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SARS-CoV-2 has a relatively high mutation rate, with the frequent emergence of new variants of concern (VOCs). Each subsequent variant is more difficult to neutralize by the sera of vaccinated individuals and convalescents. Some decrease in neutralizing activity against new SARS-CoV-2 variants has also been observed in patients vaccinated with Gam-COVID-Vac. In the present study, we analyzed the interplay between the history of a patient's repeated exposure to SARS-CoV-2 antigens and the breadth of neutralization activity. Our study includes four cohorts of patients: Gam-COVID-Vac booster vaccinated individuals (revaccinated, RV), twice-infected unvaccinated individuals (reinfected, RI), breakthrough infected (BI), and vaccinated convalescents (VC). We assessed S-protein-specific antibody levels and the ability of sera to neutralize lentiviral particles pseudotyped with Spike protein from the original Wuhan variant, as well as the Omicron variants BA.1 and BA.4/5. Individuals with hybrid immunity (BI and VC cohorts) exhibited significantly higher levels of virus-binding IgG and enhanced breadth of virus-neutralizing activity compared to individuals from either the revaccination or reinfection (RV and RI) cohorts. These findings suggest that a combination of infection and vaccination, regardless of the sequence, results in significantly higher levels of S-protein-specific IgG antibodies and the enhanced neutralization of SARS-CoV-2 variants, thereby underscoring the importance of hybrid immunity in the context of emerging viral variants. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 has a relatively high mutation rate, with the frequent emergence of new variants of concern (VOCs). Each subsequent variant is more difficult to neutralize by the sera of vaccinated individuals and convalescents. Some decrease in neutralizing activity against new SARS-CoV-2 variants has also been observed in patients vaccinated with Gam-COVID-Vac. In the present study, we analyzed the interplay between the history of a patient’s repeated exposure to SARS-CoV-2 antigens and the breadth of neutralization activity. Our study includes four cohorts of patients: Gam-COVID-Vac booster vaccinated individuals (revaccinated, RV), twice-infected unvaccinated individuals (reinfected, RI), breakthrough infected (BI), and vaccinated convalescents (VC). We assessed S-protein-specific antibody levels and the ability of sera to neutralize lentiviral particles pseudotyped with Spike protein from the original Wuhan variant, as well as the Omicron variants BA.1 and BA.4/5. Individuals with hybrid immunity (BI and VC cohorts) exhibited significantly higher levels of virus-binding IgG and enhanced breadth of virus-neutralizing activity compared to individuals from either the revaccination or reinfection (RV and RI) cohorts. These findings suggest that a combination of infection and vaccination, regardless of the sequence, results in significantly higher levels of S-protein-specific IgG antibodies and the enhanced neutralization of SARS-CoV-2 variants, thereby underscoring the importance of hybrid immunity in the context of emerging viral variants.

Viral diseases are a leading cause of worldwide yield losses in crop production. Breeding of resistance genes (R gene) into elite crop cultivars has been the standard and most cost-effective practice. However, R gene-mediated resistance is limited by the available R genes within genetic resources and in many cases, by strain specificity. Therefore, it is important to generate new and broad-spectrum antiviral strategies. The CRISPR-Cas9 (clustered regularly interspaced palindromic repeat, CRISPR-associated) editing system has been employed to confer resistance to human viruses and several plant single-stranded DNA geminiviruses, pointing out the possible application of the CRISPR-Cas9 system for virus control. Here, we demonstrate that strong viral resistance to cauliflower mosaic virus (CaMV), a pararetrovirus with a double-stranded DNA genome, can be achieved through Cas9-mediated multiplex targeting of the viral coat protein sequence. We further show that small interfering RNAs (siRNA) are produced and mostly map to the 3 end of single-guide RNAs (sgRNA), although very low levels of siRNAs map to the spacer region as well. However, these siRNAs are not responsible for the inhibited CaMV infection because there is no resistance if Cas9 is not present. We have also observed edited viruses in systematically infected leaves in some transgenic plants, with short deletions or insertions consistent with Cas9-induced DNA breaks at the sgRNA target sites in coat protein coding sequence. These edited coat proteins, in most cases, led to earlier translation stop and thus, nonfunctional coat proteins. We also recovered wild-type CP sequence in these infected transgenic plants, suggesting these edited viral genomes were packaged by wild-type coat proteins. Our data demonstrate that the CRISPR-Cas9 system can be used for virus control against plant pararetroviruses with further modifications.

