Impact of vaccination on SARS-CoV-2 evolution and immune escape variants.

Vaccines and host genetic factors can influence the SARS-CoV-2 evolution and emergence of new variants. Even vaccinated cases get affected as virus continues to evolve, raising concerns about vaccine efficacy and the emergence of immune escape variants. Here, we have analyzed 2295 whole-genome sequences of SARS-CoV-2 collected from vaccinated and unvaccinated cases to evaluate the impact of vaccines on virus diversity within hosts. Our comparative analysis revealed a significant higher incidence of intra-host single nucleotides variants (iSNVs) in vaccinated cases compared to unvaccinated ones (p value<0.0001). Furthermore, we have found that specific mutational processes, including APOBEC (C > T) mediated and ADAR1 (A > G) mediated mutations, were found more prevalent in vaccinated cases. Vaccinated cases exhibited higher accumulation of nonsynonymous mutation than unvaccinated cases. Fixed iSNVs were predominantly located in the ORF1ab and spike genes, several key omicron defining immune escape variants S477N, Q493R, Q498R, Y505H, L452R, and N501Y were identified in the RBD domain of spike gene in vaccinated cases. Our findings suggest that vaccine plays an important role in the evolution of the virus genome. The virus genome acquires random mutations due to error-prone replication of the virus, host modification through APOBEC and ADAR1 mediated editing mechanism, and oxidative stress. These mutations become fixed in the viral population due to the selective pressure imposed by vaccination.

Joint host-pathogen genomic analysis identifies hepatitis B virus mutations associated with human NTCP and HLA class I variation.

Evolutionary changes in the hepatitis B virus (HBV) genome could reflect its adaptation to host-induced selective pressure. Leveraging paired human exome and ultra-deep HBV genome-sequencing data from 567 affected individuals with chronic hepatitis B, we comprehensively searched for the signatures of this evolutionary process by conducting "genome-to-genome" association tests between all human genetic variants and viral mutations. We identified significant associations between an East Asian-specific missense variant in the gene encoding the HBV entry receptor NTCP (rs2296651, NTCP S267F) and mutations within the receptor-binding region of HBV preS1. Through in silico modeling and in vitro preS1-NTCP binding assays, we observed that the associated HBV mutations are in proximity to the NTCP variant when bound and together partially increase binding affinity to NTCP S267F. Furthermore, we identified significant associations between HLA-A variation and viral mutations in HLA-A-restricted T cell epitopes. We used in silico binding prediction tools to evaluate the impact of the associated HBV mutations on HLA presentation and observed that mutations that result in weaker binding affinities to their cognate HLA alleles were enriched. Overall, our results suggest the emergence of HBV escape mutations that might alter the interaction between HBV PreS1 and its cellular receptor NTCP during viral entry into hepatocytes and confirm the role of HLA class I restriction in inducing HBV epitope variations.

Targeting hepatitis B vaccine escape using immunogenetics in Bangladeshi infants.

Hepatitis B virus (HBV) vaccine escape mutants (VEM) are increasingly described, threatening progress in control of this virus worldwide. Here we studied the relationship between host genetic variation, vaccine immunogenicity and viral sequences implicating VEM emergence. In a cohort of 1,096 Bangladeshi children, we identified human leukocyte antigen (HLA) variants associated with response vaccine antigens. Using an HLA imputation panel with 9,448 south Asian individuals DPB1*04:01 was associated with higher HBV antibody responses (p=4.5×10-30). The underlying mechanism is a result of higher affinity binding of HBV surface antigen epitopes to DPB1*04:01 dimers. This is likely a result of evolutionary pressure at the HBV surface antigen 'a-determinant' segment incurring VEM specific to HBV. Prioritizing pre-S isoform HBV vaccines may tackle the rise of HBV vaccine evasion.

An RBD bispecific antibody effectively neutralizes a SARS-CoV-2 Omicron variant.

