Pathogen Infection Research Articles

Bivalent norovirus mRNA vaccine elicits cellular and humoral responses protecting human enteroids from GII.4 infection

Nucleoside-modified mRNA-LNP vaccines have revolutionized vaccine development against infectious pathogens due to their ability to elicit potent humoral and cellular immune responses. In this article, we present the results of the first norovirus vaccine candidate employing mRNA-LNP platform technology. The mRNA-LNP bivalent vaccine encoding the major capsid protein VP1 from GI.1 and GII.4 of human norovirus, generated high levels of neutralizing antibodies, robust cellular responses, and effectively protected human enteroids from infection by the most prevalent genotype (GII.4). These results serve as a proof of concept, demonstrating that a modified-nucleoside mRNA-LNP vaccine based on norovirus VP1 sequences can stimulate an immunogenic response in vivo and generates neutralizing antibodies capable of preventing viral infection in models of human gastrointestinal tract infection.

Polydopamine Nanoconjugate‐Coated Masks: An Efficient Barrier Against Airborne Pathogens

AbstractCurrent global burden of respiratory tract infections (RTIs) has thrown new challenges for researchers to design and develop efficient and multifaceted face masks. Airborne pathogens are highly transmissible through droplet nuclei/aerosol (<5 μm) which can be significantly reduced by wearing masks. Currently available disposable surgical masks have disparate limitations in terms of surface wettability, microbial penetration, dermal infections due to anaerobic reactions beneath the surface, discomfort, and eruption of chronic obstructive pulmonary diseases (COPD). These mask surfaces get contaminated with airborne pathogens along with commensal bacterial strains. Generation of high temperature and humidity during repetitive breathing allow bacteria to penetrate from the masks to respiratory tract causing serious complications. Till date, functionalized face masks with different microbicidal agents including nanomaterials experience restricted leaching of antimicrobial agents, cytotoxic effects, short‐lived microbicidal effects, inflammatory responses, negative impact on ecosystem, etc. To address these limitations, here, we have attempted fabrication of mussel mimetic biopolymer (PDA) and its aminoglycoside‐nanoconjugate‐coated face masks. Formation of a uniform layer on the surface not only acted as a potential barrier for infectious pathogens but also inhibited their entry, deactivated the adhered bacteria, imparted non‐immunogenic response with no harmful effects on the wearer's skin owing to the biocompatible nature and pH compatibility of PDA and their conjugates. Amongst all, PDA‐kanamycin (PDA−K)‐coated masks exhibited excellent microbicidal activity with the least bacterial infiltration efficiency.

Translation initiation or elongation inhibition triggers contrasting effects on Caenorhabditis elegans survival during pathogen infection.

Several microbial pathogens target host protein synthesis machinery, potentially limiting the innate immune responses of the host. In response, hosts trigger a defensive response, elevating immune gene transcripts. This counterintuitive response can have either beneficial or harmful effects on host survival. In this study, we conduct a comprehensive analysis of the impact of knocking down various translation initiation and elongation factors on the survival of Caenorhabditis elegans exposed to Pseudomonas aeruginosa. Intriguingly, inhibiting initiation and elongation factors has contrasting effects on C. elegans survival. Inhibiting translation initiation activates immune responses that protect the host from bacterial infection, while inhibiting translation elongation induces aberrant immune responses that, although clear the infection, are detrimental to the host. Our study reveals divergent roles of translation initiation and elongation inhibition in C. elegans survival during P. aeruginosa infection and identifies differential transcriptional reprogramming that could underlie these differences.

Impact of ocean warming on early development of the Mediterranean mussel Mytilus galloprovincialis: Effects on larval susceptibility to potential vibrio pathogens

In a global change scenario, ocean warming and pathogen infection can occur simultaneously in coastal areas, threatening marine species. Data are shown on the impact of temperature on early larvae of the Mediterranean mussel Mytilus galloprovincialis. Increasing temperatures (18-20-22 °C) altered larval phenotypes at 48 hpf and affected gene expression from eggs to 24 and 48 hpf, with shell biogenesis related genes among the most affected. The effects of temperature on larval susceptibility to infection were evaluated using Vibrio coralliilyticus, a coral pathogen increasingly associated with bivalve mortalities, whose ecology is affected by global warming. Malformations and mortalities at 48 hpf were observed at higher temperature and vibrio concentrations, with interactive effects. In non-lethal conditions, interactions on gene expression at 24 and 48 hpf were also detected. Although temperature is the main environmental driver affecting M. galloprovincialis early larvae, warming may increase the susceptibility to vibrio infection, with consequences on mussel populations.

