Identification of epidermal growth factor receptor-tyrosine kinase inhibitor targeting the VP1 pocket of human rhinovirus.

Screening a library of 1,200 preselected kinase inhibitors for anti-human rhinovirus 2 (HRV-2) activity in HeLa cells identified a class of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI) as effective virus blockers. These were based on the 4-anilinoquinazoline-7-oxypiperidine scaffold, with the most potent representative AZ5385 inhibiting the virus with EC50 of 0.35 µM. Several structurally related analogs confirmed activity in the low µM range, while interestingly, other TKIs targeting EGFR lacked anti-HRV-2 activity. To further probe this lack of association between antiviral activity and EGFR inhibition, we stained infected cells with antibodies specific for activated EGFR (Y1068) and did not observe a dependency on EGFR-TK activity. Instead, consecutive passages of HRV-2 in HeLa cells in the presence of a compound and subsequent nucleotide sequence analysis of resistant viral variants identified the S181T and T210A alterations in the major capsid VP1 protein, with both residues located in the vicinity of a known hydrophobic pocket on the viral capsid. Further characterization of the antiviral effects of AZ5385 showed a modest virus-inactivating (virucidal) activity, while anti-HRV-2 activity was still evident when the inhibitor was added as late as 10 h post infection. The RNA copy/infectivity ratio of HRV-2 propagated in AZ5385 presence was substantially higher than that of control HRV indicating that the compound preferentially targeted HRV progeny virions during their maturation in infected cells. Besides HRV, the compound showed anti-respiratory syncytial virus activity, which warrants its further studies as a candidate compound against viral respiratory infections.

Identification of four neutralizing antigenic sites on the enterovirus D68 capsid.

IMPORTANCE: Enterovirus D68 (EV-D68) is an emerging respiratory pathogen associated with acute flaccid myelitis. Currently, no approved vaccines or antiviral drugs are available. Here, we report four functionally independent neutralizing antigenic sites (I to IV) by analyses of neutralizing monoclonal antibody (MAb)-resistant mutants. Site I is located in the VP1 BC loop near the fivefold axis. Site II resides in the VP2 EF loop, and site III is situated in VP1 C-terminus; both sites are located at the south rim of the canyon. Site IV is composed of residue in VP2 ßB strand and residues in the VP3 BC loop and resides around the threefold axis. The developed MAbs targeting the antigenic sites can inhibit viral binding to cells. These findings advance the understanding of the recognition of EV-D68 by neutralizing antibodies and viral evolution and immune escape and also have important implications for the development of novel EV-D68 vaccines.

Conservation of HLA Spike Protein Epitopes Supports T Cell Cross-Protection in SARS-CoV-2 Vaccinated Individuals against the Potentially Zoonotic Coronavirus Khosta-2.

Heterologous vaccines, which induce immunity against several related pathogens, can be a very useful and rapid way to deal with new pandemics. In this study, the potential impact of licensed COVID-19 vaccines on cytotoxic and helper cell immune responses against Khosta-2, a novel sarbecovirus that productively infects human cells, was analyzed for the 567 and 41 most common HLA class I and II alleles, respectively. Computational predictions indicated that most of these 608 alleles, covering more than 90% of the human population, contain sufficient fully conserved T-cell epitopes between the Khosta-2 and SARS-CoV-2 spike-in proteins. Ninety percent of these fully conserved peptides for class I and 93% for class II HLA molecules were verified as epitopes recognized by CD8+ or CD4+ T lymphocytes, respectively. These results show a very high correlation between bioinformatic prediction and experimental assays, which strongly validates this study. This immunoinformatics analysis allowed a broader assessment of the alleles that recognize these peptides, a global approach at the population level that is not possible with experimental assays. In summary, these findings suggest that both cytotoxic and helper cell immune protection elicited by currently licensed COVID-19 vaccines should be effective against Khosta-2 virus infection. Finally, by being rapidly adaptable to future coronavirus pandemics, this study has potential public health implications.