Potent neutralizing antibodies (nAbs) against SARS-CoV-2 are a promising therapeutic against the ongoing COVID-19 pandemic. However, the continuous emergence of neutralizing antibody escape variants makes it challenging for antibody therapeutics based on monospecific nAbs. Here, we generated an IgG-like bispecific antibody (bsAb), Bi-Nab, based on a pair of human neutralizing antibodies targeting multiple and invariant sites of the spike receptor binding domain (RBD): 35B5 and 32C7. We demonstrated that Bi-Nab exhibited higher binding affinity to the Delta spike protein than its parental antibodies and presented an extended inhibition breadth of preventing RBD binding to angiotensin-converting enzyme 2 (ACE2), the cellular receptor of SARS-CoV-2. In addition, pseudovirus neutralization results showed that Bi-Nab improved the neutralization potency and breadth with a lower half maximum inhibitory concentration (IC50) against wild-type SARS-CoV-2, variants being monitored (VBMs) and variants of concern (VOCs). Notably, the IgG-like Bi-Nab enhanced the neutralizing activity against Omicron variants with potent capabilities for transmission and immune evasion in comparison with its parental monoclonal antibody (mAb) 32C7 and a cocktail (with the lowest IC50 values of 31.6 ng/mL against the Omicron BA.1 and 399.2 ng/mL against the Omicron BA.2), showing evidence of synergistic neutralization potency of Bi-Nab against the Omicron variants. Thus, Bi-Nab represents a feasible and effective strategy against SARS-CoV-2 variants of concern.

CD8 T cell-mediated depletion of HBV surface-antigen-expressing, bilineal-differentiated liver carcinoma cells generates highly aggressive escape variants.

The expression of viral antigens in chronic hepatitis B virus (HBV) infection drives continuous liver inflammation, one of the main risk factors to develop liver cancer. HBV developed immune-suppressive functions to escape from the host immune system, but their link to liver tumor development is not well understood. Here, we analyzed if and how HBV surface antigen (HBs) expression in combined hepatocellular-cholangiocarcinoma (cHCC/iCCA) cells influences their antigenicity for CD8 T cells. We randomly isolated liver tumor tissues from AlfpCre+-Trp53fl/fl/Alb-HBs+ tg mice and established primary carcinoma cell lines (pCCL) that showed a bilineal (CK7+/HNF4α+) cHCC/iCCA phenotype. These pCCL uniformly expressed HBs (HBshi), and low levels of MHC-I (MHC-Ilo), and were transiently convertible to a high antigenicity (MHC-Ihi) phenotype by IFN-γ treatment. HBshi/pCCL induced HBs/(Kb/S190-197)-specific CD8 T cells and developed slow-growing tumors in subcutaneously transplanted C57Bl/6J (B6) mice. Interestingly, pCCL-ex cells, established from HBshi/pCCL-induced and re-explanted tumors in B6 but not those in immune-deficient Rag1-/- mice showed major alterations, like an MHC-Ihi phenotype, a prominent growth-biased gene expression signature, a significantly decreased HBs expression (HBslo) and a switch to fast-growing tumors in re-transplanted B6 or PD-1-/- hosts with an unlocked PD-1/PD-L1 control system. CD8 T cell-mediated elimination of HBshi/pCCL, together with the attenuation of the negative restraints of HBs in the tumor cells, like ER-stress, reveals a novel mechanism to unleash highly aggressive HBslo/pCCL-ex immune-escape variants. Under certain conditions, HBs-specific CD8 T-cell responses thus potentiate tumor growth, an aspect that should be considered for therapeutic vaccination strategies against chronic HBV infection and liver tumors.

Post-Vaccination and Post-Infection Immunity to the Hepatitis B Virus and Circulation of Immune-Escape Variants in the Russian Federation 20 Years after the Start of Mass Vaccination.

