, elucidating how each omicron mutation affects ACE2 binding. The enhanced ACE2 binding by the omicron RBD may facilitate the omicron variant's infection of the respiratory tracts where ACE2 expression level is low. Our study provides insights into the receptor recognition and tissue tropism of the omicron variant. IMPORTANCE Despite the scarcity of the SARS-CoV-2 receptor-human angiotensin-converting enzyme 2 (ACE2)-in the respiratory tract, the omicron variant efficiently infects the respiratory tract, causing rapid and widespread infections of COVID-19. The omicron variant contains extensive mutations in the receptor-binding domain (RBD) of its spike protein that recognizes human ACE2. Here, using a combination of biochemical and X-ray crystallographic approaches, we showed that the omicron RBD binds to ACE2 with enhanced affinity and also elucidated the role of each of the omicron mutations in ACE2 binding. The enhanced ACE2 binding by the omicron RBD may contribute to the omicron variant's new viral tropism in the respiratory tract despite the low level of ACE2 expression in the tissue. These findings help us to understand tissue tropism of the omicron variant and shed light on the molecular evolution of SARS-CoV-2.

Full-genome sequences of the first two SARS-CoV-2 viruses from India.

Background & objectives: Since December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has globally affected 195 countries. In India, suspected cases were screened for SARS-CoV-2 as per the advisory of the Ministry of Health and Family Welfare. The objective of this study was to characterize SARS-CoV-2 sequences from three identified positive cases as on February 29, 2020. Methods: Throat swab/nasal swab specimens for a total of 881 suspected cases were screened by E gene and confirmed by RdRp (1), RdRp (2) and N gene real-time reverse transcription-polymerase chain reactions and next-generation sequencing. Phylogenetic analysis, molecular characterization and prediction of B- and T-cell epitopes for Indian SARS-CoV-2 sequences were undertaken. Results: Three cases with a travel history from Wuhan, China, were confirmed positive for SARS-CoV-2. Almost complete (29,851 nucleotides) genomes of case 1, case 3 and a fragmented genome for case 2 were obtained. The sequences of Indian SARS-CoV-2 though not identical showed high (~99.98%) identity with Wuhan seafood market pneumonia virus (accession number: NC 045512). Phylogenetic analysis showed that the Indian sequences belonged to different clusters. Predicted linear B-cell epitopes were found to be concentrated in the S1 domain of spike protein, and a conformational epitope was identified in the receptor-binding domain. The predicted T-cell epitopes showed broad human leucocyte antigen allele coverage of A and B supertypes predominant in the Indian population. Interpretation & conclusions: The two SARS-CoV-2 sequences obtained from India represent two different introductions into the country. The genetic heterogeneity is as noted globally. The identified B- and T-cell epitopes may be considered suitable for future experiments towards the design of vaccines and diagnostics. Continuous monitoring and analysis of the sequences of new cases from India and the other affected countries would be vital to understand the genetic evolution and rates of substitution of the SARS-CoV-2.

N-glycosylation profiles of the SARS-CoV-2 spike D614G mutant and its ancestral protein characterized by advanced mass spectrometry.

N-glycosylation plays an important role in the structure and function of membrane and secreted proteins. The spike protein on the surface of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, is heavily glycosylated and the major target for developing vaccines, therapeutic drugs and diagnostic tests. The first major SARS-CoV-2 variant carries a D614G substitution in the spike (S-D614G) that has been associated with altered conformation, enhanced ACE2 binding, and increased infectivity and transmission. In this report, we used mass spectrometry techniques to characterize and compare the N-glycosylation of the wild type (S-614D) or variant (S-614G) SARS-CoV-2 spike glycoproteins prepared under identical conditions. The data showed that half of the N-glycosylation sequons changed their distribution of glycans in the S-614G variant. The S-614G variant showed a decrease in the relative abundance of complex-type glycans (up to 45%) and an increase in oligomannose glycans (up to 33%) on all altered sequons. These changes led to a reduction in the overall complexity of the total N-glycosylation profile. All the glycosylation sites with altered patterns were in the spike head while the glycosylation of three sites in the stalk remained unchanged between S-614G and S-614D proteins.

Omicron SARS-CoV-2 variant: Unique features and their impact on pre-existing antibodies.

