Potential role of killer immunoglobulin receptor genes among individuals vaccinated against hepatitis B virus in Lebanon

AIM: To explore the role of killer immunoglobulin receptor (KIR) genes in responsiveness or non-responsiveness to vaccination against hepatitis B virus. METHODS: We recruited 101 voluntary participants between March 2010 and December 2011. Sera samples from vaccinated and non-vaccinated participants were tested for the presence of anti-HBs antibodies as a measure of protection against hepatitis B, hepatitis B surface antigen and hepatitis B core antibody as indicators of infection by enzyme-linked immunosorbent assay. KIR gene frequencies were determined by polymerase chain reaction. RESULTS: Sera samples from 99 participants were tested for the levels of anti-HBs as an indicator of protection (≥ 10 mIU/mL) following vaccination as defined by the World Health Organization international reference standard. Among the vaccinated participants, 47% (35/74) had anti-HBs titers above 100 mIU/mL, 22% (16/74) had anti-HBs ranging between 10-100 mIU/mL, and 20% (15/74) had values of less than 10 mIU/mL. We report the lack of significant association between the number of vaccine dosages and the titer of antibodies among our vaccinated participants. The inhibitory KIR2DL1, KIR2DL4, KIR3DL1, KIR3DL2, and KIR3DL were detected in more than 95%, whereas KIR2DL2, KIR2DL3, KIR2DL5 (KR2DL5A and KIR2DL5B) were expressed in 56%, 84% and 42% (25% and 29%) of participants, respectively. The observed frequency of the activating KIR genes ranged between 35% and 55% except for KIR2DS4, detected in 95% of the study participants (40.6% 2DS4*001/002; 82.2% 2DS4*003/007). KIR2DP1 pseudogene was detected in 99% of our participants, whereas KIR3DP*001/02/04 and KIR3DP1*003 had frequencies of 17% and 100%, respectively. No association between the frequency of KIR genes and anti-HBs antibodies was detected. When we compared the frequency of KIR genes between vaccinated individuals with protective antibodies titers and those who lost their protective antibody levels, we did not detect a significant difference. KIR2DL5B was significantly different among different groups of vaccinated participants (group I > 100 mIU/mL, group II 10-100 mIU/mL, group III < 10 mIU/mL and group IV with undetectable levels of protective antibodies). CONCLUSION: To our knowledge, this is the first study screening for the possible role of KIR genes among individuals vaccinated against hepatitis B virus (HBV). Our results can be used to design larger studies to better understand the role of KIR genes in protection against or susceptibility to HBV post vaccination

Targeted isolation of diverse human protective broadly neutralizing antibodies against SARS-like viruses

The emergence of current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) and potential future spillovers of SARS-like coronaviruses into humans pose a major threat to human health and the global economy. Development of broadly effective coronavirus vaccines that can mitigate these threats is needed. Here, we utilized a targeted donor selection strategy to isolate a large panel of human broadly neutralizing antibodies (bnAbs) to sarbecoviruses. Many of these bnAbs are remarkably effective in neutralizing a diversity of sarbecoviruses and against most SARS-CoV-2 VOCs, including the Omicron variant. Neutralization breadth is achieved by bnAb binding to epitopes on a relatively conserved face of the receptor-binding domain (RBD). Consistent with targeting of conserved sites, select RBD bnAbs exhibited protective efficacy against diverse SARS-like coronaviruses in a prophylaxis challenge model in vivo. These bnAbs provide new opportunities and choices for next-generation antibody prophylactic and therapeutic applications and provide a molecular basis for effective design of pan-sarbecovirus vaccines