A neonatal vaccination against the Hepatitis B virus (HBV) infection was initiated in Russia 20 years ago, with catch-up immunization for adolescents and adults under the age of 60 years launched in 2006. Here, we have assessed the humoral immunity to HBV in different regions of Russia, as well as the infection frequency following 20 years of a nationwide vaccination campaign. We have also evaluated the role of immune-escape variants in continuing HBV circulation. A total of 36,149 healthy volunteers from nine regions spanning the Russian Federation from west to east were tested for HBV surface antigen (HBsAg), antibodies to HBV capsid protein (anti-HBc), and antibodies to HBsAg (anti-HBs). HBV sequences from 481 chronic Hepatitis B patients collected from 2018-2022 were analyzed for HBsAg immune-escape variants, compared with 205 sequences obtained prior to 2010. Overall, the HBsAg detection rate was 0.8%, with this level significantly exceeded only in one study region, the Republic of Dagestan (2.4%, p < 0.0001). Among the generation vaccinated at birth, the average HBsAg detection rate was below 0.3%, ranging from 0% to 0.7% depending on the region. The anti-HBc detection rate in subjects under 20 years was 7.4%, indicating ongoing HBV circulation. The overall proportion of participants under 20 years with vaccine-induced HBV immunity (anti-HBs positive, anti-HBc negative) was 41.7% but below 10% in the Tuva Republic and below 25% in the Sverdlovsk and Kaliningrad regions. The overall prevalence of immune-escape HBsAg variants was 25.2% in sequences obtained from 2018-2022, similar to the prevalence of 25.8% in sequences collected prior to 2010 (p > 0.05). The population dynamics of immune-escape variants predicted by Bayesian analysis have remained stable over the last 20 years, indicating the absence of vaccine-driven positive selection. In contrast, the wild-type HBV population size experienced a rapid decrease starting in the mid-1990s, following the introduction of mass immunization, but it subsequently began to recover, reaching pre-vaccination levels by 2020. Taken together, these data indicate that it is gaps in vaccination, and not virus evolution, that may be responsible for the continued virus circulation despite 20 years of mass vaccination.

Comprehensive engineering of a therapeutic neutralizing antibody targeting SARS-CoV-2 spike protein to neutralize escape variants.

The emergence of escape variants of SARS-CoV-2 carrying mutations in the spike protein poses a challenge for therapeutic antibodies. Here, we show that through the comprehensive engineering of the variable region of the neutralizing monoclonal antibody 5A6, the engineered antibody, 5A6CCS1, is able to neutralize SARS-CoV-2 variants that escaped neutralization by the original 5A6 antibody. In addition to the improved affinity against variants, 5A6CCS1 was also optimized to achieve high solubility and low viscosity, enabling a high concentration formulation for subcutaneous injection. In cynomolgus monkeys, 5A6CCS1 showed a long plasma half-life and good subcutaneous bioavailability through engineering of the variable and constant region. These data demonstrate that 5A6CCS1 is a promising antibody for development against SARS-CoV-2 and highlight the importance of antibody engineering as a potential method to counteract escape variants.

[Ten years of the National Reference Center for hepatitis B viruses and hepatitis D viruses in Giessen, Germany: activities and experiences]. / 10 Jahre Nationales Referenzzentrum für Hepatitis-B-Viren und Hepatitis-D-Viren in Gießen: Tätigkeiten und Erfahrungen.

The National Reference Center (NRC) for hepatitis B viruses (HBV) and hepatitis D viruses (HDV) has been located at the Institute of Medical Virology of the Justus Liebig University (JLU) in Giessen, Germany, since its establishment in 2011. This paper describes the NRC's areas of activity and related experience.The NRC offers comprehensive consulting services on all diagnostic and clinical aspects of acute and chronic HBV and HDV infections for the Public Health Service (ÖGD), diagnostic laboratories, clinics, research institutes, and physicians in private practice. Uncertain diagnostic findings can be analyzed and interpreted and epidemiological correlations clarified with the HBV/HDV special diagnostics established at the NRC using state-of-the-art molecular, biochemical, and genetic laboratory tools. The NRC has access to a strain collection of many well-characterized and cloned HBV/HDV isolates, allowing comparative analysis and evaluation of antiviral resistance mutations and immune escape variants. Together with its national and international partner institutions, the NRC initiates and supervises, among other things, interlaboratory studies for the diagnosis of HBV resistance and immune escape for the establishment and validation of international World Health Organization (WHO) standards and for the improvement of quantitative HDV genome determination. The NRC actively participates in current recommendations and guidelines on HBV and HDV and the recommendations of medical societies. It also highlights current HBV/HDV-relevant aspects with contributions in the form of national and international lectures as well as original articles and comments in national and international journals.

Comprehensive mapping of binding hot spots of SARS-CoV-2 RBD-specific neutralizing antibodies for tracking immune escape variants.