Aspergillus aureoterreus, A. pseudonomiae, A. siamensis, and A. flocculosus-ochraceopetaliformis as Four New Pathogens of Eye Infection in DNA Barcoding Markers

Background: The use of a morphological method to determine Aspergillus species in routine laboratory investigation has always been challenged with low sensitivity and low specificity. Time-consuming specimen preparation and insufficient expertise are the crucial reasons. DNA barcoding markers have been introduced for identification as another gold standard method. Objective: To investigate DNA barcoding markers in identifying Aspergillus strains isolated from patients with eye infection. Methods: This study extracted 12 DNA samples of 12 Aspergillus strains. DNA barcoding genes, i.e., ITS, betatubulin (BT2), calmodulin (CAL), and actin (ACT) genes, were amplified and sequenced by polymerase chain reaction and Sanger's sequencing methods. All gene sequences were compared to those of the type or reference species collected from public databases. The species were identified with phylogenetic analyses in free applications from public websites. Results: ITS, BT2 and CAL, and ACT identified 5, 6, and 3 species and 3, 2, and 1 species complex of Aspergillus, respectively. DNA barcoding with multi-locus sequence typing revealed four new pathogens of human eye infection, including A. aureoterreus, A. pseudonomiae, A. siamensis, and A. flocculosus-ochraceopetaliformis. Conclusion: The DNA barcoding markers identified Aspergillus species with better specificity than the conventional method. The markers and techniques need to be used as tools for routine Aspergillus identification.

Insight into the new infection pathway resulting from above-ground pathogen infection of grapevine crown gall.

Grapevine crown gall (GCG), a soil-borne plant disease caused by tumorigenic Allorhizobium vitis (TAV) (=tumorigenic Rhizobium vitis) strains, poses a significant threat to grapevines worldwide. Recently, outbreaks of GCG have been reported in several vineyards, necessitating investigation into potential alternative infection pathways beyond soil transmission. The spatiotemporal distribution of GCG in vineyards from 2020 to 2022 was analyzed using the binary power law (BPL) model, with variations in quadrat shapes. Both total and newly observed diseased plants exhibited an aggregated distribution, indicating that new infections clustered around existing diseased plants, with secondary infections appearing as independent cluster points. This study provides evidence that infected pruning tools can transmit the pathogen to healthy grapevines and that TAV inoculation through spraying contributes more to GCG incidence than planting in infected soil alone. This represents the first documented case of secondary above-ground TAV infection contributing to GCG in commercial vineyards.

Role of N6-methyladenosine methylation in head and neck cancer and its regulation of innate immune pathways.

N6-methyladenosine (m6A) is considered the most prevalent methylation modification in messenger RNA (mRNA) that critically impacts head and neck cancer (HNC) pathogenesis and development. Alterations of m6A methylation related proteins are closely related to the progression, therapeutic effect, and prognosis of HNC. The human innate immune system activates immune pathways through pattern recognition receptors, which can not only resist pathogen infection, but also play a vital role in tumor immunity. Emerging evidence has confirmed that m6A methylation affects the activation of innate immune pathways such as TLR, cGAS-STING, and NLR by regulating RNA metabolism, revealing its potential mechanisms in the innate immune response of tumor cells. However, the relevant research is still in its infancy. This review elaborates the biological significance of RNA m6A methylation in HNC and discusses its potential regulatory relationship with TLR, cGAS-STING, and NLR pathways, providing a new perspective for in-depth understanding of the role of RNA methylation in the innate immune mechanism and therapeutic application of HNC.

Diabetic Microenvironment‐Unlocked BioHJzyme with H2S Evolution for Robust Anti‐Pathogens and Hyperinflammatory Wound Regeneration Through TGF‐β/Smad Pathway