Abstract Background We previously developed drug-like peptide triazoles (PTs) that target HIV-1 Envelope (Env) gp120, potently inhibit viral entry, and irreversibly inactivate virions. Here, we investigated potential mechanisms of viral escape from this promising class of HIV-1 entry inhibitors. Results HIV-1 resistance to cyclic (AAR029b) and linear (KR13) PTs was obtained by dose escalation in viral passaging experiments. High-level resistance for both inhibitors developed slowly (relative to escape from gp41-targeted C-peptide inhibitor C37) by acquiring mutations in gp120 both within (Val255) and distant to (Ser143) the putative PT binding site. The similarity in the resistance profiles for AAR029b and KR13 suggests that the shared IXW pharmacophore provided the primary pressure for HIV-1 escape. In single-round infectivity studies employing recombinant virus, V255I/S143N double escape mutants reduced PT antiviral potency by 150- to 3900-fold. Curiously, the combined mutations had a much smaller impact on PT binding affinity for monomeric gp120 (four to ninefold). This binding disruption was entirely due to the V255I mutation, which generated few steric clashes with PT in molecular docking. However, this minor effect on PT affinity belied large, offsetting changes to association enthalpy and entropy. The escape mutations had negligible effect on CD4 binding and utilization during entry, but significantly altered both binding thermodynamics and inhibitory potency of the conformationally-specific, anti-CD4i antibody 17b. Moreover, the escape mutations substantially decreased gp120 shedding induced by either soluble CD4 or AAR029b. Conclusions Together, the data suggest that the escape mutations significantly modified the energetic landscape of Env’s prefusogenic state, altering conformational dynamics to hinder PT-induced irreversible inactivation of Env. This work therein reveals a unique mode of virus escape for HIV-1, namely, resistance by altering the intrinsic conformational dynamics of the Env trimer.

Autophagy is a crucial and conserved homeostatic mechanism for early defense against viral infections. Recent studies indicate that coronaviruses (CoVs) have evolved various strategies to evade the autophagy–lysosome pathway. In this minireview, we describe the source of double-membrane vesicles during CoV infection, which creates a microenvironment that promotes viral RNA replication and virion synthesis and protects the viral genome from detection by the host. Firstly, CoVs hijack autophagy initiation through non-structural proteins and open-reading frames, leading to the use of non-nucleated phagophores and omegasomes for autophagy-derived double-membrane vesicles. Contrastingly, membrane rearrangement by hijacking ER-associated degradation machinery to form ER-derived double-membrane vesicles independent from the typical autophagy process is another important routine for the production of double-membrane vesicles. Furthermore, we summarize the molecular mechanisms by which CoV non-structural proteins and open-reading frames are used to intercept autophagic flux and thereby evade host clearance and innate immunity. A comprehensive understanding of the above mechanisms may contribute to developing novel therapies and clinical drugs against coronavirus disease 2019 (COVID-19) in the future. [ABSTRACT FROM AUTHOR]

Autophagy is a crucial and conserved homeostatic mechanism for early defense against viral infections. Recent studies indicate that coronaviruses (CoVs) have evolved various strategies to evade the autophagy–lysosome pathway. In this minireview, we describe the source of double-membrane vesicles during CoV infection, which creates a microenvironment that promotes viral RNA replication and virion synthesis and protects the viral genome from detection by the host. Firstly, CoVs hijack autophagy initiation through non-structural proteins and open-reading frames, leading to the use of non-nucleated phagophores and omegasomes for autophagy-derived double-membrane vesicles. Contrastingly, membrane rearrangement by hijacking ER-associated degradation machinery to form ER-derived double-membrane vesicles independent from the typical autophagy process is another important routine for the production of double-membrane vesicles. Furthermore, we summarize the molecular mechanisms by which CoV non-structural proteins and open-reading frames are used to intercept autophagic flux and thereby evade host clearance and innate immunity. A comprehensive understanding of the above mechanisms may contribute to developing novel therapies and clinical drugs against coronavirus disease 2019 (COVID-19) in the future.