Severe Acute Respiratory Coronavirus (SARS-CoV-2) has been emerging in the form of different variants since its first emergence in early December 2019. A new Variant of Concern (VOC) named the Omicron variant (B.1.1.529) was reported recently. This variant has a large number of mutations in the S protein. To date, there exists a limited information on the Omicron variant. Here we present the analyses of mutation distribution, the evolutionary relationship of Omicron with previous variants, and probable structural impact of mutations on antibody binding. Our analyses show the presence of 46 high prevalence mutations specific to Omicron. Twenty-three of these are localized within the spike (S) protein and the rest localized to the other 3 structural proteins of the virus, the envelope (E), membrane (M), and nucleocapsid (N). Phylogenetic analysis showed that the Omicron is closely related to the Gamma (P.1) variant. The structural analyses showed that several mutations are localized to the region of the S protein that is the major target of antibodies, suggesting that the mutations in the Omicron variant may affect the binding affinities of antibodies to the S protein.

SARS-CoV-2 spike protein displays sequence similarities with paramyxovirus surface proteins; a bioinformatics study.

Recent emergence of SARS-CoV-2 and associated COVID-19 pandemic have posed a great challenge for the scientific community. In this study, we performed bioinformatic analyses on SARS-CoV-2 protein sequences, trying to unravel potential molecular similarities between this newly emerged pathogen with non-coronavirus ssRNA viruses. Comparing the proteins of SARS-CoV-2 with non-coronavirus positive and negative strand ssRNA viruses revealed multiple sequence similarities between SARS-CoV-2 and non-coronaviruses, including similarities between RNA-dependent RNA-polymerases and helicases (two highly-conserved proteins). We also observed similarities between SARS-CoV-2 surface (i.e. spike) protein with paramyxovirus fusion proteins. This similarity was restricted to a segment of spike protein S2 subunit which is involved in cell fusion. We next analyzed spike proteins from SARS-CoV-2 "variants of concern" (VOCs) and "variants of interests" (VOIs) and found that some of these variants show considerably higher spike-fusion similarity with paramyxoviruses. The 'spike-fusion' similarity was also observed for some pathogenic coronaviruses other than SARS-CoV-2. Epitope analysis using experimentally verified data deposited in Immune Epitope Database (IEDB) revealed that several B cell epitopes as well as T cell and MHC binding epitopes map within the spike-fusion similarity region. These data indicate that there might be a degree of convergent evolution between SARS-CoV-2 and paramyxovirus surface proteins which could be of pathogenic and immunological importance.

A monoclonal antibody that neutralizes SARS-CoV-2 variants, SARS-CoV, and other sarbecoviruses.

The repeated emergence of highly pathogenic human coronaviruses as well as their evolving variants highlight the need to develop potent and broad-spectrum antiviral therapeutics and vaccines. By screening monoclonal antibodies (mAbs) isolated from COVID-19-convalescent patients, we found one mAb, 2-36, with cross-neutralizing activity against SARS-CoV. We solved the cryo-EM structure of 2-36 in complex with SARS-CoV-2 or SARS-CoV spike, revealing a highly conserved epitope in the receptor-binding domain (RBD). Antibody 2-36 neutralized not only all current circulating SARS-CoV-2 variants and SARS-COV, but also a panel of bat and pangolin sarbecoviruses that can use human angiotensin-converting enzyme 2 (ACE2) as a receptor. We selected 2-36-escape viruses in vitro and confirmed that K378 T in SARS-CoV-2 RBD led to viral resistance. Taken together, 2-36 represents a strategic reserve drug candidate for the prevention and treatment of possible diseases caused by pre-emergent SARS-related coronaviruses. Its epitope defines a promising target for the development of a pan-sarbecovirus vaccine.

SARS-CoV-2 B.1.617 Indian variants: Are electrostatic potential changes responsible for a higher transmission rate?