AbstractDeferred diabetic skin healing is an ever‐growing complication owing to the hyperglycemic microenvironment, which accelerates the generation of advanced glycated end products (AGEs) and provides a hotbed for pathogenic infection. Here, the H2S‐evolving bio‐heterojunction enzyme (BioHJzyme), which is consisted by MXene/FeS2 and glucose oxidase (GOx) is devised. It presents glutathione peroxidase (GPx)‐ and peroxidase (POD)‐mimetic antibacterial activity for anti‐pathogens and wound regeneration by AGEs depression. The GOx catalyzes glucose, resulting in reducing the bacterial nutrient and supplying H2O2. The POD‐mimetic activity of the BioHJzyme catalyzes the H2O2 to hydroxyl radical (•OH) with a turnover number of 4.45 × 10−1 s−1, while the GPx‐mimetic activity of it consumes glutathione for further •OH accumulation. The anti‐pathogens can be enhanced by near infrared laser (NIR) irradiation owing to the efficient separation of electron‐hole pairs originated from the heterostructure, which presents NIR‐activatable •OH and 1O2 production. Moreover, the BioHJzyme evolves H2S in acidic environment, acting as an H2S donor, which protects cells around the wound from oxidative damage and AGEs, rescues mitochondrial respiration, improves the extracellular matrix deposition and ameliorates dysfunction of fibroblasts for diabetic skin regeneration through TGF‐β/Smad pathway. The work provides a proof‐of‐concept for bacteria‐invaded diabetic wound regeneration via H2S‐evolving BioHJzyme.

The evolution of plant responses underlying specialized metabolism in host-pathogen interactions.

In the course of plant evolution from aquatic to terrestrial environments, land plants (embryophytes) acquired a diverse array of specialized metabolites, including phenylpropanoids, flavonoids and cuticle components, enabling adaptation to various environmental stresses. While embryophytes and their closest algal relatives share candidate enzymes responsible for producing some of these compounds, the complete genetic network for their biosynthesis emerged in embryophytes. In this review, we analysed genomic data from chlorophytes, charophytes and embryophytes to identify genes related to phenylpropanoid, flavonoid and cuticle biosynthesis. By integrating published research, transcriptomic data and metabolite studies, we provide a comprehensive overview on how these specialized metabolic pathways have contributed to plant defence responses to pathogens in non-vascular bryophytes and vascular plants throughout evolution. The evidence suggests that these biosynthetic pathways have provided land plants with a repertoire of conserved and lineage-specific compounds, which have shaped immunity against invading pathogens. The discovery of additional enzymes and metabolites involved in bryophyte responses to pathogen infection will provide evolutionary insights into these versatile pathways and their impact on environmental terrestrial challenges.This article is part of the theme issue 'The evolution of plant metabolism'.

Urinary tract infection pathogens diagnosed in Basra city hospitals

Background. Urinary tract infection (UTI) is the growth of pathological bacteria within the urinary tract, and is one of the most common types of bacterial infections worldwide. Bacterial resistance to antibiotics is a major public health concern. Antibiotic-resistant bacterial infections exacerbate the situation in developed, developing and underdeveloped countries. Objective. The aim of this research is to identify the frequency of different bacterial pathogens and their sensitivity to different types of antibiotics in patients suffering from UTI in Basra city hospitals. Method. This cross-sectional study included 505 samples positive for bacterial growth. It was conducted by data extraction from hospital laboratory records, where information was obtained about bacterial examination of urine samples taken from patients with urinary tract infection. The samples were cultured and antibiotic sensitivity was tested by laboratory workers, in order to determine the appropriate treatment for patients. Results and conclusions. The average age of patients was 43.5 years, of whom (60.4%) were females. The most common bacteria was Escherichia coli (40.6%). In antibiotic susceptibility testing process, gentamicin (5.7%), ciprofloxacin (4.7%), trimethoprim (4.7%), trimethoprim/sulfamethoxazole (4.1%), and cefepime (3.9%) were the most frequently used antibiotics, while cefotetan (0.06%), ampicillin-sulbactam (0.09%), mupirocin (0.2%), cefazolin (0.2%), fosfomycin and amoxicillin (0.2%) were the least used. The highest sensitivity shown by cultured bacteria was to linezolid (95.5%), ertapenem (91.7%), teicoplanin (79.4%), nitrofurantoin (75.2%) and amikacin (72.4%). The highest resistance shown by cultured bacteria was to cefazolin (100%), oxacillin (94.6%), fusidic acid (91.2%), ampicillin (90.4%), and amoxicillin (88.2%).