Immune Escape Adaptive Mutations in Hemagglutinin Are Responsible for the Antigenic Drift of Eurasian Avian-Like H1N1 Swine Influenza Viruses.

The continuous antigenic variation of influenza A viruses remains a major hurdle for vaccine selection; however, the molecular determinants and mechanisms of antigenic change remain largely unknown. In this study, two escape mutants were generated by serial passages of the Eurasian avian-like H1N1 swine influenza virus (EA H1N1 SIV) A/swine/Henan/11/2005 (HeN11) in the presence of two neutralizing monoclonal antibodies (mAbs) against the hemagglutinin (HA) protein, which were designated HeN11-2B6-P5 and HeN11-4C7-P8, respectively. The HeN11-2B6-P5 mutant simultaneously harbored the N190D and I230M substitutions in HA, whereas HeN11-4C7-P8 harbored the M269R substitution in HA (H3 numbering). The effects of each of these substitutions on viral antigenicity were determined by measuring the neutralization and hemagglutination inhibition (HI) titers with mAbs and polyclonal sera raised against the representative viruses. The results indicate that residues 190 and 269 are key determinants of viral antigenic variation. In particular, the N190D mutation had the greatest antigenic impact, as determined by the HI assay. Further studies showed that both HeN11-2B6-P5 and HeN11-4C7-P8 maintained the receptor-binding specificity of the parent virus, although the single mutation N190D decreased the binding affinity for the human-type receptor. The replicative ability in vitro of HeN11-2B6-P5 was increased, whereas that of HeN11-4C7-P8 was decreased. These findings extend our understanding of the antigenic evolution of influenza viruses under immune pressure and provide insights into the functional effects of amino acid substitutions near the receptor-binding site and the interplay among receptor binding, viral replication, and antigenic drift. IMPORTANCE The antigenic changes that occur continually in the evolution of influenza A viruses remain a great challenge for the effective control of disease outbreaks. Here, we identified three amino acid substitutions (at positions 190, 230, and 269) in the HA of EA H1N1 SIVs that determine viral antigenicity and result in escape from neutralizing monoclonal antibodies. All three of these substitutions have emerged in nature. Of note, residues 190 and 230 have synergistic effects on receptor binding and antigenicity. Our findings provide a better understanding of the functional effects of amino acid substitutions in HA and their consequences for the antigenic drift of influenza viruses.

Antigenic Architecture of the H7N2 Influenza Virus Hemagglutinin Belonging to the North American Lineage.

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.

Emergence of Avian coronavirus Escape Mutants Under Suboptimal Antibody Titers.

To perform a quasispecies assessment of the effect of vaccine combinations and antibody titers on the emergence of Avian coronavirus (AvCoV) escape mutants, 5-week-old males from a commercial chicken breeder lineage were vaccinated intramuscularly with one dose of a monovalent (genotype GI-1) or a bivalent (genotypes GI-1 and GI-11 (n = 40 birds/group) AvCoV vaccine. Seven birds were kept as controls. Six weeks later, pools of sera of each group were prepared and incubated at virus neutralization doses of 10 and 10-1 with the Beaudette strain (GI-1) of AvCoV in VERO cells. Rescued viruses were then submitted to genome-wide deep sequencing for subconsensus variant detection. After treatment with serum from birds vaccinated with the bivalent vaccine at a titer of 10-1, an F307I variant was detected in the spike glycoprotein that mapped to an important neutralizing region, which indicated an escape mutant derived from natural selection. Further variants were detected in nonstructural proteins and non-coding regions that are not targets of neutralizing antibodies and might be indicators of genetic drift. These results indicate that the evolution of AvCoV escape mutants after vaccination depends on the type of vaccine strain and the antibody titer and must be assessed based on quasispecies rather than consensus dominant sequences only because quasispecies may be otherwise undetected.