BACKGROUND: The receptor-binding domain (RBD) variants of SARS-CoV-2 could impair antibody-mediated neutralization of the virus by host immunity; thus, prospective surveillance of antibody escape mutants and understanding the evolution of RBD are urgently needed. METHODS: Using the single B cell cloning technology, we isolated and characterized 93 RBD-specific antibodies from the memory B cells of four COVID-19 convalescent individuals in the early stage of the pandemic. Then, global RBD alanine scanning with a panel of 19 selected neutralizing antibodies (NAbs), including several broadly reactive NAbs, was performed. Furthermore, we assessed the impact of single natural mutation or co-mutations of concern at key positions of RBD on the neutralization escape and ACE2 binding function by recombinant proteins and pseudoviruses. RESULTS: Thirty-three amino acid positions within four independent antigenic sites (1 to 4) of RBD were identified as valuable indicators of antigenic changes in the RBD. The comprehensive escape mutation map not only confirms the widely circulating strains carrying important immune escape RBD mutations such as K417N, E484K, and L452R, but also facilitates the discovery of new immune escape-enabling mutations such as F486L, N450K, F490S, and R346S. Of note, these escape mutations could not affect the ACE2 binding affinity of RBD, among which L452R even enhanced binding. Furthermore, we showed that RBD co-mutations K417N, E484K, and N501Y present in B.1.351 appear more resistant to NAbs and human convalescent plasma from the early stage of the pandemic, possibly due to an additive effect. Conversely, double mutations E484Q and L452R present in B.1.617.1 variant show partial antibody evasion with no evidence for an additive effect. CONCLUSIONS: Our study provides a global view of the determinants for neutralizing antibody recognition, antigenic conservation, and RBD conformation. The in-depth escape maps may have value for prospective surveillance of SARS-CoV-2 immune escape variants. Special attention should be paid to the accumulation of co-mutations at distinct major antigenic sites. Finally, the new broadly reactive NAbs described here represent new potential opportunities for the prevention and treatment of COVID-19.

SARS-CoV-2 variants and the so-called resistance to vaccines.

RNA viruses (except retroviruses) replicate by the action of an RNA-dependent RNA polymerase, which lacks a proofreading exonuclease and, consequently, errors may occur in each replication giving place to viral mutants. Depending on their fitness, these mutants either become extinct or thrive, spawning variants that escape the immune system. The most important SARS-CoV-2 mutations are those that alter the amino acid sequence in the viral S protein because this protein holds the key for the virus to enter the human cell. The more viruses replicate, the more they mutate, and the more likely it is that dominant resistant variants will appear. In such cases, more stringent measures for community protection will be required. Vaccines and polyclonal antibodies, which induce a response directed towards several sites along the S protein, would maintain effective protection against SARS-CoV-2 variants. Furthermore, vaccines appear to induce an increased helper and cytotoxic T-cell response, which may also be a biomarker of protection. In densely populated areas with insufficient protection measures, the virus spreads freely, thus increasing the likelihood of generating escape mutants. India and Manaus exemplify this situation. Natural evolution selects the mutants that multiply most efficiently without eliminating the host, thus facilitating their spread. Contrastingly, the circulation of viruses of high virulence and lethality (Ebola, hantavirus) that eliminate the host remain limited to certain geographic areas, without further dissemination. Therefore, it would be expected that SARS-CoV-2 will evolve into more infectious and less virulent variants.

Los virus ARN, excepto los retrovirus, se replican por acción de una ARN polimerasa ARN-dependiente que carece de exonucleasa correctora y, en consecuencia, en cada replicación puede cometer errores. Así se originan mutantes que, según su menor o mayor fitness, se extinguen o bien prosperan y originan variantes que escapan al sistema inmune. Las mutaciones de SARS-CoV-2 más importantes son las que alteran la proteína viral S, porque ella tiene la llave de ingreso del virus a la célula humana. Cuanto más se replican los virus, más mutan, y se hace más probable que aparezcan variantes resistentes dominantes. En esos casos, se requerirá una aplicación más estricta de las medidas de protección de la comunidad. Las vacunas y los anticuerpos policlonales, que inducen una respuesta dirigida hacia toda la proteína S, mantendrían protección efectiva contra las variantes del SARSCoV-2. Además, las vacunas inducirían una mayor respuesta de células T helper y citotóxicas, lo que puede ser un biomarcador de protección. En áreas densamente pobladas con escasas medidas de protección, el virus se difunde libremente y aumenta la probabilidad de mutaciones de escape. India y Manaos ejemplifican esa situación. La evolución natural selecciona las mutantes que se reproducen con mayor eficiencia sin eliminar al huésped, lo que facilita la propagación. En cambio, la circulación de virus de alta virulencia y letalidad (Ebola, hantavirus), que eliminan al huésped, se circunscribe a determinadas áreas geográficas, sin mayor difusión. Por lo tanto, sería esperable que SARS-CoV-2 evolucione a variantes más infecciosas y menos virulentas.