Lineage B.1.617+, also known as G/452R.V3 and now denoted by WHO with the Greek letters δ and κ, is a recently described SARS-CoV-2 variant under investigation first identified in October 2020 in India. As of May 2021, three sublineages labeled as B.1.617.1 (κ), B.1.617.2 (δ), and B.1.617.3 have been already identified, and their potential impact on the current pandemic is being studied. This variant has 13 amino acid changes, three in its spike protein, which are currently of particular concern: E484Q, L452R, and P681R. Here, we report a major effect of the mutations characterizing this lineage, represented by a marked alteration of the surface electrostatic potential (EP) of the receptor-binding domain (RBD) of the spike protein. Enhanced RBD-EP is particularly noticeable in the B.1.617.2 (δ) sublineage, which shows multiple replacements of neutral or negatively charged amino acids with positively charged amino acids. We here hypothesize that this EP change can favor the interaction between the B.1.617+ RBD and the negatively charged ACE2, thus conferring a potential increase in the virus transmission.

Novel nucleocapsid protein-targeting phenanthridine inhibitors of SARS-CoV-2.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is unprecedented in human history. As a major structural protein, nucleocapsid protein (NPro) is critical to the replication of SARS-CoV-2. In this work, 17 NPro-targeting phenanthridine derivatives were rationally designed and synthesized, based on the crystal structure of NPro. Most of these compounds can interact with SARS-CoV-2 NPro tightly and inhibit the replication of SARS-CoV-2 in vitro. Compounds 12 and 16 exhibited the most potent anti-viral activities with 50% effective concentration values of 3.69 and 2.18 µM, respectively. Furthermore, site-directed mutagenesis of NPro and Surface Plasmon Resonance (SPR) assays revealed that 12 and 16 target N-terminal domain (NTD) of NPro by binding to Tyr109. This work found two potent anti-SARS-CoV-2 bioactive compounds and also indicated that SARS-CoV-2 NPro-NTD can be a target for new anti-virus agents.

Remdesivir: Quo vadis?

Remdesivir (GS-5734, Veklury®) has remained the only antiviral drug formally approved by the US FDA for the treatment of Covid-19 (SARS-CoV-2 infection). Its key structural features are the fact that it is a C-nucleoside (adenosine) analogue, contains a 1'-cyano function, and could be considered as a ProTide based on the presence of a phosphoramidate group. Its antiviral spectrum and activity in animal models have been well established and so has been its molecular mode of action as a delayed chain terminator of the viral RdRp (RNA-dependent RNA polymerase). Its clinical efficacy has been evaluated, but needs to be optimized with regard to timing, dosage and duration of treatment, and route of administration. Safety, toxicity and pharmacokinetics need to be further addressed, and so are its potential combinations with other drugs such as corticosteroids (i.e. dexamethasone) and ribavirin.

Destruction mechanisms of ozone over SARS-CoV-2.

In this pandemic SARS-CoV-2 crisis, any attempt to contain and eliminate the virus will also stop its spread and consequently decrease the risk of severe illness and death. While ozone treatment has been suggested as an effective disinfection process, no precise mechanism of action has been previously reported. This study aimed to further investigate the effect of ozone treatment on SARS-CoV-2. Therefore, virus collected from nasopharyngeal and oropharyngeal swab and sputum samples from symptomatic patients was exposed to ozone for different exposure times. The virus morphology and structure were monitored and analyzed through Atomic Force Microscopy (AFM), Transmission Electron Microscopy (TEM), Atomic Absorption Spectroscopy (AAS), and ATR-FTIR. The obtained results showed that ozone treatment not only unsettles the virus morphology but also alters the virus proteins' structure and conformation through amino acid disturbance and Zn ion release from the virus non-structural proteins. These results could provide a clearer pathway for virus elimination and therapeutics preparation.

ZoomQA: residue-level protein model accuracy estimation with machine learning on sequential and 3D structural features.

MOTIVATION: The Estimation of Model Accuracy problem is a cornerstone problem in the field of Bioinformatics. As of CASP14, there are 79 global QA methods, and a minority of 39 residue-level QA methods with very few of them working on protein complexes. Here, we introduce ZoomQA, a novel, single-model method for assessing the accuracy of a tertiary protein structure/complex prediction at residue level, which have many applications such as drug discovery. ZoomQA differs from others by considering the change in chemical and physical features of a fragment structure (a portion of a protein within a radius $r$ of the target amino acid) as the radius of contact increases. Fourteen physical and chemical properties of amino acids are used to build a comprehensive representation of every residue within a protein and grade their placement within the protein as a whole. Moreover, we have shown the potential of ZoomQA to identify problematic regions of the SARS-CoV-2 protein complex. RESULTS: We benchmark ZoomQA on CASP14, and it outperforms other state-of-the-art local QA methods and rivals state of the art QA methods in global prediction metrics. Our experiment shows the efficacy of these new features and shows that our method is able to match the performance of other state-of-the-art methods without the use of homology searching against databases or PSSM matrices. AVAILABILITY: http://zoomQA.renzhitech.com.