Selective mycobacterial capture with ultraviolet-polymerized poly-dimethyldiallyl chloride functionalized surfaces

Tuberculosis (TB) is the top cause of death from a single infectious pathogen after COVID-19. Despite molecular diagnostic advances, two-thirds of the 10 million annual TB cases are still diagnosed using direct smear microscopy which has ~50% sensitivity. To increase the analytical performance of smear microscopy, we developed and characterized a novel polymer (Polydiallyldimethylammonium chloride [PDADMAC]) engraftment on inexpensive polystyrene (PS) specifically functionalized for mycobacterial capture. Engraftment is achieved via UV photopolymerization of DADMAC monomer on plasma-activated PS. The platform was tested on sputum from presumptive TB cases in Kampala, Uganda (n = 50), with an increased overall sensitivity of 81.8% (27/33) vs. fluorescent smear microscopy 57% (19/33) compared to a molecular (Cepheid GeneXpert MTB/RIF) gold standard. Frugal smear diagnostic innovation that is rapid and does not require dedicated instrumentation may offer an important solution to bridge the TB diagnostic gap.Graphical

Exploring the probiotic potential of Lactiplantibacillus pentosus SM1: Resistance, anti-microbial activity, anti-biofilm, cytotoxic activity, and safety properties

Lactiplantibacillus pentosus SM1 displayed notable resistance to acidic environments, with over 76% survival after 3 h in low pH conditions, and demonstrated viability in simulated gastrointestinal fluids. The Lpb. pentosus SM1 exhibited surface hydrophobicity, auto-aggregation, co-aggregation, and adhesion values of 59.60%, 38.80%, 44.90%, and 13.10% respectively. The anti-adhesion capability of Lpb. pentosus SM1 against Listeria monocytogenes adhesion to Caco-2 cells was demonstrated by its ability to compete with, inhibit, and displace at rates of 54.10%, 48.80%, and 23.90%, respectively. The Lpb. pentosus SM1 exhibited broad-spectrum anti-microbial activity, particularly against Gram-positive bacteria, and inhibited biofilm formation. The strain also showcased antioxidant activity, effectively scavenging DPPH (57.60%) and ABTS (60.54%) radicals. Notably, it absorbed 51.60% of cholesterol, highlighting its potential for cholesterol-lowering benefits. The cytotoxicity values were measured at 36.57 mg/mL for HT-29 and 38.20 mg/mL for Hela cancer cell lines. Importantly, Lpb. pentosus SM1 was free of hemolytic activity and biogenic amines, demonstrating a favorable safety profile. Lpb. pentosus SM1 exhibited the strongest resistance to ampicillin, while its resistance to erythromycin was the weakest, with inhibition zones measuring 15.50 mm and 26.40 mm, respectively. These findings suggest that Lpb. pentosus SM1 has promising applications as a probiotic candidate for promoting gut health and preventing pathogenic infections.

Serine ubiquitination of SQSTM1 regulates NFE2L2-dependent redox homeostasis

ABSTRACT The KEAP1-NFE2L2 axis is essential for the cellular response against metabolic and oxidative stress. KEAP1 is an adaptor protein of CUL3 (cullin 3) ubiquitin ligase that controls the cellular levels of NFE2L2, a critical transcription factor of several cytoprotective genes. Oxidative stress, defective autophagy and pathogenic infections activate NFE2L2 signaling through phosphorylation of the autophagy receptor protein SQSTM1, which competes with NFE2L2 for binding to KEAP1. Here we show that phosphoribosyl-linked serine ubiquitination of SQSTM1 catalyzed by SidE effectors of Legionella pneumophila controls NFE2L2 signaling and cell metabolism upon Legionella infection. Serine ubiquitination of SQSTM1 sterically blocks its binding to KEAP1, resulting in NFE2L2 ubiquitination and degradation. This reduces NFE2L2-dependent antioxidant synthesis in the early phase of infection. Levels of serine ubiquitinated SQSTM1 diminish in the later stage of infection allowing the expression of NFE2L2-target genes; causing a differential regulation of the host metabolome and proteome in a NFE2L2-dependent manner. Abbreviation: ARE: antioxidant response element; Dup: deubiquitinase specific for phosphoribosyl-linked serine ubiquitination; ER: endoplasmic reticulum; h.p.i: hours post infection; HIF1A/HIF-1α: hypoxia inducible factor 1 subunit alpha; KEAP1: kelch like ECH associated protein 1; KIR: KEAP1-interacting region; LIR: LC3-interacting region; NES: nuclear export signal; NFKB/NF-κB: nuclear factor kappa B; NLS: nuclear localization signal; NFE2L2/Nrf2: NFE2 like bZIP transcription factor 2; PB1 domain: Phox1 and Bem1 domain; PR-Ub: phosphoribosyl-linked serine ubiquitination; ROS: reactive oxygen species; SQSTM1/p62: sequestosome 1; tBHQ: tertiary butylhydroquinone; TUBE2: tandem ubiquitiin binding entity 2; UBA domain: ubiquitin-associated domain.

