Search Results (77)

In Nigeria, hepatitis B virus (HBV) infection remains a significant public health issue. The emergence of immune escape mutants (IEMs), basal core promoters, and precore (BCP/PC) mutants among asymptomatic individuals has enabled the continuous evolution of the virus in the country. In this study, we used Sanger sequencing of the S gene and the BCP/PC region to investigate the genetic diversity, phylogenetic relationships, and mutational profiles of HBV strains detected in two regions in Nigeria. A total of 178 HBsAg-positive samples confirmed by ELISA underwent viral DNA extraction and PCR amplification of the surface and BCP/PC genes, and 76 and 60 sequences were found to be exploitable for S and BCP/PC genes, respectively, which were used for HBV genotyping and mutational analysis. We detected various mutations in the major hydrophilic loop (target of neutralizing antibodies), including vaccine escape mutants (VEMs) (L127P/R, S140T/L, and G145A), HBV immunoglobulin resistance mutants (T131N, S143T, and W156R), and mutations previously reported in patients with reactivated infections (T115N, G159A/R, and F161Y). We also identified a high proportion of C1741T in 34/42 (81%) along with A1762T or G1764A mutation in 14/42 (33%) and 18/42 (43%) as the dominant variants in the BCP region. The predominant classical PC G1896A and G1899A variants were identified in 26/42 (62%) and 17/42 (40%) participants in this study. Two HBV genotypes were identified (A and E). However, HBV genotype E was the most frequently identified genotype, and is still the dominant strain circulating in Nigeria. We report the circulation of HBV IEMs and the preponderance of BCP and classical PC variants among asymptomatic carriers. Our findings suggest that the spread of these HBV mutant variants among asymptomatic carriers may have an impact on the effectiveness of diagnostic immunoassays and the success of HBsAg-based vaccinations. This highlights the need for robust surveillance. Full article

Background: Parvovirus B19 (B19V) can cause severe relapsing episodes of pure red cell aplasia in immunocompromised individuals, which are commonly treated with intravenous immunoglobulins (IVIGs). Few data are available on B19V intra-host evolution and the role of humoral immune selection. Here, we report the dynamics of genomic mutations and subsequent protein changes during relapsing infection. Methods: Longitudinal plasma samples from immunocompromised patients with relapsing B19V infection in the period 2011–2019 were analyzed using whole-genome sequencing to evaluate intra-host evolution. The impact of mutations on the 3D viral protein structure was predicted by deep neural network modeling. Results: Of the three immunocompromised patients with relapsing infections for 3 to 9 months, one patient developed two consecutive nonsynonymous mutations in the VP1/2 region: T372S/T145S and Q422L/Q195L. The first mutation was detected in multiple B19V IgG-seropositive follow-up samples and resolved after IgG seroreversion. Computational prediction of the VP1 3D structure of this mutant showed a conformational change in the proximity of the antibody binding domain. No conformational changes were predicted for the other mutations detected. Discussion: Analysis of relapsing B19V infections showed mutational changes occurring over time. Resulting amino acid changes were predicted to lead to a conformational capsid protein change in an IgG-seropositive patient. The impact of humoral response and IVIG treatment on B19V infections should be further investigated to understand viral evolution and potential immune escape. Full article