Reactivation of occult hepatitis B virus infection in a renal transplant recipient.

We report a case of hepatitis B virus (HBV) reactivation in a renal transplant recipient. Reactivation manifested as an occult infection with detectable HBV-DNA and negativity for hepatitis B surface antigen (HBsAg). The anti-HBs antibody titre was above the protective threshold and continued to rise, to 951.36 mIU/ml, after HBV reactivation. Sequencing revealed multiple vaccine- and diagnostic-escape mutations in the major hydrophilic region of HBsAg. This case demonstrates both reactivation of an HBV escape mutant in a vaccinated patient and host immunity after virus mutation.

Escaping stochastic extinction of mutant virus: Temporal pattern of emergence of drug resistance within a host.

After infecting a host, a viral strain may increase rapidly within the body and produce mutants with a faster proliferation rate than the virus itself. However, most of the mutants become extinct because of the stochasticity caused by the small number of infected cells. In addition, the mean growth rate of a mutant strain decreases with time because the number of susceptible target cells is reduced by the original strain. In this study, we calculated the fraction of mutants that can escape stochastic extinction, based on a continuous-time branching process with a time-dependent growth rate. We analyzed two cases differing in the mode of viral transmission: (1) an infected cell transmits the virus through cell-to-cell contact with a susceptible target cell; (2) an infected cell releases numerous free viral particles that subsequently infect susceptible target cells. The chance for a mutant strain to be established decreases with time after infection of the original type, and it may oscillate before convergence at the stationary value. We then calculated the probability of escaping stochastic extinction for a drug-resistant mutant when a patient received an antiviral drug that suppressed the original strain. Combining the rate of mutant production from the original strain and the chance of escaping stochastic extinction, the number of emerging drug-resistant mutations may have two peaks: one soon after the infection of the original type and the second at the start of antiviral drug administration.

Molecular epidemiology of hepatitis B virus mutants associated with vaccine escape, drug resistance and diagnosis failure.

The massive implementation of the vaccine and antiviral agents against hepatitis B virus (HBV), targeting the envelope and viral polymerase genes, induces a selection pressure that might lead to the emergence of variants that impair the effectiveness of the vaccine, diagnostic methods and antiviral therapy. The aim of this study was to evaluate the prevalence of HBV vaccine escape mutants (VEMs), diagnostic failure mutants (DFMs) and treatment resistance mutants (ARMs) among individuals from Buenos Aires, Argentina. HBV surface antigen and polymerase sequences obtained from serum samples of 530 HBV-infected individuals were analysed. Samples belonged to genotypes A (28.1%), D (13.6%) and F (58.3%). VEMs, DMFs and ARMs were present in 40 (7.5%), 57 (10.7%) and 27 (5.1%) samples within the studied population. Additionally, eight nonpreviously reported VEMs and nine DFMs were identified. VEMs and DFMs were biased by genotype, being higher in genotype D (33.3% and 33.3%) compared to genotype A (6% and 17.4%) and genotype F (2.3% and 2.3%) (P > 0.001). On the contrary, there was no association between the presence of ARMs and HBV genotype (P = 0.324). VEMs, DFMs and ARMs create public health concerns. The current study provided valuable information about mutants in surface antigen and polymerase in HBV-infected patients from Argentina where HBV-F is the most prevalent genotype. Consequently, it constitutes an important reference for Latin American clinicians in order to optimize the management of HBV-infected patients.

Next-generation sequencing analyses of the emergence and maintenance of mutations in CTL epitopes in HIV controllers with differential viremia control.