Host miRNA and mRNA profiles during in DEF and duck after DHAV-1 infection

DHAV-1 is a highly infectious pathogen that can cause acute hepatitis in ducklings. MicroRNA (miRNA) plays an essential regulatory role in virus response. We characterized and compared miRNA and mRNA expression profiles in duck embryonic fibroblasts (DEF) and the liver of ducklings infected with DHAV-1. DHAV-1 infected DEF was divided into infection group (D group) and blank group (M group), and DHAV-1 infected duckling group was divided into infection group (H group) and blank group (N group). D vs. M have 130 differentially expressed (DE) miRNA (DEM) and 2204 differentially expressed (DE) mRNA (DEG), H vs. N have 72 DEM and 1976 DEG. By the intersection of D vs. M and H vs. N comparisons, 15 upregulated DEM, 5 downregulated DEM, 340 upregulated DEG and 50 downregulated DEG were found with both in vivo and in vitro DHAV-1 infection. In particular, we identified the same DE miRNA target genes and functional annotations of DE mRNA. We enriched with multiple gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, which may have important roles in viral virulence, host immunity, and metabolism. We selected miR-155, which is co-upregulated, and found that miR-155 targets SOCS1 to inhibit DHVA-1 replication.

Preparation and application of polycaprolactone/β-cyclodextrin/gamma-Decalactone nanofiber composites in citrus postharvest diseases

Citrus fruits are highly susceptible to pathogenic fungal infections after harvesting, which causes serious economic losses. Therefore, it's necessary to develop new antifungal packaging. In this study, gamma-Decanolactone (DL) was successfully encapsulated in a polycaprolactone (PCL)/β-cyclodextrin (β-CD) composite system using electrostatic spinning technology. PCL/β-CD was compounded in different ratios, the ratio was screened through other indicators such as fiber morphologies and mechanical properties. Then, antifungal mats were prepared by adding different concentrations of DL to the PCL/β-CD solution. The results showed that when the mixture ratio of PCL/β-CD was 6:1 and loaded with 6 % DL, the antifungal felt had strong mechanical, significantly inhibiting the growth of three citrus pathogens (P. digitatum, P. italicum and G. candidum), released DL for up to 204 h and effectively reduced the morbidity rate of citrus fruits. Therefore, the antifungal pad prepared in this study has great potential in the field of citrus disease control.

Sugar-Coated: Can Multivalent Glycoconjugates Improve upon Nature's Design?

Multivalent interactions between receptors and glycans play an important role in many different biological processes, including pathogen infection, self-recognition, and the immune response. The growth in the number of tools and techniques toward the assembly of multivalent glycoconjugates means it is possible to create synthetic systems that more and more closely resemble the diversity and complexity we observe in nature. In this Perspective we present the background to the recognition and binding enabled by multivalent interactions in nature, and discuss the strategies used to construct synthetic glycoconjugate equivalents. We highlight key discoveries and the current state of the art in their applications to glycan arrays, vaccines, and other therapeutic and diagnostic tools, with an outlook toward some areas we believe are of most interest for future work in this area.

Antimicrobial Sub-MIC induces Staphylococcus aureus biofilm formation without affecting the bacterial count

BackgroundBiofilm formation is an essential virulence factor that creates a highly protected growth mode for Staphylococcus aureus (S. aureus) to survive in any hostile environment. Antibiotic sub-minimal inhibitory concentration (sub-MIC) may modulate the biofilm formation ability of bacterial pathogens, thereby affecting bacterial pathogenesis and infection outcomes. Intense antimicrobial therapy to treat biofilm-associated infections can control the pathogenic infection aggravation but cannot guarantee its complete eradication.ObjectiveThis study aimed to assess the sub-MICs effect of 5 different antimicrobial classes on biofilm-forming capacity among Staphylococcus aureus clinical isolates using three different biofilm quantitation techniques.MethodsIn this study, the effects of 5 different antimicrobial agents, namely, azithromycin, gentamicin, ciprofloxacin, doxycycline, and imipenem, at sub-MICs of 12.5%, 25%, and 50% were tested on 5 different clinical isolates of S. aureus. The biofilms formed in the absence and presence of different antimicrobial sub-MICs were then assessed using the following three different techniques: the crystal violet (CV) staining method, the quantitative PCR (qPCR) method, and the spread plate method (SPM).ResultsBiofilm formation was significantly induced in 64% of the tested conditions using the CV technique. On the other hand, the qPCR quantifying the total bacterial count and the SPM quantifying the viable bacterial count showed significant induction only in 24% and 17.3%, respectively (Fig. 1). The difference between CV and the other techniques indicates an increase in biofilm biomass without an increase in bacterial growth. As expected, sub-MICs did not reduce the viable cell count, as shown by the SPM. The CV staining method revealed that sub-MICs of imipenem and ciprofloxacin had the highest significance rate (80%) showing an inductive effect on the biofilm development. On the other hand, doxycycline, azithromycin, and gentamicin displayed lower significance rates of 73%, 53%, and 47%, respectively.ConclusionExposure to sub-MIC doses of antimicrobial agents induces the biofilm-forming capacity of S. aureus via increasing the total biomass without significantly affecting the bacterial growth of viable count.