Severe acute respiratory syndrome coronavirus 2 has undergone several mutations since 2020, and novel variants continue to emerge to this day. The immune escape ability of the emerging mutants is enhanced and results in robust transmissibility. The neutralizing ability of the antibodies produced in the human body during previous infections is decreased against some of these mutants, which poses a severe challenge to the preventive and therapeutic effectiveness of vaccines and antibody drugs. The nucleocapsid protein is one of the main structural proteins of the coronavirus and plays an important role in the life cycle of the novel coronavirus. This protein is one of the key targets for drug development, and the first major step in drug development is to obtain pure nucleocapsid proteins. However, since nucleocapsid proteins have a nucleic acid-binding function and automatically undergo liquid–liquid phase separation and agglomeration, the purification of full-length nucleocapsids is challenging. In this context, a set of easy-to-operate processes was developed in this study for the purification of nucleocapsid proteins. Finally, a pure full-length nucleocapsid protein without nucleic acid contamination was obtained, which exhibited significantly enhanced accessibility for structural and functional virological studies, vaccine development, and related research applications. Further, the nucleic acid-binding domain of the nucleocapsid protein was targeted, and potential severe acute respiratory syndrome coronavirus 2 inhibitors were identified using virtual screening and biolayer interferometry technology. Notably, the eukaryotically expressed nucleocapsid protein demonstrated a significantly greater binding affinity for Light Green SF Yellowish (K_D = 119.7 nM) compared to that demonstrated by its prokaryotic counterpart (K_D = 19.9 × 10³ nM). The findings of this study suggest the importance of considering both protein source and post-translational modifications of the target proteins to be used in drug screening workflows. Therefore, this compound not only represents a novel therapeutic candidate for COVID-19 but also a critical tool for elucidating antiviral mechanisms. Full article

The three-year COVID-19 pandemic ‘has’ caused a wide range of medical, social, political, and financial implications. Since the end of 2020, various mutations and variations in SARS-CoV-2 strains, along with the immune escape phenomenon, have emerged. There is an urgent need to identify a relatively stable target for the development of universal vaccines and drugs that can effectively combat both SARS-CoV-2 strains and their mutants. Currently, the main focus in treating SARS-CoV-2 lies in disrupting the virus’s life cycle. The main protease (M^pro) is closely associated with virus replication and maturation and plays a crucial role in the early stages of infection. Consequently, it has become an important target for the development of SARS-CoV-2-specific drugs. This review summarizes the recent research progress on the novel coronavirus’s main proteases, including the pivotal role of M^pro in the virus’s life cycle, the structure and catalytic mechanism of M^pro, the self-maturation mechanism of M^pro, the role of M^pro in virus immune escape, the current methods of developing antiviral drugs targeting M^pro, and the key drugs that have successfully entered clinical trials. The aim is to provide researchers involved in the development of antiviral drugs targeting M^pro with systematic and comprehensive information. Full article

Enhancer of zeste homolog 2 (EZH2) is a methyltransferase involved in cell cycle regulation, cell differentiation, and cell death and plays a role in modulating the immune response. Although it mainly functions by catalyzing the tri-methylation of H3 histone on K27 (H3K27), to inhibit the transcription of target genes, EZH2 can directly methylate several transcription factors or form complexes with them, regulating their functions. EZH2 expression/activity is often dysregulated in cancer, contributing to carcinogenesis and immune escape, thereby representing an important target in anti-cancer therapy. This review summarizes some of the mechanisms through which EZH2 regulates the expression and function of tumor suppressor genes and oncogenic molecules such as STAT3, mutant p53, and c-Myc and how it modulates the anti-cancer immune response. The influence of posttranslational modifications on EZH2 activity and stability and the possible strategies leading to its inhibition are also reviewed. Full article