Background: We previously developed drug-like peptide triazoles (PTs) that target HIV-1 Envelope (Env) gp120, potently inhibit viral entry, and irreversibly inactivate virions. Here, we investigated potential mechanisms of viral escape from this promising class of HIV-1 entry inhibitors. Results: HIV-1 resistance to cyclic (AAR029b) and linear (KR13) PTs was obtained by dose escalation in viral passaging experiments. High-level resistance for both inhibitors developed slowly (relative to escape from gp41-targeted C-peptide inhibitor C37) by acquiring mutations in gp120 both within (Val255) and distant to (Ser143) the putative PT binding site. The similarity in the resistance profiles for AAR029b and KR13 suggests that the shared IXW pharmacophore provided the primary pressure for HIV-1 escape. In single-round infectivity studies employing recombinant virus, V255I/S143N double escape mutants reduced PT antiviral potency by 150- to 3900-fold. Curiously, the combined mutations had a much smaller impact on PT binding affinity for monomeric gp120 (four to ninefold). This binding disruption was entirely due to the V255I mutation, which generated few steric clashes with PT in molecular docking. However, this minor effect on PT affinity belied large, offsetting changes to association enthalpy and entropy. The escape mutations had negligible effect on CD4 binding and utilization during entry, but significantly altered both binding thermodynamics and inhibitory potency of the conformationally-specific, anti-CD4i antibody 17b. Moreover, the escape mutations substantially decreased gp120 shedding induced by either soluble CD4 or AAR029b. Conclusions: Together, the data suggest that the escape mutations significantly modified the energetic landscape of Env's prefusogenic state, altering conformational dynamics to hinder PT-induced irreversible inactivation of Env. This work therein reveals a unique mode of virus escape for HIV-1, namely, resistance by altering the intrinsic conformational dynamics of the Env trimer. [ABSTRACT FROM AUTHOR]

The ΦX174 reduction effect describes a plasmid-based inhibitory phenomenon that mimics the superinfection inhibition found in wild phage populations. In this effect, when a portion of the ΦX174 genome – the 3′ end of the pilot protein gene (H), the 5' end of the replication gene (A), and the H-A intergenic region – is present on a plasmid in the host cell, almost complete protection from phage infection occurs. Here we demonstrate that only the phage pilot protein H portion of the plasmid is sufficient for the observed inhibition, that protein synthesis is necessary for inhibition to occur, that inserting the entire H gene in the plasmid may also impart a blocking effect, and that partial to complete recovery from this inhibition is possible with minimal viral evolution. [ABSTRACT FROM AUTHOR]

Newcastle disease virus (NDV) is a major cause of infectious mortality and morbidity in poultry worldwide. It is an enveloped virus with two outer-membrane proteins—haemagglutinin-neuraminidase (HN) and fusion protein (F)—that induce neutralizing antibodies. All NDV strains belong to one serotype. Yet, NDV vaccines, derived from genotype II, do not fully prevent infection or shedding of viruses from other genotypes. The aim of this study was to test if an updated vaccine is required. For this purpose, NDVs isolated from infected, albeit heavily vaccinated, flocks were genetically and immunologically characterized. Amino acid differences in F and HN protein sequences were identified between the vaccine strain and each of the isolates, some specifically at the neutralization sites. Whereas all tested isolates showed similar haemagglutination-inhibition (HI) titres, 100-100,000 times higher antibody-to-virus ratios were needed to neutralize viral propagation in embryos by the field isolates versus the vaccine strain. As a result, a model and an equation were developed to explain the phenomenon of escape in one-serotype viruses and to calculate the HI values needed for protection, depending on variation rate at key positions. In conclusion, to confer full protection against NDVs that differ from the vaccine strain at the neutralizing epitopes, very high levels of antibodies should be raised and maintained to compensate for the reduction in the number of effective epitopes; alternatively, an adjusted attenuated vaccine should be developed—a task made possible in the current era of reverse vaccinology. [ABSTRACT FROM AUTHOR]