BACKGROUND: Despite the low level of viral replication in HIV controllers (HICs), studies have reported viral mutations related to escape from cytotoxic T-lymphocyte (CTL) response in HIV-1 plasma sequences. Thus, evaluating the dynamics of the emergence of CTL-escape mutants in HICs reservoirs is important for understanding viremia control. To analyze the HIV-1 mutational profile and dynamics of CTL-escape mutants in HICs, we selected 11 long-term non-progressor individuals and divided them into the following groups: (1) viremic controllers (VCs; n = 5) and (2) elite controllers (ECs; n = 6). For each individual, we used HIV-1 proviral DNA from PBMCs related to earliest (VE) and latest (VL) visits to obtain gag and nef sequences using the Illumina HiSeq system. The consensus of each mapped gene was used to assess viral divergence, and next-generation sequencing data were employed to identify SNPs and variations within and flanking CTL epitopes. RESULTS: Divergence analysis showed higher values for nef compared to gag among the HICs. EC and VC groups showed similar divergence rates for both genes. Analysis of the number of SNPs showed that VCs present more variability in both genes. Synonymous/non-synonymous mutation ratios were < 1 for gag among ECs and for nef among ECs and VCs, exhibiting a predominance of non-synonymous mutations. Such mutations were observed in regions encoding CTL-restricted epitopes in all individuals. All ECs presented non-synonymous mutations in CTL epitopes but generally at low frequency (< 1%); all VCs showed a high number of mutations, with significant frequency changes between VE and VL visits. A higher frequency of internal mutations was observed for gag epitopes, with significant changes across visits compared to Nef epitopes, indicating a pattern associated with differential genetic pressure. CONCLUSIONS: The high genetic conservation of HIV-1 gag and nef among ECs indicates that the higher level of viremia control restricts the evolution of both genes. Although viral replication levels in HICs are low or undetectable, all individuals exhibited CTL epitope mutations in proviral gag and nef variants, indicating that potential CTL escape mutants are present in HIC reservoirs and that situations leading to a disequilibrium of the host-virus relationship can result in the spread of CTL-escape variants.

Escape of Tick-Borne Flavivirus from 2'-C-Methylated Nucleoside Antivirals Is Mediated by a Single Conservative Mutation in NS5 That Has a Dramatic Effect on Viral Fitness.

Tick-borne encephalitis virus (TBEV) causes a severe and potentially fatal neuroinfection in humans. Despite its high medical relevance, no specific antiviral therapy is currently available. Here we demonstrate that treatment with a nucleoside analog, 7-deaza-2'-C-methyladenosine (7-deaza-2'-CMA), substantially improved disease outcomes, increased survival, and reduced signs of neuroinfection and viral titers in the brains of mice infected with a lethal dose of TBEV. To investigate the mechanism of action of 7-deaza-2'-CMA, two drug-resistant TBEV clones were generated and characterized. The two clones shared a signature amino acid substitution, S603T, in the viral NS5 RNA-dependent RNA polymerase (RdRp) domain. This mutation conferred resistance to various 2'-C-methylated nucleoside derivatives, but no cross-resistance was seen with other nucleoside analogs, such as 4'-C-azidocytidine and 2'-deoxy-2'-beta-hydroxy-4'-azidocytidine (RO-9187). All-atom molecular dynamics simulations revealed that the S603T RdRp mutant repels a water molecule that coordinates the position of a metal ion cofactor as 2'-C-methylated nucleoside analogs approach the active site. To investigate its phenotype, the S603T mutation was introduced into a recombinant TBEV strain (Oshima-IC) generated from an infectious cDNA clone and into a TBEV replicon that expresses a reporter luciferase gene (Oshima-REP-luc2A). The mutants were replication impaired, showing reduced growth and a small plaque size in mammalian cell culture and reduced levels of neuroinvasiveness and neurovirulence in rodent models. These results indicate that TBEV resistance to 2'-C-methylated nucleoside inhibitors is conferred by a single conservative mutation that causes a subtle atomic effect within the active site of the viral NS5 RdRp and is associated with strong attenuation of the virus.IMPORTANCE This study found that the nucleoside analog 7-deaza-2'-C-methyladenosine (7-deaza-2'-CMA) has high antiviral activity against tick-borne encephalitis virus (TBEV), a pathogen that causes severe human neuroinfections in large areas of Europe and Asia and for which there is currently no specific therapy. Treating mice infected with a lethal dose of TBEV with 7-deaza-2'-CMA resulted in significantly higher survival rates and reduced the severity of neurological signs of the disease. Thus, this compound shows promise for further development as an anti-TBEV drug. It is important to generate drug-resistant mutants to understand how the drug works and to develop guidelines for patient treatment. We generated TBEV mutants that were resistant not only to 7-deaza-2'-CMA but also to a broad range of other 2'-C-methylated antiviral medications. Our findings suggest that combination therapy may be used to improve treatment and reduce the emergence of drug-resistant viruses during nucleoside analog therapy for TBEV infection.