Even though COVID-19 is no longer the primary focus of the global scientific community, its high mutation rate (nearly 30 substitutions per year) poses a threat of a potential comeback. Effective vaccines have been developed and administered to the population, ending the pandemic. Nonetheless, reinfection by newly emerging subvariants, particularly the latest JN.1 strain, remains common. The rapid mutation of this virus demands a fast response from the scientific community in case of an emergency. While the immune escape of earlier variants was extensively investigated, one still needs a comprehensive understanding of how specific mutations, especially in the newest subvariants, influence the antigenic escape of the pathogen. Here, we tested comprehensive in silico approaches to identify methods for fast and accurate prediction of antibody neutralization by various mutants. As a benchmark, we modeled the complexes of the murine antibody 2B04, which neutralizes infection by preventing the SARS-CoV-2 spike glycoprotein’s association with angiotensin-converting enzyme (ACE2). Complexes with the wild-type, B.1.1.7 Alpha, and B.1.427/429 Epsilon SARS-CoV-2 variants were used as positive controls, while complexes with the B.1.351 Beta, P.1 Gamma, B.1.617.2 Delta, B.1.617.1 Kappa, BA.1 Omicron, and the newest JN.1 Omicron variants were used as decoys. Three essentially different algorithms were employed: forced placement based on a template, followed by two steps of extended molecular dynamics simulations; protein–protein docking utilizing PIPER (an FFT-based method extended for use with pairwise interaction potentials); and the AlphaFold 3.0 model for complex structure prediction. Homology modeling was used to assess the 3D structure of the newly emerged JN.1 Omicron subvariant, whose crystallographic structure is not yet available in the Protein Database. After a careful comparison of these three approaches, we were able to identify the pros and cons of each method. Protein–protein docking yielded two false-positive results, while manual placement reinforced by molecular dynamics produced one false positive and one false negative. In contrast, AlphaFold resulted in only one doubtful result and a higher overall accuracy-to-time ratio. The reasons for inaccuracies and potential pitfalls of various approaches are carefully explained. In addition to a comparative analysis of methods, some mechanisms of immune escape are elucidated herein. This provides a critical foundation for improving the predictive accuracy of vaccine efficacy against new viral subvariants, introducing accurate methodologies, and pinpointing potential challenges. Full article

Since the onset of the COVID-19 pandemic, a variety of diagnostic approaches, including RT-qPCR, RAPID, and LFA, have been adopted, with RT-qPCR emerging as the gold standard. However, a significant challenge in COVID-19 diagnostics is the wide range of symptoms presented by patients, necessitating early and accurate diagnosis for effective management. Although RT-qPCR is a precise molecular technique, it is not immune to false-negative results. In contrast, CRISPR-based detection methods for SARS-CoV-2 offer several advantages: they are cost-effective, time-efficient, highly sensitive, and specific, and they do not require sophisticated instruments. These methods also show promise for scalability, enabling diagnostic tests. CRISPR technology can be customized to target any genomic region of interest, making it a versatile tool with applications beyond diagnostics, including therapeutic development. The CRISPR/Cas systems provide precise gene targeting with immense potential for creating next-generation diagnostics and therapeutics. One of the key advantages of CRISPR/Cas-based therapeutics is the ability to perform multiplexing, where different sgRNAs or crRNAs can target multiple sites within the same gene, reducing the likelihood of viral escape mutants. Among the various CRISPR systems, CRISPR/Cas13 and CARVER (Cas13-assisted restriction of viral expression and readout) are particularly promising. These systems can target a broad range of single-stranded RNA viruses, making them suitable for the diagnosis and treatment of various viral diseases, including SARS-CoV-2. However, the efficacy and safety of CRISPR-based therapeutics must be thoroughly evaluated in pre-clinical and clinical settings. While CRISPR biotechnologies have not yet been fully harnessed to control the current COVID-19 pandemic, there is an optimism that the limitations of the CRISPR/Cas system can be overcome soon. This review discusses how CRISPR-based strategies can revolutionize disease diagnosis and therapeutic development, better preparing us for future viral threats. Full article