New Insights into the Mechanisms of Antiviral Resistance and Action

Due to the error-prone nature of viral replication, antiviral drugs can select for drug-resistant strains. Understanding the determinants for drug resistance is useful for the preclinical evaluation of antivirals, and provides important insight into the biochemical nature of the target. The first part of this dissertation describes studies designed to investigate properties of antiviral drug resistance using a well-established drug target from the influenza A virus as a model protein. The second part of the dissertation mainly focuses on the validation of a novel drug target for a re-emerging virus using biochemical and viral replication assays. Influenza A viruses are pernicious human pathogens that lead to seasonal and pandemic outbreaks. Amantadine and rimantadine are two FDA-approved drugs that target a viral proton channel called M2. These two therapeutics are no longer used due to the emergence of drug resistance. The two principal mutations that give rise to resistance in circulating strains are the S31N and V27A found within the pore of M2. Structural characterization of M2 has guided efforts to target these individual drug resistant channels through the conjugation of the adamantane group, and it was shown that the best V27A inhibitor has in vivo activity, and that the best S31N inhibitor displayed favorable pharmacokinetic properties. Although the determinants of drug resistance for amantadine and rimantadine have been thoroughly investigated, similar studies have not been performed for inhibitors of the V27A and S31N variants. Due to the fine-tuned function and structure of M2, I have hypothesized that V27A and S31N inhibitors would have a high genetic barrier to drug resistance. Using representative V27A and S31N inhibitors, I performed serial passage experiments in cell culture to select for drug-resistant influenza A viruses. Sequencing of the M2 gene segment identified mutations in the channel that gave rise to resistance, which were confirmed using recombinant viruses. In collaboration, functional assays and molecular dynamics simulations were performed to characterize the biophysical and biochemical properties of each mutation. Overall, I discovered that there are at least three mutational strategies for resistance that are selected: 1) mutations in pore-lining residues, 2) mutations in residues lining the interhelical region, and 3) mutations in the C-end of the channel below the tryptophan gate. Analysis of each mutation reveals unique effects on viral fitness, channel function, structural flexibility, pore hydration, and pore size. These studies reveal the utility of cell culture passage experiments in evaluating drug resistance, and provide insights into the mutational landscape of M2 in actively replicating influenza A viruses. The second part of this dissertation mainly focuses on the validation of a drug target from enterovirus D68, a re-emerging virus linked with neurological disease in children. No antivirals are currently available for EV-D68 infections, and few viral proteins have been validated as drug targets. I performed sequence alignment analysis which suggested that the D68 2A gene segment may encode a viral cysteine protease, and may therefore represent an attractive target for antiviral development. To test this hypothesis, we developed a protein expression protocol to obtain the protein for functional experiments. Using a fluorescence resonance energy transfer (FRET)-based assay, the expressed 2A protein was used to confirm and characterize the protease activity. I subsequently identified telaprevir—an FDA-approved peptide mimetic used to treat HCV infections—as a potent inhibitor of enterovirus D68 by targeting the 2A protein. By using drug resistance selection, I was able to confirm the antiviral activity by telaprevir was due to 2A inhibition. The enteroviral replication assays I utilized for testing telaprevir activity were further applied to study other enteroviruses and protein targets. Overall, by using a combination of biochemical functional assays, cellular antiviral assays, and molecular modeling, we have provided new insights into the mechanism of action and resistance for novel antiviral targets

Chemical Genomics Approach Leads to the Identification of Hesperadin, an Aurora B Kinase Inhibitor, as a Broad-Spectrum Influenza Antiviral

Influenza viruses are respiratory pathogens that are responsible for annual influenza epidemics and sporadic influenza pandemics. Oseltamivir (Tamiflu((R))) is currently the only FDA-approved oral drug that is available for the prevention and treatment of influenza virus infection. However, its narrow therapeutic window, coupled with the increasing incidence of drug resistance, calls for the next generation of influenza antivirals. In this study, we discovered hesperadin, an aurora B kinase inhibitor, as a broad-spectrum influenza antiviral through forward chemical genomics screening. Hesperadin inhibits multiple human clinical isolates of influenza A and B viruses with single to submicromolar efficacy, including oseltamivir-resistant strains. Mechanistic studies revealed that hesperadin inhibits the early stage of viral replication by delaying the nuclear entry of viral ribonucleoprotein complex, thereby inhibiting viral RNA transcription and translation as well as viral protein synthesis. Moreover, a combination of hesperadin with oseltamivir shows synergistic antiviral activity, therefore hesperadin can be used either alone to treat infections by oseltamivir-resistant influenza viruses or used in combination with oseltamivir to delay resistance evolution among oseltamivir-sensitive strains. In summary, the discovery of hesperadin as a broad-spectrum influenza antiviral offers an alternative to combat future influenza epidemics and pandemics.University of Arizona; NIH [AI 119187, T32 GM008804]Open Access Journal.This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

An M2-V27A channel blocker demonstrates potent in&nbsp;vitro and in&nbsp;vivo antiviral activities against amantadine-sensitive and -resistant influenza A viruses