First whole genome sequencing and analysis of human parechovirus type 3 causing a healthcare-associated outbreak among neonates in Hungary.

In November 2023, the National Reference Laboratory for Enteroviruses (Budapest, Hungary) received stool, pharyngeal swab and cerebrospinal fluid samples from five newborns suspected of having human parechovirus (PEV-A) infection. The neonates were born in the same hospital and presented with fever and sepsis-like symptoms at 8-9 days of age, and three of them showed symptoms consistent with central nervous system involvement. PEV-A positivity was confirmed by quantitative reverse transcription polymerase chain reaction. To determine the PEV-A genotype responsible for the infections, fecal samples of four neonates were subjected to metagenomic sequencing. For further analyses, amplicon-based whole genome sequencing was performed directly from the clinical samples. On the basis of whole genome analysis, sequences were allocated to PEV-A genotype 3 (PEV-A3) and consensus sequences were identical. Two ambiguities were identified in the viral protein 1 (VP1) region of all sequences at a frequency of 17.7-53.7%, indicating the simultaneous presence of at least two quasispecies in the clinical samples. The phylogenetic analysis and similarity plotting showed that all sequences clustered without any topological inconsistencies between the P1 capsid and P2, P3 non-capsid regions, suggesting that recombination events during evolution were unlikely. Our findings suggest that the apparent cluster of cases were microbiologically related, and the results may also inform future investigations on the evolution and pathogenicity of PEV-A3 infections.

Conservation of molecular responses upon viral infection in the non-vascular plant Marchantia polymorpha

After plants transitioned from water to land around 450 million years ago, they faced novel pathogenic microbes. Their colonization of diverse habitats was driven by anatomical innovations like roots, stomata, and vascular tissue, which became central to plant-microbe interactions. However, the impact of these innovations on plant immunity and pathogen infection strategies remains poorly understood. Here, we explore plant-virus interactions in the bryophyte Marchantia polymorpha to gain insights into the evolution of these relationships. Virome analysis reveals that Marchantia is predominantly associated with RNA viruses. Comparative studies with tobacco mosaic virus (TMV) show that Marchantia shares core defense responses with vascular plants but also exhibits unique features, such as a sustained wound response preventing viral spread. Additionally, general defense responses in Marchantia are equivalent to those restricted to vascular tissues in Nicotiana, suggesting that evolutionary acquisition of developmental innovations results in re-routing of defense responses in vascular plants.

Structure-guided mutations in CDRs for enhancing the affinity of neutralizing SARS-CoV-2 nanobody

The optimization of antibodies to attain the desired levels of affinity and specificity holds great promise for the development of next generation therapeutics. This study delves into the refinement and engineering of complementarity-determining regions (CDRs) through in silico affinity maturation followed by binding validation using isothermal titration calorimetry (ITC) and pseudovirus-based neutralization assays. Specifically, it focuses on engineering CDRs targeting the epitopes of receptor-binding domain (RBD) of the spike protein of SARS-CoV-2. A structure-guided virtual library of 112 single mutations in CDRs was generated and screened against RBD to select the potential affinity-enhancing mutations. Protein-protein docking analysis identified 32 single mutants of which nine mutants were selected for molecular dynamics (MD) simulations. Subsequently, biophysical ITC studies provided insights into binding affinity, and consistent with in silico findings, six mutations that demonstrated better binding affinity than native nanobody were further tested in vitro for neutralization activity against SARS-CoV-2 pseudovirus. Leu106Thr mutant was found to be most effective in virus-neutralization with IC50 values of ∼0.03 μM, as compared to the native nanobody (IC50 ∼0.77 μM). Thus, in this study, the developed computational pipeline guided by structure-aided interface profiles and thermodynamic analysis holds promise for the streamlined development of antibody-based therapeutic interventions against emerging variants of SARS-CoV-2 and other infectious pathogens.