Despite advances in vaccination and therapies for coronavirus disease, challenges remain due to reduced antibody longevity and the emergence of virulent variants like Omicron (BA.1) and its subvariants (BA.1.1, BA.2, BA.3, and BA.5). This study explored the potential of adoptive immunotherapy and harnessing the protective abilities using virus-specific T cells (VSTs). Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) VSTs were generated by stimulating donor-derived peripheral blood mononuclear cells with spike, nucleocapsid, and membrane protein peptide mixtures. Phenotypic characterization, including T-cell receptor (TCR) vβ and pentamer analyses, was performed on the ex vivo-expanded cells. We infected human leukocyte antigen (HLA)-partially matched human Calu-3 cells with various authentic SARS-CoV-2 strains in a Biosafety Level 3 facility and co-cultured them with VSTs. VSTs exhibited a diverse TCR vβ repertoire, confirming their ability to target a broad range of SARS-CoV-2 antigens from both the ancestral and mutant strains, including Omicron BA.1 and BA.5. These ex vivo-expanded cells exhibited robust cytotoxicity and low alloreactivity against HLA-partially matched SARS-CoV-2-infected cells. Their cytotoxic effects were consistent across variants, targeting conserved spike and nucleocapsid epitopes. Our findings suggest that third-party partial HLA-matching VSTs could counter immune-escape mechanisms posed by emerging variants of concern. Full article

The p53 protein is the master regulator of cellular integrity, primarily due to its tumor-suppressing functions. Approximately half of all human cancers carry mutations in the TP53 gene, which not only abrogate the tumor-suppressive functions but also confer p53 mutant proteins with oncogenic potential. The latter is achieved through so-called gain-of-function (GOF) mutations that promote cancer progression, metastasis, and therapy resistance by deregulating transcriptional networks, signaling pathways, metabolism, immune surveillance, and cellular compositions of the microenvironment. Despite recent progress in understanding the complexity of mutp53 in neoplastic development, the exact mechanisms of how mutp53 contributes to cancer development and how they escape proteasomal and lysosomal degradation remain only partially understood. In this review, we address recent findings in the field of oncogenic functions of mutp53 specifically regarding, but not limited to, its implications in metabolic pathways, the secretome of cancer cells, the cancer microenvironment, and the regulating scenarios of the aberrant proteasomal degradation. By analyzing proteasomal and lysosomal protein degradation, as well as its connection with autophagy, we propose new therapeutical approaches that aim to destabilize mutp53 proteins and deactivate its oncogenic functions, thereby providing a fundamental basis for further investigation and rational treatment approaches for TP53-mutated cancers. Full article

Neutralizing antibodies targeting the spike (S) protein of SARS-CoV-2, elicited either by natural infection or vaccination, are crucial for protection against the virus. Nonetheless, the emergence of viral escape mutants presents ongoing challenges by contributing to breakthrough infections. To define the evolution trajectory of SARS-CoV-2 within the immune population, we co-incubated replication-competent rVSV/SARS-CoV-2/GFP chimeric viruses with sera from COVID-19 convalescents. Our findings revealed that the E484D mutation contributes to increased viral resistant against both convalescent and vaccinated sera, while the L1265R/H1271Y double mutation enhanced viral infectivity in 293T-hACE2 and Vero cells. These findings suggest that under the selective pressure of polyclonal antibodies, SARS-CoV-2 has the potential to accumulate mutations that facilitate either immune evasion or greater infectivity, facilitating its adaption to neutralizing antibody responses. Although the mutations identified in this study currently exhibit low prevalence in the circulating SARS-CoV-2 populations, the continuous and meticulous surveillance of viral mutations remains crucial. Moreover, there is an urgent necessity to develop next-generation antibody therapeutics and vaccines that target diverse, less mutation-prone antigenic sites to ensure more comprehensive and durable immune protection against SARS-CoV-2. Full article