Las variantes de SARS-CoV-2 y la llamada resistencia a las vacunas / SARS-CoV-2 variants and the so-called resistance to vaccines

Resumen Los virus ARN, excepto los retrovirus, se replican por acción de una ARN polimerasa ARN-dependiente que carece de exonucleasa correctora y, en consecuencia, en cada replicación puede co meter errores. Así se originan mutantes que, según su menor o mayor fitness, se extinguen o bien prosperan y originan variantes que escapan al sistema inmune. Las mutaciones de SARS-CoV-2 más importantes son las que alteran la proteína viral S, porque ella tiene la llave de ingreso del virus a la célula humana. Cuanto más se replican los virus, más mutan, y se hace más probable que aparezcan variantes resistentes dominantes. En esos casos, se requerirá una aplicación más estricta de las medidas de protección de la comunidad. Las vacunas y los anticuerpos policlonales, que inducen una respuesta dirigida hacia toda la proteína S, mantendrían protec ción efectiva contra las variantes del SARS-CoV-2. Además, las vacunas inducirían una mayor respuesta de células T helper y citotóxicas, lo que puede ser un biomarcador de protección. En áreas densamente pobladas con escasas medidas de protección, el virus se difunde libremente y aumenta la probabilidad de mutaciones de escape. India y Manaos ejemplifican esa situación. La evolución natural selecciona las mutantes que se repro ducen con mayor eficiencia sin eliminar al huésped, lo que facilita la propagación. En cambio, la circulación de virus de alta virulencia y letalidad (Ebola, hantavirus), que eliminan al huésped, se circunscribe a determinadas áreas geográficas, sin mayor difusión. Por lo tanto, sería esperable que SARS-CoV-2 evolucione a variantes más infecciosas y menos virulentas.

Abstract RNA viruses (except retroviruses) replicate by the action of an RNA-dependent RNA polymerase, which lacks a proofreading exo nuclease and, consequently, errors may occur in each replication giving place to viral mutants. Depending on their fitness, these mutants either become extinct or thrive, spawning variants that escape the immune system. The most important SARS-CoV-2 mutations are those that alter the amino acid sequence in the viral S protein because this protein holds the key for the virus to enter the human cell. The more viruses replicate, the more they mutate, and the more likely it is that dominant resistant variants will appear. In such cases, more stringent measures for community protection will be required. Vaccines and polyclonal antibodies, which induce a response directed towards several sites along the S protein, would maintain effective protection against SARS-CoV-2 vari ants. Furthermore, vaccines appear to induce an increased helper and cytotoxic T-cell response, which may also be a biomarker of protection. In densely populated areas with insufficient protection measures, the virus spreads freely, thus increasing the likelihood of generating escape mutants. India and Manaus exemplify this situation. Natural evolution selects the mutants that multiply most efficiently without eliminating the host, thus facilitating their spread. Contrastingly, the circulation of viruses of high virulence and lethality (Ebola, hantavirus) that elimi nate the host remain limited to certain geographic areas, without further dissemination. Therefore, it would be expected that SARS-CoV-2 will evolve into more infectious and less virulent variants.

Characterization and genetic analysis of recent and emergent virulent newcastle disease viruses in Egypt.