Receptor binding, immune escape, and protein stability direct the natural selection of SARS-CoV-2 variants.

Emergence of new severe acute respiratory syndrome coronavirus 2 variants has raised concerns related to the effectiveness of vaccines and antibody therapeutics developed against the unmutated wildtype virus. Here, we examined the effect of the 12 most commonly occurring mutations in the receptor-binding domain of the spike protein on its expression, stability, activity, and antibody escape potential. Stability was measured using thermal denaturation, and the activity and antibody escape potential were measured using isothermal titration calorimetry in terms of binding to the human angiotensin-converting enzyme 2 and to neutralizing human antibody CC12.1, respectively. Our results show that mutants differ in their expression levels. Of the eight best-expressed mutants, two (N501Y and K417T/E484K/N501Y) showed stronger affinity to angiotensin-converting enzyme 2 compared with the wildtype, whereas four (Y453F, S477N, T478I, and S494P) had similar affinity and two (K417N and E484K) had weaker affinity than the wildtype. Compared with the wildtype, four mutants (K417N, Y453F, N501Y, and K417T/E484K/N501Y) had weaker affinity for the CC12.1 antibody, whereas two (S477N and S494P) had similar affinity, and two (T478I and E484K) had stronger affinity than the wildtype. Mutants also differ in their thermal stability, with the two least stable mutants showing reduced expression. Taken together, these results indicate that multiple factors contribute toward the natural selection of variants, and all these factors need to be considered to understand the evolution of the virus. In addition, since not all variants can escape a given neutralizing antibody, antibodies to treat new variants can be chosen based on the specific mutations in that variant.

Molecular basis for kinin selectivity and activation of the human bradykinin receptors.

Bradykinin and kallidin are endogenous kinin peptide hormones that belong to the kallikrein-kinin system and are essential to the regulation of blood pressure, inflammation, coagulation and pain control. Des-Arg10-kallidin, the carboxy-terminal des-Arg metabolite of kallidin, and bradykinin selectively activate two G protein-coupled receptors, type 1 and type 2 bradykinin receptors (B1R and B2R), respectively. The hyperactivation of bradykinin receptors, termed 'bradykinin storm', is associated with pulmonary edema in COVID-19 patients, suggesting that bradykinin receptors are important targets for COVID-19 intervention. Here we report two G protein-coupled complex structures of human B1R and B2R bound to des-Arg10-kallidin and bradykinin, respectively. Combined with functional analysis, our structures reveal the mechanism of ligand selectivity and specific activation of the bradykinin receptor. These findings also provide a framework for guiding drug design targeting bradykinin receptors for the treatment of inflammation, cardiovascular disorders and COVID-19.

SARS-CoV-2 S2P spike ages through distinct states with altered immunogenicity.

The SARS-CoV-2 spike is the primary target of virus-neutralizing antibodies and critical to the development of effective vaccines against COVID-19. Here, we demonstrate that the prefusion-stabilized two-proline "S2P" spike-widely employed for laboratory work and clinical studies-unfolds when stored at 4 °C, physiological pH, as observed by electron microscopy (EM) and differential scanning calorimetry, but that its trimeric, native-like conformation can be reacquired by low pH treatment. When stored for approximately 1 week, this unfolding does not significantly alter antigenic characteristics; however, longer storage diminishes antibody binding, and month-old spike elicits virtually no neutralization in mice despite inducing high ELISA-binding titers. Cryo-EM structures reveal the folded fraction of spike to decrease with aging; however, its structure remains largely similar, although with varying mobility of the receptor-binding domain. Thus, the SARS-CoV-2 spike is susceptible to unfolding, which affects immunogenicity, highlighting the need to monitor its integrity.