Lactate dehydrogenase A (LDHA) primarily catalyzes the conversion between lactic acid and pyruvate, serving as a key enzyme in the aerobic glycolysis pathway of sugar in tumor cells. LDHA plays a crucial role in the occurrence, development, progression, invasion, metastasis, angiogenesis, and immune escape of tumors. Consequently, LDHA not only serves as a biomarker for tumor diagnosis and prognosis but also represents an ideal target for tumor therapy. Although LDHA inhibitors show great therapeutic potential, their development has proven to be challenging. In the development of LDHA inhibitors, the key active sites of LDHA are emphasized. Nevertheless, there is a relative lack of research on the amino acid residues around the active center of LDHA. Therefore, in this study, we investigated the amino acid residues around the active center of LDHA. Through structure comparison analysis, five key amino acid residues (Ala30, Met41, Lys131, Gln233, and Ala259) were identified. Subsequently, the effects of these five residues on the enzymatic properties of LDHA were investigated using site-directed mutagenesis. The results revealed that the catalytic activities of the five mutants varied to different degrees in both the reaction from lactic acid to pyruvate and pyruvate to lactic acid. Notably, the catalytic activities of LDHA^M41G and LDHA^K131I were improved, particularly in the case of LDHA^K131I. The results of the molecular dynamics analysis of LDHA^K131I explained the reasons for this phenomenon. Additionally, the optimum temperature of LDHA^M41G and LDHA^Q233M increased from 35 °C to 40 °C, whereas in the reverse reaction, the optimum temperature of LDHA^M41G and LDHA^K131I decreased from 70 °C to 60 °C. These findings indicate that Ala30, Met41, Lys131, Gln233, and Ala259 exert diverse effects on the catalytic activity and optimum temperature of LHDA. Therefore, these amino acid residues, in addition to the key catalytic site of the active center, play a crucial role. Considering these residues in the design and screening of LDHA inhibitors may lead to the development of more effective inhibitors. Full article

A mutator is a variant in a population of organisms whose mutation rate is higher than the average mutation rate in the population. For genetic and population dynamics reasons, mutators are produced and survive with much greater frequency than anti-mutators (variants with a lower-than-average mutation rate). This strong asymmetry is a consequence of both fundamental genetics and natural selection; it can lead to a ratchet-like increase in the mutation rate. The rate at which mutators appear is, therefore, a parameter that should be of great interest to evolutionary biologists generally; for example, it can influence: (1) the survival duration of a species, especially asexual species (which are known to be short-lived), (2) the evolution of recombination, a process that can ameliorate the deleterious effects of mutator abundance, (3) the rate at which cancer appears, (4) the ability of pathogens to escape immune surveillance in their hosts, (5) the long-term fate of mitochondria, etc. In spite of its great relevance to basic and applied science, the rate of mutation to a mutator phenotype continues to be essentially unknown. The reasons for this gap in our knowledge are largely methodological; in general, a mutator phenotype cannot be observed directly, but must instead be inferred from the numbers of some neutral “marker” mutation that can be observed directly: different mutation-rate variants will produce this marker mutation at different rates. Here, we derive the expected distribution of the numbers of the marker mutants observed, accounting for the fact that some of the mutants will have been produced by a mutator phenotype that itself arose by mutation during the growth of the culture. These developments, together with previous enhancements of the Luria–Delbrück assay (by one of us, dubbed the “Jones protocol”), make possible a novel experimental protocol for estimating the rate of mutation to a mutator phenotype. Simulated experiments using biologically reasonable parameters that employ this protocol show that such experiments in the lab can give us fairly accurate estimates of the rate of mutation to a mutator phenotype. Although our ability to estimate mutation-to-mutator rates from simulated experiments is promising, we view this study as a proof-of-concept study and an important first step towards practical empirical estimation. Full article

The North American low pathogenic H7N2 avian influenza A viruses, which lack the 220-loop in the hemagglutinin (HA), possess dual receptor specificity for avian- and human-like receptors. The purpose of this work was to determine which amino acid substitutions in HA affect viral antigenic and phenotypic properties that may be important for virus evolution. By obtaining escape mutants under the immune pressure of treatment with monoclonal antibodies, antigenically important amino acids were determined to be at positions 125, 135, 157, 160, 198, 200, and 275 (H3 numbering). These positions, except 125 and 275, surround the receptor binding site. The substitutions A135S and A135T led to the appearance of an N-glycosylation site at 133N, which reduced affinity for the avian-like receptor analog and weakened binding with tested monoclonal antibodies. Additionally, the A135S substitution is associated with the adaptation of avian viruses to mammals (cat, human, or mouse). The mutation A160V decreased virulence in mice and increased affinity for the human-type receptor analog. Conversely, substitution G198E, in combination with 157N or 160E, displayed reduced affinity for the human-type receptor analog. Full article