In bacteria and archaea, the clustered regularly interspaced short palindromic repeats (CRISPR) and associated proteins (Cas) confer adaptive immunity against exogenous DNA elements. This CRISPR-Cas system has been turned into an effective tool for editing of eukaryotic DNA genomes. Pathogenic viruses that have a double-stranded DNA (dsDNA) genome or that replicate through a dsDNA intermediate can also be targeted with this DNA editing tool. Here, we review how CRISPR-Cas was used in novel therapeutic approaches against the human immunodeficiency virus type-1 (HIV-1), focusing on approaches that aim to permanently inactivate all virus genomes or to prevent viral persistence in latent reservoirs. [ABSTRACT FROM AUTHOR]

We characterized 55 influenza A(H9N2) viruses isolated in Pakistan during 2014-2016 and found that the hemagglutinin gene is of the G1 lineage and that internal genes have differentiated into a variety of novel genotypes. Some isolates had up to 4-fold reduction in hemagglutination inhibition titers compared with older viruses. Viruses with hemagglutinin A180T/V substitutions conveyed this antigenic diversity and also caused up to 3,500-fold greater binding to avian-like and >20-fold greater binding to human-like sialic acid receptor analogs. This enhanced binding avidity led to reduced virus replication in primary and continuous cell culture. We confirmed that altered receptor-binding avidity of H9N2 viruses, including enhanced binding to human-like receptors, results in antigenic variation in avian influenza viruses. Consequently, current vaccine formulations might not induce adequate protective immunity in poultry, and emergence of isolates with marked avidity for human-like receptors increases the zoonotic risk. [ABSTRACT FROM AUTHOR]

Purpose of Review: The purpose of this study is to summarize recent advances in the use of broadly neutralizing antibodies (bNAbs) as therapeutics in human clinical trials and in non-human primate (NHP) models. We seek to highlight lessons from these studies with an emphasis on consequences to the virus and immune system. Recent Findings: In the past 10 years, advances in HIV-1 trimer structure and B cell isolation methods have precipitated the identification of 'new-generation' anti-HIV antibodies with broad and potent neutralization. In the past 2 years, the concept of using these bNAbs as therapeutic tools has moved from NHP models into human clinical trials. These trials have investigated the effects of bNAb infusions into patients chronically infected with HIV-1, while the NHP model has investigated treatment during acute infection. Summary: Through this work, the relationship between in vitro breadth and potency and in vivo clinical effect, although unresolved, is gradually being elucidated. These results emphasize the need for combination antibody therapy. [ABSTRACT FROM AUTHOR]

Highlights: [•] HIV-infected pregnant women have evidence of HBV escape compared to uninfected women. [•] One in six HBV-infected pregnant women is HBeAg seropositive, regardless of HIV status. [•] These data support the call to implement a birth dose of HBV vaccine. [ABSTRACT FROM AUTHOR]

In addition to CD8+ cytotoxic T lymphocyte (CTL) responses, neutralizing antibodies contribute substantially to the long-term immune control of noncytopathic viruses, as demonstrated during infection with the lymphocytic choriomeningitis virus (LCMV). The high virus load during the initial phase of an infection and the ability of this RNA virus to spontaneously acquire mutations are important prerequisites for escaping an ongoing immune response. In this context, LCMV escape from the humoral response by single point mutations in neutralizing envelope protein determinants may occur, particularly during conditions of CTL deficiency, leading to virus persistence. [ABSTRACT FROM AUTHOR]