Low immunogenic bio-nanocapsule based on hepatitis B virus escape mutants.

Bio-nanocapsules (BNCs) consisting of hepatitis B virus surface antigen (HBsAg) L proteins and phospholipids are used as efficient non-viral carriers for liver-specific delivery of genes and drugs. Considering the administration to HB vaccinees and HB patients, endogenous anti-HBsAg immunoglobulins (HBIGs) may reduce the delivery efficacy and prevent repetitive administration. Therefore, low immunogenic BNCs were generated by inserting two point mutations in the HBsAg L protein, which were found in HBV escape mutants. Escape mutant-type BNC (emBNC) showed 50% lower HBIG binding capacity than that of parental BNC (wtBNC). It induced HBIG production to a lesser extent than that associated with wtBNC in BALB/c mice. The emBNC could accumulate into human hepatocyte-derived tumor in mice pre-treated with HBIGs. The complex of emBNC and cationic liposomes could deliver plasmid DNA to HepG2 cells efficiently in the presence of HBIGs. Thus, emBNC could evade HBIG-neutralizing antibodies, expanding the clinical utility of BNC-based nanomedicine.

Eradicating hepatitis B virus: The critical role of preventing perinatal transmission.

Prevention of hepatitis B virus (HBV) transmission from infected mothers to their newborns is critical to HBV control and eventual eradication. Mother-to-child perinatal transmission causes the highest chronic carrier rate (>85%) with a high rate of subsequent chronic liver disease and hepatocellular carcinoma. This risk is reduced by 90% with HBV vaccine given along with hepatitis B immune globulin (HBIG) starting at birth. New analyses of our data from US trials of HBIG and HBV vaccine in high-risk infants revealed better efficacy with yeast-recombinant vaccine than plasma-derived vaccine, especially in preventing late onset infections, with evidence that vaccine prevented transmission of maternal HBV infection with the glycine to arginine mutation in surface antigen codon 145 (sG145R). Most late infections with sG145R were in vaccine non-responders, suggesting escape from HBIG rather than from vaccine-induced antibody. Our findings also help explain survey results from Taiwan following universal childhood immunization implemented in the mid-1980s. We conclude that current vaccines will remain effective against surface antigen mutants. Anti-viral drugs in high-risk pregnant women, in combination with newborn HBIG and vaccine, show promise for eliminating residual breakthrough neonatal infections, critical to meeting WHO 2030 goals and for eradicating HBV.

The impact of hepatitis B virus precore/core gene carboxyl terminal mutations on viral biosynthesis and the host immune response.