Respiratory syncytial virus (RSV) is the most common cause of upper and lower respiratory tract infections in infants and young children. Virus-specific monoclonal antibodies (mAbs) can be used for diagnosis, prophylaxis, and research of RSV pathogenesis. A panel of 16 anti-RSV mAbs was obtained from mice immunized by RSV strain Long. Half of them had virus-neutralizing activity. According to Western blot all of these mAbs effectively bound native oligomeric (homodimeric and homotrimeric) forms of the RSV fusion (F) protein. Only five of the mAbs interacted with the monomeric form, and only one of these possessed neutralizing activity. None of these mAbs, nor the commercial humanized neutralizing mAb palivizumab, reacted with the denaturated F protein. Thus, interaction of all these mAbs with F protein had clear conformational dependence. Competitive ELISA and neutralization assays allowed the identification of nine antigenic target sites for the interaction of mAb with the F protein. Five partially overlapping sites may represent a complex spatial structure of one antigenic determinant, including one neutralizing and four non-neutralizing epitopes. Four sites (three neutralizing and one non-neutralizing) were found to be distinct. As a result of virus cultivation RSV–A, strain Long, in the presence of a large amount of one of the neutralizing mAbs, an escape mutant with a substitution, N240S, in the F protein, was obtained. Thus, it was shown for the first time that position 240 is critical for the protective effect of an anti-RSV antibody. To assess the ability of these mAbs to interact with modern RSV strains circulating in St. Petersburg (Russia) between 2014 and 2022, 73 RSV-A and 22 RSV-B isolates were analyzed. Six mAbs were directed to conserved epitopes of the F protein as they interacted most efficiently with both RSV subtypes in a fixed cell-ELISA and could be used for diagnostic assays detecting RSV. Full article

Several mutations in the surface (S), basal core promoter (BCP), and precore (PC) genes of the hepatitis B virus have been linked to inaccurate diagnosis and the development of immune escape mutants (IEMs) of the infection, which can lead to chronic infection. Understanding the prevalence and spread of these mutations is critical in the global effort to eliminate HBV. Blood samples were collected from 410 people in Osun and Ekiti states, southwest Nigeria, between 2019 and 2021. Participants were drawn from a group of asymptomatic people who were either blood donors, outpatients, or antenatal patients with no record of HBV infection at the medical outpatients’ unit of the hospital. DNA was extracted from plasma using a Qiagen DNEasy kit, followed by nested PCR targeting HBV S and BCP/PC genes. The Sanger sequencing method was used to sequence the positive PCR amplicons, which were further analyzed for IEMs, BCP, and PC mutations. HBV-DNA was detected in 12.4% (51/410) of individuals. After DNA amplification and purification, 47.1% (24) of the S gene and 76.5% (39) of the BCP/PC gene amplicons were successfully sequenced. Phylogenetic analysis showed that all the HBV sequences obtained in this study were classified as HBV genotype E. Mutational analysis of the major hydrophilic region (MHR) and a-determinant domain of S gene sequences revealed the presence of three immune escape mutations: two samples harbored a T116N substitution, six samples had heterogenous D144A/N/S/H substitution, and one sample had a G145E substitution, respectively. The BCP/PC region analysis revealed a preponderance of major BCP mutants, with the prevalence of BCP double substitutions ranging from 38.5% (A1762T) to 43.6% (G1764A). Previously reported classical PC mutant variants were observed in high proportion, including G1896A (33.3%) and G1899A (12.8%) mutations. This study confirms the strong presence of HBV genotype E in Nigeria, the ongoing circulation of HBV IEMs, and a high prevalence of BCP/PC mutants in the cohorts. This has implications for diagnosis and vaccine efficacy for efficient management and control of HBV in the country. Full article