The molecular constraints affecting Zika virus (ZIKV) evolution are not well understood. To investigate ZIKV genetic flexibility, we used transposon mutagenesis to add 15-nucleotide insertions ...throughout the ZIKV MR766 genome and subsequently deep sequenced the viable mutants. Few ZIKV insertion mutants replicated, which likely reflects a high degree of functional constraints on the genome. The NS1 gene exhibited distinct mutational tolerances at different stages of the screen. This result may define regions of the NS1 protein that are required for the different stages of the viral life cycle. The ZIKV structural genes showed the highest degree of insertional tolerance. Although the envelope (E) protein exhibited particular flexibility, the highly conserved envelope domain II (EDII) fusion loop of the E protein was intolerant of transposon insertions. The fusion loop is also a target of pan-flavivirus antibodies that are generated against other flaviviruses and neutralize a broad range of dengue virus and ZIKV isolates. The genetic restrictions identified within the epitopes in the EDII fusion loop likely explain the sequence and antigenic conservation of these regions in ZIKV and among multiple flaviviruses. Thus, our results provide insights into the genetic restrictions on ZIKV that may affect the evolution of this virus.
Zika virus recently emerged as a significant human pathogen. Determining the genetic constraints on Zika virus is important for understanding the factors affecting viral evolution. We used a genome-wide transposon mutagenesis screen to identify where mutations were tolerated in replicating viruses. We found that the genetic regions involved in RNA replication were mostly intolerant of mutations. The genes coding for structural proteins were more permissive to mutations. Despite the flexibility observed in these regions, we found that epitopes bound by broadly reactive antibodies were genetically constrained. This finding may explain the genetic conservation of these epitopes among flaviviruses.
Measles virus undergoes error-prone replication like other RNA viruses, but over time, it has remained antigenically monotypic. The constraints on the virus that prevent the emergence of antigenic ...variants are unclear. As a first step in understanding this question, we subjected the measles virus genome to unbiased insertional mutagenesis, and viruses that could tolerate insertions were rescued. Only insertions in the nucleoprotein, phosphoprotein, matrix protein, as well as intergenic regions were easily recoverable. Insertions in the glycoproteins of measles virus were severely under-represented in our screen. Host immunity depends on developing neutralizing antibodies to the hemagglutinin and fusion glycoproteins; our analysis suggests that these proteins occupy very little evolutionary space and therefore have difficulty changing in the face of selective pressures. We propose that the inelasticity of these proteins prevents the sequence variation required to escape antibody neutralization in the host, allowing for long-lived immunity after infection with the virus.
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•An insertional mutagenesis screen of the measles virus genome is performed•Some genomic regions are tolerant of insertions, but the F or H glycoproteins are not•In comparison to other viruses, the glycoproteins of measles are highly inflexible•The inflexibility of measles F and H may contribute to measles antigenic stability
It is unclear why measles virus is antigenically monotypic. Fulton et al. perform a viral mutagenesis screen and find that the glycoproteins (the major immune response targets) are exceptionally inflexible. These data suggest that the intrinsic rigidity of the glycoproteins is an important constraint on the development of antigenic diversity.
Antibodies targeting the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) present a promising approach to combat the coronavirus disease 2019 (COVID-19) pandemic; ...however, concerns remain that mutations can yield antibody resistance. We investigated the development of resistance against four antibodies to the spike protein that potently neutralize SARS-CoV-2, individually as well as when combined into cocktails. These antibodies remain effective against spike variants that have arisen in the human population. However, novel spike mutants rapidly appeared after in vitro passaging in the presence of individual antibodies, resulting in loss of neutralization; such escape also occurred with combinations of antibodies binding diverse but overlapping regions of the spike protein. Escape mutants were not generated after treatment with a noncompeting antibody cocktail.
Monoclonal antibodies against SARS-CoV-2 are a clinically validated therapeutic option against COVID-19. Because rapidly emerging virus mutants are becoming the next major concern in the fight ...against the global pandemic, it is imperative that these therapeutic treatments provide coverage against circulating variants and do not contribute to development of treatment-induced emergent resistance. To this end, we investigated the sequence diversity of the spike protein and monitored emergence of virus variants in SARS-COV-2 isolates found in COVID-19 patients treated with the two-antibody combination REGEN-COV, as well as in preclinical in vitro studies using single, dual, or triple antibody combinations, and in hamster in vivo studies using REGEN-COV or single monoclonal antibody treatments. Our study demonstrates that the combination of non-competing antibodies in REGEN-COV provides protection against all current SARS-CoV-2 variants of concern/interest and also protects against emergence of new variants and their potential seeding into the population in a clinical setting.
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•REGEN-COV retains neutralization potency against current variants of concern/interest•In vitro escape studies can predict emergence of viral variants in animals and humans•3 noncompeting mAb in combination reduce variant risk compared to a combination of 2•Treatment with REGEN-COV in humans does not lead to emergence of viral variants
Treatment with monoclonal antibody combinations limits generation of SARS-CoV-2 escape variants in humans and model animals and protects against current variants of concern.
