Characteristics of escape mutations from occult hepatitis B virus infected patients with hematological malignancies in South Egypt

AIM To investigate the prevalence and virological characteristics of occult hepatitis B virus(HBV) infections in patients with hematological malignancies in South Egypt.METHODS Serum samples were collected from 165 patients with hematological malignancies to monitor titers of HBV DNA, hepatitis B surface antigen(HBs Ag), and antibodies to HBV core(anti-HBc) and surface antigens. Serum samples negative for HBs Ag and positive for anti-HBc were subjected to nucleic acid extraction and HBV DNA detection by real-time polymerase chain reaction. DNA sequences spanning the S region were analyzed in cases with occult HBV infection. In vitro comparative study of constructed 1.24-fold wild type and S protein mutant HBV genotype D clones was further performed. RESULTS HBV DNA was detected in 23(42.6%) of 54 patients with hematological malignancies who were HBsA g negative, but anti-HBc positive, suggesting the presence of occult HBV infection. The complete HBV genome was retrieved from 6 occult HBV patients, and P120 T and S143 L were detected in 3 and 2 cases, respectively. Site directed mutagenesis was done to produce 1.24-fold genotype D clones with amino acid mutations T120 and L143. The in vitro analyses revealed that a lower level of extracellular HBsA g was detected by chemiluminescence enzyme immunoassay(CLEIA) with the clone containing T120 mutation, compared with the wild type or the clone with S143 L mutation despite the similar levels of extracellular and intracellular HBs Ag detected by Western blot. Southern blot experiments showed that the levels of intracellular HBV DNA were not different between these clones. CONCLUSION Occult HBV infection is common in patients with hematological malignancies and associated with P120 T and S143 L mutations. 120 T mutation impairs the detection of HBsA g by CLEIA.

Adaptive immune response during hepatitis C virus infection

Hepatitis C virus(HCV)infection affects about 170 million people worldwide and it is a major cause of liver cirrhosis and hepatocellular carcinoma.HCV is a hepatotropic non-cytopathic virus able to persist in a great percentage of infected hosts due to its ability to escape from the immune control.Liver damage and disease progression during HCV infection are driven by both viral and host factors.Specifically,adaptive immune response carries out an essential task in controllingnon-cytopathic viruses because of its ability to recognize infected cells and to destroy them by cytopathic mechanisms and to eliminate the virus by non-cytolytic machinery.HCV is able to impair this response by several means such as developing escape mutations in neutralizing antibodies and in T cell receptor viral epitope recognition sites and inducing HCV-specific cytotoxic T cell anergy and deletion.To impair HCV-specific T cell reactivity,HCV affects effector T cell regulation by modulating T helper and Treg response and by impairing the balance between positive and negative co-stimulatory molecules and between pro-and antiapoptotic proteins.In this review,the role of adaptive immune response in controlling HCV infection and the HCV mechanisms to evade this response are reviewed.

An antibody cocktail with broadened mutational resistance and effective protection against SARS-CoV-2

Neutralizing antibodies have been proven to be highly effective in treating mild and moderate COVID-19 patients,but continuous emergence of SARS-CoV-2 variants poses significant challenges.Antibody cocktail treatments reduce the risk of escape mutants and resistance.In this study,a new cocktail composed of two highly potent neutralizing antibodies(HB27 and H89Y)was developed,whose binding epitope is different from those cocktails that received emergency use authorization.This cocktail showed more potent and balanced neutralizing activities(IC_(50)0.9–11.3 ng mL^(-1))against a broad spectrum of SARS-CoV-2 variants over individual HB27 or H89Y antibodies.Furthermore,the cocktail conferred more effective protection against the SARS-CoV-2 Beta variant in an aged murine model than monotherapy.It was shown to prevent SARS-CoV-2 mutational escape in vitro and effectively neutralize 61 types of pseudoviruses harbouring single amino acid mutation originated from variants and escape strains of Bamlanivimab,Casirivimab and Imdevimab with IC_(50)of 0.6–65 ng mL^(-1).Despite its breadth of variant neutralization,the HB27+H89Y combo and EUA cocktails lost their potencies against Omicron variant.Our results provide important insights that new antibody cocktails covering different epitopes are valuable tools to counter virus mutation and escape,highlighting the need to search for more conserved epitopes to combat Omicron.

Rational design of unrestricted pRN1 derivatives and their application in the construction of a dual plasmid vector system for Saccharolobus islandicus

Saccharolobus islandicus REY15A represents one of the very few archaeal models with versatile genetic tools,which include efficient genome editing,gene silencing,and robust protein expression systems.However,plasmid vectors constructed for this crenarchaeon thus far are based solely on the pRN2 cryptic plasmid.Although this plasmid coexists with pRN1 in its original host,early attempts to test pRN1-based vectors consistently failed to yield any stable host-vector system for Sa.islandicus.We hypothesized that this failure could be due to the occurrence of CRISPR immunity against pRN1 in this archaeon.We identified a putative target sequence in orf904 encoding a putative replicase on pRN1(target N1).Mutated targets(N1a,N1b,and N1c)were then designed and tested for their capability to escape the host CRISPR immunity by using a plasmid inter-ference assay.The results revealed that the original target triggered CRISPR immunity in this archaeon,whereas all three mutated targets did not,indicating that all the designed target mutations evaded host immunity.These mutated targets were then incorporated into orf904 individually,yielding corresponding mutated pRN1 backbones with which shuttle plasmids were constructed(pN1aSD,pN1bSD,and pN1cSD).Sa.islandicus transformation revealed that pN1aSD and pN1bSD were functional shuttle vectors,but pN1cSD lost the capability for replication.These results indicate that the missense mutations in the conserved helicase domain in pN1c inactivated the replicase.We further showed that pRN1-based and pRN2-based vectors were stably maintained in the archaeal cells either alone or in combination,and this yielded a dual plasmid system for genetic study with this important archaeal model.