Kinetics of hepatitis B surface antigen quasispecies during REP 2139-Ca therapy in HBeAg-positive chronic HBV infection.

Chronic HBV infection results in various clinical manifestations due to different levels of immune response. In recent years, hepatitis B treatment has improved by long-term administration of nucleos(t)ide analogues (NUCs) and peg-interferon. Nucleic acid polymers (NAPs; REP 2139-Ca and REP 2139-Mg) are new antiviral drugs that block the assembly of subviral particles, thus preventing the release of HBsAg and allowing its clearance and restoration of functional control of infection when combined with various immunotherapies. In the REP 102 study (NCT02646189), 9 of 12 patients showed substantial reduction of HBsAg and seroconversion to anti-HBs in response to REP 2139-Ca, whereas 3 of 12 patients did not show responses (>1 log reduction of HBsAg and HBV DNA from baseline). We characterized the dynamic changes of HBV quasispecies (QS) within the major hydrophilic region (MHR) of the 'pre-S/S' open reading frame including the 'a' determinant in responders and nonresponders of the REP 102 study and four untreated matched controls. HBV QS complexity at baseline varied slightly between responders and nonresponders (P = .28). However, these responders showed significant decline in viral complexity (P = .001) as REP 2139-Ca therapy progressed but no significant change in complexity was observed among the nonresponders (P = .99). The MHR mutations were more frequently observed in responders than in nonresponders and matched controls. No mutations were observed in 'a' determinant of major QS population which may interfere with the detection of HBsAg by diagnostic assays. No specific mutations were found within the MHR which could explain patients' poor HBsAg response during REP 2139-Ca therapy.

Modular epitope binding predicts influenza quasispecies dominance and vaccine effectiveness: Application to 2018/19 season.

The modular binding sites on the influenza A(H3N2) hemagglutinin protein are under significant pressure to acquire mutations in order to evade human antibody recognition. Analysis of these hemagglutinin epitopes in the strains circulating during 2017/18 and early 2018/19 identified the emergence of a new antigenic cluster that has grown from 4% of circulating strains to 11%. We regressed our module-based antigenic distance, pepitope, with A(H3N2) vaccine effectiveness from recent studies conducted by the US Centers for Disease Control and Prevention (r2 = 0.92), and we used this to estimate that the 2018/19 vaccines will protect against most circulating A(H3N2) strains. The pEpitope model is useful for A(H3N2) influenza vaccine virus selection and development, and it has the potential to aid national or regional regulatory authorities in making geographically localized decisions.

Cyclical adaptation of measles virus quasispecies to epithelial and lymphocytic cells: To V, or not to V.

Measles virus (MeV) is dual-tropic: it replicates first in lymphatic tissues and then in epithelial cells. This switch in tropism raises the question of whether, and how, intra-host evolution occurs. Towards addressing this question, we adapted MeV either to lymphocytic (Granta-519) or epithelial (H358) cells. We also passaged it consecutively in both human cell lines. Since passaged MeV had different replication kinetics, we sought to investigate the underlying genetic mechanisms of growth differences by performing deep-sequencing analyses. Lymphocytic adaptation reproducibly resulted in accumulation of variants mapping within an 11-nucleotide sequence located in the middle of the phosphoprotein (P) gene. This sequence mediates polymerase slippage and addition of a pseudo-templated guanosine to the P mRNA. This form of co-transcriptional RNA editing results in expression of an interferon antagonist, named V, in place of a polymerase co-factor, named P. We show that lymphocytic-adapted MeV indeed produce minimal amounts of edited transcripts and V protein. In contrast, parental and epithelial-adapted MeV produce similar levels of edited and non-edited transcripts, and of V and P proteins. Raji, another lymphocytic cell line, also positively selects V-deficient MeV genomes. On the other hand, in epithelial cells V-competent MeV genomes rapidly out-compete the V-deficient variants. To characterize the mechanisms of genome re-equilibration we rescued four recombinant MeV carrying individual editing site-proximal mutations. Three mutations interfered with RNA editing, resulting in almost exclusive P protein expression. The fourth preserved RNA editing and a standard P-to-V protein expression ratio. However, it altered a histidine involved in Zn2+ binding, inactivating V function. Thus, the lymphocytic environment favors replication of V-deficient MeV, while the epithelial environment has the opposite effect, resulting in rapid and thorough cyclical quasispecies re-equilibration. Analogous processes may occur in natural infections with other dual-tropic RNA viruses.

Applying Unique Molecular Identifiers in Next Generation Sequencing Reveals a Constrained Viral Quasispecies Evolution under Cross-Reactive Antibody Pressure Targeting Long Alpha Helix of Hemagglutinin.

To overcome yearly efforts and costs for the production of seasonal influenza vaccines, new approaches for the induction of broadly protective and long-lasting immune responses have been developed in the past decade. To warrant safety and efficacy of the emerging crossreactive vaccine candidates, it is critical to understand the evolution of influenza viruses in response to these new immune pressures. Here we applied unique molecular identifiers in next generation sequencing to analyze the evolution of influenza quasispecies under in vivo antibody pressure targeting the hemagglutinin (HA) long alpha helix (LAH). Our vaccine targeting LAH of hemagglutinin elicited significant seroconversion and protection against homologous and heterologous influenza virus strains in mice. The vaccine not only significantly reduced lung viral titers, but also induced a well-known bottleneck effect by decreasing virus diversity. In contrast to the classical bottleneck effect, here we showed a significant increase in the frequency of viruses with amino acid sequences identical to that of vaccine targeting LAH domain. No escape mutant emerged after vaccination. These results not only support the potential of a universal influenza vaccine targeting the conserved LAH domains, but also clearly demonstrate that the well-established bottleneck effect on viral quasispecies evolution does not necessarily generate escape mutants.

Early changes in quasispecies variant after antiviral therapy for chronic hepatitis B.

Hepatitis B virus (HBV) polymerase gene is targeted by nucleos(t)ide analogues (NUC), but it is unclear how HBV quasispecies of whole genome changes during early period of NUC treatment. To understand the unknown region of drug sensitivity and treatment resistance, HBV quasispecies of whole genome during early period of NUC treatment was examined using ultra­deep sequencing. Eleven patients with chronic HBV infection who received NUC treatment were enrolled in the current study. Viral DNA was extracted from serum samples before and early period of NUC treatment. Polymerase chain reaction analysis was subsequently performed on the DNA products. The viral quasispecies of the entire genome was analyzed by ultra­deep sequencing. The regions and positions corresponding to the changes in the quasispecies were investigated before and early period of NUC treatment. The secondary structure changes were predicted by mutations/substitutions detected using Lasergene Protean v14.1 software. The frequency of quasispecies variants increased significantly in the polymerase domain from before to early period of NUC treatment (3.08±1.28 vs. 3.51±1.47%, P<0.008), particularly the reverse transcription (RT) domain (3.76±1.25 vs. 4.52±1.37%, P<0.012). In addition, increased variation detected from HBsAg domain showed statistically significant during NUC treatment (6.81±3.26 vs. 7.81±3.26%, P<0.040). The amino acid (aa) mutations/substitutions were detected and compared from before to early period of treatment. Interestingly, most of them were located in the RT region (RT1 motif: aa21­aa51) and small S region in the early duration of NUC treatment. Furthermore, several mutation patterns, such as cI97L and cP130T showed alterations in the secondary structure and predicted antigenicity of HBV protein. Although the HBV whole genome can be affected by NUC treatment, RT 1 motif region and small S region are more sensitive to the early period of NUC treatment. This study suggested the initial changes of HBV quasispecies might affect the long­term drug sensitivity and resistance to NUC treatment.