Extensive vaccination against Newcastle disease virus (NDV) induced more selective immune pressure from hosts that enhanced the evolutionary process of NDV. Herein, we characterized 13 recently isolated NDV isolates from vaccinated chicken flocks during 2014-2017. Sequence analysis of F gene showed the presence of 112 RRQKRF117 velogenic cleavage motif in 11 isolates, whereas the other two isolates (Ck/ME3/Eg/16 and Ck/ME5/Eg/16) showed the monobasic motif 112 GGRQGRL117 . Interestingly, isolate Ck/ME5/Eg/16 showed 100% and 99.82% nucleotide identity with the LaSota F gene hypervariable region and full-length gene, respectively. On the other hand, isolate Ck/ME3/Eg/16 revealed natural recombination with strains NDV/Ck/Egypt/3/2006 and NDV/Teal/VRLCU/Egypt/2015 that indicates re-emergence of that old strain. Interestingly, all 13 isolates showed high intracerebral pathogenicity (ICPI) and mean death time (MDT) despite the presence of lentogenic motif in both Ck/ME5/Eg/16 and Ck/ME3/Eg/16 isolates. Comparative analysis of F antigenic epitopes in our isolates with other published sequences from Egypt revealed high sequence conservation; recent isolates had one fixed amino acid substitution (K78R) and a novel V168I substitution, whereas a D170N substitution was detected in older strains (NDV-EG-35-2014 and NDV-KFR-B7-2012). Taken together, our results support the first isolation of virulent NDV isolates with a lentogenic motif; isolate Ck/ME5/Eg/16 might be generated in nature from LaSota live vaccine, whereas isolate Ck/ME3/Eg/16 is emergent from NDV/Ck/Egypt/3/2006. We conclude that the current diagnostic evaluation of the virulence of NDV isolates by characteristic amino acid residues at the F protein cleavage site is insufficient. There is a need to link virologic and epidemiologic data together, and routine and emergency LaSota vaccination protocols should be carefully and optimally applied, with regards to the timing and presence of co-infecting agents in the field.

Substantial Antigenic Drift in the Hemagglutinin Protein of Swine Influenza A Viruses.

The degree of antigenic drift in swine influenza A viruses (swIAV) has historically been regarded as minimal compared to that of human influenza A virus strains. However, as surveillance activities on swIAV have increased, more isolates have been characterized, revealing a high level of genetic and antigenic differences even within the same swIAV lineage. The objective of this study was to investigate the level of genetic drift in one enzootically infected swine herd over one year. Nasal swabs were collected monthly from sows (n = 4) and piglets (n = 40) in the farrowing unit, and from weaners (n = 20) in the nursery. Virus from 1-4 animals were sequenced per month. Analyses of the sequences revealed that the hemagglutinin (HA) gene was the main target for genetic drift with a substitution rate of 7.6 × 10-3 substitutions/site/year and evidence of positive selection. The majority of the mutations occurred in the globular head of the HA protein and in antigenic sites. The phylogenetic tree of the HA sequences displayed a pectinate typology, where only a single lineage persists and forms the ancestor for subsequent lineages. This was most likely caused by repeated selection of a single immune-escape variant, which subsequently became the founder of the next wave of infections.

Correcting for Population Stratification Reduces False Positive and False Negative Results in Joint Analyses of Host and Pathogen Genomes.

Studies of host genetic determinants of pathogen sequence variations can identify sites of genomic conflicts, by highlighting variants that are implicated in immune response on the host side and adaptive escape on the pathogen side. However, systematic genetic differences in host and pathogen populations can lead to inflated type I (false positive) and type II (false negative) error rates in genome-wide association analyses. Here, we demonstrate through a simulation that correcting for both host and pathogen stratification reduces spurious signals and increases power to detect real associations in a variety of tested scenarios. We confirm the validity of the simulations by showing comparable results in an analysis of paired human and HIV genomes.

HCV Genotype 6a Escape From and Resistance to Velpatasvir, Pibrentasvir, and Sofosbuvir in Robust Infectious Cell Culture Models.