BACKGROUND: We aimed to elucidate the impact of hepatitis B virus (HBV) precore/core gene mutations on spontaneous hepatitis B e antigen (HBeAg) seroconversion, HBV biosynthesis, and the human immune responses in chronic HBV-infected patients. METHODS: We analyzed the HBV precore/core gene sequences by cloning method in 33 chronic HBV-infected patients during the inflammatory phase before spontaneous HBeAg seroconversion. The impact of the most prevalent mutant on HBeAg biosynthesis was assessed by Western blotting and native agarose gel analysis in Huh7 cells, and the human immune responses were assessed by in vitro stimulation of CD3+ CD8+ T lymphocytes of chronic HBV-infected subjects. RESULTS: The P135Q and G1896A were the most prevalent mutants before HBeAg seroconversion, acting as markers of HBeAg seroconversion (hazard ratios = 2.75 and 4.50; P = .01 and <.001, respectively). The P135Q mutants displayed decreased HBeAg secretion and HBV capsid molecular weight, while showing increased 22 kD HBeAg proprotein accumulation in Huh7 cells. The P135Q mutant peptide induced less interferon-γ expression in CD3+ CD8+ T lymphocytes in HBeAg-negative subjects compared to the wild-type peptide (P = .03). CONCLUSIONS: The HBV P135Q mutant emergence during the inflammatory phase was associated with HBeAg seroconversion. It was associated with altered HBV capsid assembly, HBeAg biosynthesis, and reduced human immune responses following HBeAg seroconversion.

Preparation and characterization of mouse-derived monoclonal antibodies against the hemagglutinin of the H1N1 influenza virus.

H1N1 influenza virus is a significant global public health concern. Monoclonal antibodies (mAbs) targeting specific viral proteins such as hemagglutinin (HA) have become an important therapeutic strategy, offering highly specific targeting to block viral transmission and infection. This study focused on the development of mAbs targeting HA of the A/Victoria/2570/2019 (H1N1pdm09, VIC-19) strain by utilizing hybridoma technology to produce two mAbs with high binding capacity. Notably, mAb 2B2 has demonstrated a strong affinity for HA proteins in recent H1N1 influenza vaccine strains. In vitro assessments showed that both mAbs exhibited broad-spectrum hemagglutination inhibition and potent neutralizing effects against various vaccine strains of H1N1pdm09 viruses. 2B2 was also effective in animal models, offering both preventive and therapeutic protection against infections caused by recent H1N1 strains, highlighting its potential for clinical application. By individually co-cultivating each of the aforementioned mAbs with the virus in chicken embryos, four amino acid substitution sites in HA (H138Q, G140R, A141E/V, and D187E) were identified in escape mutants, three in the antigenic site Ca2, and one in Sb. The identification of such mutations is pivotal, as it compels further investigation into how these alterations could undermine the binding efficacy and neutralization capacity of antibodies, thereby impacting the design and optimization of mAb therapies and influenza vaccines. This research highlights the necessity for continuous exploration into the dynamic interaction between viral evolution and antibody response, which is vital for the formulation of robust therapeutic and preventive strategies against influenza.

Immune-Escape Mutations Are Prevalent among Patients with a Coexistence of HBsAg and Anti-HBs in a Tertiary Liver Center in the United States.

The concurrent seropositivity of HBsAg and anti-HBs has been described among patients with chronic hepatitis B (CHB), but its prevalence is variable. HBV S-gene mutations can affect the antigenicity of HBsAg. Patients with mutations in the 'α' determinant region of the S gene can develop severe HBV reactivation under immunosuppression. In this study at a tertiary liver center in the United States, we evaluated the frequency and virological characteristics of the HBsAg mutations among CHB patients with the presence of both HBsAg and anti-HBs. In this cohort, 45 (2.1%) of 2178 patients were identified to have a coexistence of HBsAg and anti-HBs, and 24 had available sera for the genome analysis of the Pre-S1, Pre-S2, and S regions. The frequency of mutations in the S gene was significantly higher among those older than 50 years (mean 8.5 vs. 5.4 mutations per subject, p = 0.03). Twelve patients (50%) had mutations in the 'α' determinant region of the S gene. Mutations at amino acid position 126 were most common in eight subjects. Three had a mutation at position 133. Only one patient had a mutation at position 145-the classic vaccine-escape mutation. Despite the universal HBV vaccination program, the vaccine-escape mutant is rare in our cohort of predominantly Asian patients.

Identification of key antigenic sites in hemagglutinin of H10N3 avian influenza virus.