Adamantanes such as amantadine (1) and rimantadine (2) are FDA-approved anti-influenza drugs that act by inhibiting the wild-type M2 proton channel from influenza A viruses, thereby inhibiting the uncoating of the virus. Although adamantanes have been successfully used for more than four decades, their efficacy was curtailed by emerging drug resistance. Among the limited number of M2 mutants that confer amantadine resistance, the M2-V27A mutant was found to be the predominant mutant under drug selection pressure, thereby representing a high profile antiviral drug target. Guided by molecular dynamics simulations, we previously designed first-in-class M2-V27A inhibitors. One of the potent lead compounds, spiroadamantane amine (3), inhibits both the M2-WT and M2-V27A mutant with IC50 values of 18.7 and 0.3&nbsp;μM, respectively, in in&nbsp;vitro electrophysiological assays. Encouraged by these findings, in this study we further examine the in&nbsp;vitro and in&nbsp;vivo antiviral activity of compound 3 in inhibiting both amantadine-sensitive and -resistant influenza A viruses. Compound 3 not only had single to sub-micromolar EC50 values against M2-WT- and M2-V27A-containing influenza A viruses in antiviral assays, but also rescued mice from lethal viral infection by either M2-WT- or M2-V27A-containing influenza A viruses. In addition, we report the design of two analogs of compound 3, and one was found to have improved in&nbsp;vitro antiviral activity over compound 3. Collectively, this study represents the first report demonstrating the in&nbsp;vivo antiviral efficacy of inhibitors targeting M2 mutants. The results suggest that inhibitors targeting drug-resistant M2 mutants are promising antiviral drug candidates worthy of further development

Chemical Genomics Approach Leads to the Identification of Hesperadin, an Aurora B Kinase Inhibitor, as a Broad-Spectrum Influenza Antiviral

Influenza viruses are respiratory pathogens that are responsible for annual influenza epidemics and sporadic influenza pandemics. Oseltamivir (Tamiflu((R))) is currently the only FDA-approved oral drug that is available for the prevention and treatment of influenza virus infection. However, its narrow therapeutic window, coupled with the increasing incidence of drug resistance, calls for the next generation of influenza antivirals. In this study, we discovered hesperadin, an aurora B kinase inhibitor, as a broad-spectrum influenza antiviral through forward chemical genomics screening. Hesperadin inhibits multiple human clinical isolates of influenza A and B viruses with single to submicromolar efficacy, including oseltamivir-resistant strains. Mechanistic studies revealed that hesperadin inhibits the early stage of viral replication by delaying the nuclear entry of viral ribonucleoprotein complex, thereby inhibiting viral RNA transcription and translation as well as viral protein synthesis. Moreover, a combination of hesperadin with oseltamivir shows synergistic antiviral activity, therefore hesperadin can be used either alone to treat infections by oseltamivir-resistant influenza viruses or used in combination with oseltamivir to delay resistance evolution among oseltamivir-sensitive strains. In summary, the discovery of hesperadin as a broad-spectrum influenza antiviral offers an alternative to combat future influenza epidemics and pandemics.University of Arizona; NIH [AI 119187, T32 GM008804]Open Access Journal.This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

In Vitro Pharmacokinetic Optimizations of AM2-S31N Channel Blockers Led to the Discovery of Slow-Binding Inhibitors with Potent Antiviral Activity against Drug-Resistant Influenza A Viruses

Influenza viruses are respiratory pathogens that are responsible for both seasonal influenza epidemics and occasional influenza pandemics. The narrow therapeutic window of oseltamivir, coupled with the emergence of drug resistance, calls for the next-generation of antivirals. With our continuous interest in developing AM2-S31N inhibitors as oral influenza antivirals, we report here the progress of optimizing the in vitro pharmacokinetic (PK) properties of AM2-S31N inhibitors. Several AM2-S31N inhibitors, including compound <b>10b</b>, were discovered to have potent channel blockage, single to submicromolar antiviral activity, and favorable in vitro PK properties. The antiviral efficacy of compound <b>10b</b> was also synergistic with oseltamivir carboxylate. Interestingly, binding kinetic studies (<i>K</i>_d, <i>K</i>_on, and <i>K</i>_off) revealed several AM2-S31N inhibitors that have similar <i>K</i>_d values but significantly different <i>K</i>_on and <i>K</i>_off values. Overall, this study identified a potent lead compound (<b>10b</b>) with improved in vitro PK properties that is suitable for the in vivo mouse model studies