BACKGROUND & AIMS: Chronic liver diseases caused by hepatitis C virus (HCV) genotype 6 are prevalent in Asia, and millions of people require treatment with direct-acting antiviral regimens, such as NS5A inhibitor velpatasvir combined with the NS5B polymerase inhibitor sofosbuvir. We developed infectious cell culture models of HCV genotype 6a infection to study the effects of these inhibitors and the development of resistance. METHODS: The consensus sequences of strains HK2 (MG717925) and HK6a (MG717928), originating from serum of patients with chronic HCV infection, were determined by Sanger sequencing of genomes amplified by reverse-transcription polymerase chain reaction. In vitro noninfectious full-length clones of these 6a strains were subsequently adapted in Huh7.5 cells, primarily by using substitutions identified in JFH1-based Core-NS5A and Core-NS5B genotype 6a recombinants. We studied the efficacy of NS5A and NS5B inhibitors in concentration-response assays. We examined the effects of long-term culture of Huh7.5 cells incubated with velpatasvir and sofosbuvir singly or combined following infection with passaged full-length HK2 or HK6a recombinant viruses. Resistance-associated substitutions (RAS) were identified by Sanger and next-generation sequencing, and their effects on viral fitness and in drug susceptibility were determined in reverse-genetic experiments. RESULTS: Adapted full-length HCV genotype 6a recombinants HK2cc and HK6acc had fast propagation kinetics and high infectivity titers. Compared with an HCV genotype 1a recombinant, HCV genotype 6a recombinants of strains HK2 and HK6a were equally sensitive to daclatasvir, elbasvir, velpatasvir, pibrentasvir, and sofosbuvir, but less sensitive to ledipasvir, ombitasvir, and dasabuvir. Long-term exposure of HCV genotype 6a-infected Huh7.5 cells with a combination of velpatasvir and sofosbuvir resulted in clearance of the virus, but the virus escaped the effects of single inhibitors via emergence of the RAS L31V in NS5A (conferring resistance to velpatasvir) and S282T in NS5B (conferring resistance to sofosbuvir). Engineered recombinant genotype 6a viruses with single RAS mediated resistance to velpatasvir or sofosbuvir. HCV genotype 6a viruses with RAS NS5A-L31V or NS5B-S282T were however, able to propagate and escape in Huh7.5 cells exposed to the combination of velpatasvir and sofosbuvir. Further, HCV genotype 6a with NS5A-L31V was able to propagate and escape in the presence of pibrentasvir with emergence of NS5A-L28S, conferring a high level of resistance to this inhibitor. CONCLUSIONS: Strains of HCV genotype 6a isolated from patients can be adapted to propagate in cultured cells, permitting studies of the complete life cycle for this important genotype. The combination of velpatasvir and sofosbuvir is required to block propagation of original HCV genotype 6a, which quickly becomes resistant to single inhibitors via the rapid emergence and persistence of RAS. These features of HCV genotype 6a could compromise treatment.

Xer1-independent mechanisms of Vpma phase variation in Mycoplasma agalactiae are triggered by Vpma-specific antibodies.

Despite their small genomes mycoplasmas maintain large multigene families devoted to surface antigenic variation. Although implicated as important factors for mycoplasma pathogenicity and persistence, the role of these antigenic switches in host immune evasion has never been unequivocally proven in these minimalist microbes. Mycoplasma agalactiae exhibits antigenic variation due to Xer1-mediated site-specific DNA inversions of vpma genes encoding abundant multiple surface lipoproteins. To evaluate the biological significance of Vpma oscillations the xer1 recombinase gene has been disrupted in earlier studies to abolish Vpma switching and to generate stable phase-locked mutants (PLMs) steadily expressing a single Vpma product. However, in previous animal infection studies, surprisingly these PLMs switched to new different Vpma phenotypes. The aim of the current study was to demonstrate the influence of anti-Vpma antibodies on change of Vpma expression in PLMs as well as on the wildtype strain. In in vitro assays it is shown that wild type M. agalactiae escapes the negative effects of Vpma-specific antibodies by high-frequency Xer1-mediated switching to alternative Vpma phenoytpes. Even for Xer1-disrupted PLMs that stably expressed the same Vpma for several in vitro generations, the presence of the corresponding Vpma-specific antibody caused repression of the target Vpma and induction of new Vpma phenotypes by novel complex vpma rearrangements like intragenic deletions and gene chimeras. These Xer1-independent vpma recombinations correlated very well with similar PLM switches observed in vivo in an earlier independent study, clearly demonstrating that Vpma phase variation is necessary to express 'Vpma immune evasion proteins' in order to escape the immune response and to survive in the immunocompetent host. The data clearly demonstrate that although the Xer1 recombinase is the sole factor responsible for Vpma switching of wild type M. agalactiae in vitro, other alternative molecular switches operate in its absence under the selective pressure of the immune response. Furthermore, this evasion from the immune attack of the host involves complex vpma rearrangements, a causal relationship that was so far never demonstrated for M. agalactiae, thereby illustrating novel features of its regulation under immune pressure. The results are anticipated to have a direct impact on understanding the in vivo role of surface antigenic variation systems and the immune evasion tactics of other pathogenic mycoplasma species.