The H10 avian influenza viruses (AIV) have been detected in both birds and mammals. Recently, the cases of human infection with H10N8 and H10N3 in China pose high risk to public health. However, the antigenic sites in hemagglutinin (HA) of H10 are poorly understood. In this study, 3 monoclonal antibodies (MAb), designated as 1F4, 6B3 and 6G12, against the HA protein of the H10N3 strain A/chicken/Taizhou/498/2021(H10N3) (TZ498), were first generated. All of these MAb could effectively inhibit TZ498 in haemagglutination inhibition assay and microneutralization assay. Four novel antigenic sites at positions 135, 208, 227, and 266 (H10 numbering) were identified in the HA of TZ498 through escape mutants selected by these 3 MAb. Moreover, natural mutations at positions 135 and 227 were found in the H10 field strains. All these not only provide novel insights into the molecular markers for monitoring the antigenic variation of H10 but also be helpful for developing efficient control strategies against H10.

Renal transplantation from hepatitis B surface antigen (HBsAg)-positive donors to HBsAg-negative recipients: a case of post-transplant fulminant hepatitis associated with an extensively mutated hepatitis B virus strain and review of the current literature.

PURPOSE: The purpose of this study was to present a fatal case of fulminant hepatitis B (FHB) that developed in a renal transplant recipient, immunized against hepatitis B, 1 year post transplantation. METHODS: Polymerase chain reaction amplification and full genome sequencing were performed to investigate whether specific mutations were associated with hepatitis B virus (HBV) transmission and FHB. RESULTS: Molecular analysis revealed multiple mutations in various open reading frames of HBV, the most important being the G145R escape mutation and a frameshift mutation-insertion (1838insA) within the pre-C/C reading frame. CONCLUSIONS: Our results highlight the possibility of developing FHB, despite previous immunization against HBV or administration of hyperimmune gammaglobulin, because of the selection of escape virus mutants. The current literature and guidelines regarding renal transplantation from hepatitis B surface antigen (HBsAg)-positive to HBsAg-negative patients were also reviewed.

Identification of key residues of B cell epitopes in hemagglutinin of H6 influenza A virus.

IMPORTANCE: Since the escape immunity of influenza A viruses (IAVs) is mainly caused by the continuous antigenic variations in HA, the identification of key antigenic epitopes is crucial for better understanding of the escape immunity and vaccine development for IAVs. The antigenic sites of several HA subtypes, including H1, H3, H5, and H9, have been well characterized, whereas those of H6 subtype are poorly understood. Here, we mapped nine key residues of antigenic epitopes in H6 through escape mutants using a panel of MAbs. Moreover, MAbs 4C2 and 6E3, targeting 140 and 89 residues, respectively, could protect mice against lethal challenge of MA E-Teal/417. These key residues of antigenic epitopes identified here provide the molecular targets for further elucidating the antigenic evolution of H6 and better preparing the vaccine against H6 IAV.

A combination of two resistance mechanisms is critical for tick-borne encephalitis virus escape from a broadly neutralizing human antibody.

Tick-borne encephalitis virus (TBEV) is a flavivirus that causes human neuroinfections and represents a growing health problem. The human monoclonal antibody T025 targets envelope protein domain III (EDIII) of TBEV and related tick-borne flaviviruses, potently neutralizing TBEV in vitro and in preclinical models, representing a promising candidate for clinical development. We demonstrate that TBEV escape in the presence of T025 or T028 (another EDIII-targeting human monoclonal antibody) results in virus variants of reduced pathogenicity, characterized by distinct sets of amino acid changes in EDII and EDIII that are jointly needed to confer resistance. EDIII substitution K311N impairs formation of a salt bridge critical for T025-epitope interaction. EDII substitution E230K is not on the T025 epitope but likely induces quaternary rearrangements of the virus surface because of repulsion of positively charged residues on the adjacent EDI. A combination of T025 and T028 prevents virus escape and improves neutralization.