Background & Aim Hepatitis D virus (HDV) superinfection of patients with chronic HBV infection results in rapid progression to liver cirrhosis. Little is known about HDV-specific T cells and how they contribute to the antiviral immune response and liver disease pathogenesis. Methods We isolated peripheral blood mononuclear cells from 28 patients with chronic HDV and HBV infection, identified HDV-specific CD8+ T-cell epitopes, and characterized HDV-specific CD8+ T cells. We associated these with HDV sequence variations and clinical features of patients. Results We identified 6 CD8+ T-cell epitopes; several were restricted by multiple HLA class I alleles. HDV-specific CD8+ T cells were as frequent as HBV-specific CD8+ T cells but were less frequent than T cells with specificity for cytomegalovirus, Epstein–Barr virus, or influenza virus. The ex vivo frequency of activated HDV-specific CD8+ T cells correlated with transaminase activity. CD8+ T-cell production of interferon gamma after stimulation with HDV peptides correlated inversely with HDV titer. HDV-specific CD8+ T cells did not express the terminal differentiation marker CD57, and fewer HDV-specific than Epstein–Barr virus–specific CD8+ T cells were 2B4+CD160+PD1+, a characteristic of exhausted cells. Approximately half of the HDV-specific CD8+ T cells had a memory-like PD1+CD127+TCF1hiT-betlow phenotype, which associated with HDV sequence variants with reduced HLA binding and reduced T-cell activation. Conclusions CD8+ T cells isolated from patients with chronic HDV and HBV infection recognize HDV epitopes presented by multiple HLA molecules. The subset of activated HDV-specific CD8+ T cells targets conserved epitopes and likely contributes to disease progression. The subset of memory-like HDV-specific CD8+ T cells is functional but unable to clear HDV because of the presence of escape variants. ClinicalTrials.gov , Numbers: NCT02511431 , NCT00023322 , NCT01495585 , and NCT00001971. GenBank accession, Number: MK333199-333226 Graphical abstract [ABSTRACT FROM AUTHOR]

Background & Aim: Hepatitis D virus (HDV) superinfection of patients with chronic HBV infection results in rapid progression to liver cirrhosis. Little is known about HDV-specific T cells and how they contribute to the antiviral immune response and liver disease pathogenesis., Methods: We isolated peripheral blood mononuclear cells from 28 patients with chronic HDV and HBV infection, identified HDV-specific CD8 + T-cell epitopes, and characterized HDV-specific CD8 + T cells. We associated these with HDV sequence variations and clinical features of patients., Results: We identified 6 CD8 + T-cell epitopes; several were restricted by multiple HLA class I alleles. HDV-specific CD8 + T cells were as frequent as HBV-specific CD8 + T cells but were less frequent than T cells with specificity for cytomegalovirus, Epstein-Barr virus, or influenza virus. The ex vivo frequency of activated HDV-specific CD8 + T cells correlated with transaminase activity. CD8 + T-cell production of interferon gamma after stimulation with HDV peptides correlated inversely with HDV titer. HDV-specific CD8 + T cells did not express the terminal differentiation marker CD57, and fewer HDV-specific than Epstein-Barr virus-specific CD8 + T cells were 2B4 + CD160 + PD1 + , a characteristic of exhausted cells. Approximately half of the HDV-specific CD8 + T cells had a memory-like PD1 + CD127 + TCF1 hi T-bet low phenotype, which associated with HDV sequence variants with reduced HLA binding and reduced T-cell activation., Conclusions: CD8 + T cells isolated from patients with chronic HDV and HBV infection recognize HDV epitopes presented by multiple HLA molecules. The subset of activated HDV-specific CD8 + T cells targets conserved epitopes and likely contributes to disease progression. The subset of memory-like HDV-specific CD8 + T cells is functional but unable to clear HDV because of the presence of escape variants. ClinicalTrials.gov, Numbers: NCT02511431, NCT00023322, NCT01495585, and NCT00001971. GenBank accession, Number: MK333199-333226., (Copyright © 2019 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Summary Apex broadly neutralizing HIV antibodies (bnAbs) recognize glycans and protein surface close to the 3-fold axis of the envelope (Env) trimer and are among the most potent and broad Abs described. The evolution of apex bnAbs from one donor (CAP256) has been studied in detail and many Abs at different stages of maturation have been described. Using diverse engineering tools, we investigated the involvement of glycan recognition in the development of the CAP256.VRC26 Ab lineage. We found that sialic acid-bearing glycans were recognized by germline-encoded and somatically mutated residues on the Ab heavy chain. This recognition provided an “anchor” for the Abs as the core protein epitope varies, prevented complete neutralization escape, and eventually led to broadening of the response. These findings illustrate how glycan-specific maturation enables a human Ab to cope with pathogen escape mechanisms and will aid in optimization of immunization strategies to induce V2 apex bnAb responses. [ABSTRACT FROM AUTHOR]