Performance of HBsAg point-of-care tests for detection of diagnostic escape-variants in clinical samples.

BACKGROUND: Hepatitis B viruses (HBV) harboring mutations in the a-determinant of the Hepatitis B surface antigen (HBsAg) are associated with reduced reactivity of HBsAg assays. OBJECTIVES: To evaluate the sensitivity and specificity of three HBsAg point-of-care tests for the detection of HBsAg of viruses harboring HBsAg mutations. STUDY DESIGN: A selection of 50 clinical plasma samples containing HBV with HBsAg mutations was used to evaluate the performance of three HBsAg point-of-care tests (Vikia(®), bioMérieux, Marcy-L'Étoile, France. Alere Determine HBsAg™, Iverness Biomedical Innovations, Köln, Germany. Quick Profile™, LumiQuick Diagnostics, California, USA) and compared to the ARCHITECT HBsAg Qualitative(®) assay (Abbott Laboratories, Sligo, Ireland). RESULTS: The sensitivity of the point-of-care tests ranged from 98% to 100%. The only false-negative result occurred using the Quick Profile™ assay with a virus harboring a D144A mutation. CONCLUSIONS: The evaluated point-of-care tests revealed an excellent sensitivity in detecting HBV samples harboring HBsAg mutations.

IFNγ producing CD8+ T cells modified to resist major immune checkpoints induce regression of MHC class I-deficient melanomas.

Tumors with reduced expression of MHC class I (MHC-I) molecules may be unrecognized by tumor antigen-specific CD8+ T cells and thus constitute a challenge for cancer immunotherapy. Here we monitored development of autochthonous melanomas in TiRP mice that develop tumors expressing a known tumor antigen as well as a red fluorescent protein (RFP) reporter knock in gene. The latter permits non-invasive monitoring of tumor growth by biofluorescence. One developing melanoma was deficient in cell surface expression of MHC-I, but MHC-I expression could be rescued by exposure of these cells to IFNγ. We show that CD8+ T cells specific for tumor antigen/MHC-I were efficient at inducing regression of the MHC-I-deficient melanoma, provided that the T cells were endowed with properties permitting their migration into the tumor and their efficient production of IFNγ. This was the case for CD8+ T cells transfected to express an active form of STAT5 (STAT5CA). The amount of IFNγ produced ex vivo from T cells present in tumors after adoptive transfer of the CD8+ T cells was correlated with an increase in surface expression of MHC-I molecules by the tumor cells. We also show that these CD8+ T cells expressed PD-1 and upregulated its ligand PDL-1 on melanoma cells within the tumor. Despite upregulation of this immunosuppressive pathway, efficient IFNγ production in the melanoma microenvironment was found associated with resistance of STAT5CA-expressing CD8+ T cells to inhibition both by PD-1/PDL-1 engagement and by TGFß1, two main immune regulatory mechanisms hampering the efficiency of immunotherapy in patients.

Hepatitis B virus PreS/S gene variants: pathobiology and clinical implications.

The emergence and takeover of hepatitis B virus (HBV) variants carrying mutation(s) in the preS/S genomic region is a fairly frequent event that may occur spontaneously or may be the consequence of immunoprophylaxis or antiviral treatments. Selection of preS/S mutants may have relevant pathobiological and clinical implications. Both experimental data and studies in humans show that several specific mutations in the preS/S gene may induce an imbalance in the synthesis of the surface proteins and their consequent retention within the endoplasmic reticulum (ER) of the hepatocytes. The accumulation of mutated surface proteins may cause ER stress with the consequent induction of oxidative DNA damage and genomic instability. Viral mutants with antigenically modified surface antigen may be potentially infectious to immune-prophylaxed patients and may account for cases of occult HBV infection. In addition, preS/S variants were reported to be associated with cases of fulminant hepatitis as well as of fibrosing cholestatic hepatitis, and they are associated with cirrhosis and hepatocellular carcinoma development.