Influenza A and B viruses can continuously evade humoral immune responses by developing mutations in the globular head of the hemagglutinin (HA) that prevent antibody binding. However, the influenza ...B virus HA over time displays less antigenic variation despite being functionally and structurally similar to the influenza A virus HA. To determine if the influenza B virus HA is under constraints that limit its antigenic variation, we performed a transposon screen to compare the mutational tolerance of the currently circulating influenza A virus HAs (H1 and H3 subtypes) and influenza B virus HAs (B/Victoria87 and B/Yamagata88 antigenic lineages). A library of insertional mutants for each HA was generated and deep sequenced after passaging to determine where insertions were tolerated in replicating viruses. The head domains of both viruses tolerated transposon mutagenesis, but the influenza A virus head was more tolerant to insertions than the influenza B virus head domain. Furthermore, all five of the known antigenic sites of the influenza A virus HA were tolerant of 15 nucleotide insertions, while insertions were detected in only two of the four antigenic sites in the influenza B virus head domain. Our analysis demonstrated that the influenza B virus HA is inherently less tolerant of transposon-mediated insertions than the influenza A virus HA. The reduced insertional tolerance of the influenza B virus HA may reveal genetic restrictions resulting in a lower capacity for antigenic evolution.
Influenza viruses cause seasonal epidemics and result in significant human morbidity and mortality. Influenza viruses persist in the human population through generating mutations in the hemagglutinin head domain that prevent antibody recognition. Despite the similar selective pressures on influenza A and B viruses, influenza A virus displays a higher rate and breadth of antigenic variability than influenza B virus. A transposon mutagenesis screen was used to examine if the reduced antigenic variability of influenza B virus was due to inherent differences in mutational tolerance. This study demonstrates that the influenza A virus head domain and the individual antigenic sites targeted by humoral responses are more tolerant to insertions than those of influenza B virus. This finding sheds light on the genetic factors controlling the antigenic evolution of influenza viruses.
Monoclonal antibodies can provide important pre- or post-exposure protection against infectious disease for those not yet vaccinated or in individuals that fail to mount a protective immune response ...after vaccination. Inmazeb (REGN-EB3), a three-antibody cocktail against Ebola virus, lessened disease and improved survival in a controlled trial. Here, we present the cryo-EM structure at 3.1 Å of the Ebola virus glycoprotein, determined without symmetry averaging, in a simultaneous complex with the antibodies in the Inmazeb cocktail. This structure allows the modeling of previously disordered portions of the glycoprotein glycan cap, maps the non-overlapping epitopes of Inmazeb, and illuminates the basis for complementary activities and residues critical for resistance to escape by these and other clinically relevant antibodies. We further provide direct evidence that Inmazeb protects against the rapid emergence of escape mutants, whereas monotherapies even against conserved epitopes do not, supporting the benefit of a cocktail versus a monotherapy approach.
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•Inmazeb antibody cocktail targets three discrete epitopes on Ebola virus GP trimer•High-resolution structure maps in detail the three contact sites•Inmazeb-GP structure defines GP glycan cap regions•Unlike monotherapies, Inmazeb cocktail prevents rapid selection of drug-resistant virus
Rayaprolu et al. combine structural and functional analysis to reveal the molecular details of the Inmazeb antibody cocktail, the first FDA-approved therapeutic effective against Ebola virus disease. They show that targeting non-overlapping epitopes of pathogens is an effective strategy to combat escape mutations and mitigate the risk of drug-induced viral resistance.
Influenza B virus is a human pathogen responsible for significant health and economic burden. Research into this pathogen has been limited by the lack of reporter viruses. Here we describe the ...development of both a replication-competent fluorescent influenza B reporter virus and bioluminescent influenza B reporter virus. Furthermore, we demonstrate these reporter viruses can be used to quickly monitor viral growth and permit the rapid screening of antiviral compounds and neutralizing antibodies.
Single-stranded RNA viruses are major human pathogens responsible for significant morbidity and mortality. Although all RNA viruses undergo error-prone replication, these viruses display different ...levels of antigenic variation over time. For example, influenza viruses rapidly generate mutations that prevent antibody recognition, while viruses like Zika and measles viruses remain serologically monotypic. This thesis was undertaken to determine if inherent differences in the tolerance for amino acid diversity lead to varying degrees of viral antigenic development. To address this question, a transposon mutagenesis approach was taken to examine the mutational tolerance of the antigenic domains of influenza, measles, and Zika viruses. We observed that the major antigenic targets of these viruses displayed different levels of tolerance to transposon mediated insertions. The influenza virus hemagglutinin was exceptionally tolerant of such mutations. However, the serologically monotypic measles virus was intolerant of, or highly attenuated by, insertions in the surface glycoproteins. Additionally, the majority of the Zika virus envelope protein was intolerant to insertions, except for a region known to be highly variable among flaviviruses of the same serotype. The contrasting tolerance of these glycoproteins to transposon insertions may be indicative of their capacity to bear the amino acid substitutions required for antigenic drift. The overall finding explains why influenza virus is antigenically diverse and can undergo rapid antigenic drift, while measles and Zika viruses are monotypic. Furthermore, the conserved stalk domain of the influenza virus hemagglutinin is less tolerant of transposon- induced mutations than the globular head of this protein. The genetic restrictions observed in the stalk domain may explain its conservation and the recent success of stalk- directed vaccine constructs to induce broadly neutralizing antibodies to multiple subtypes. Together, these findings shed light on the genetic factors controlling the antigenic evolution of these important pathogens and identify optimal epitopes unlikely to tolerate the mutations required for antigenic variation.
Necrotizing enterocolitis (NEC) is the leading cause of morbidity and mortality in premature infants. One of the most devastating complications of NEC is the development of NEC-induced brain injury, ...which manifests as impaired cognition that persists beyond infancy and which represents a proinflammatory activation of the gut-brain axis. Given that oral administration of the human milk oligosaccharides (HMOs) 2'-fucosyllactose (2'-FL) and 6'-sialyslactose (6'-SL) significantly reduced intestinal inflammation in mice, we hypothesized that oral administration of these HMOs would reduce NEC-induced brain injury and sought to determine the mechanisms involved. We now show that the administration of either 2'-FL or 6'-SL significantly attenuated NEC-induced brain injury, reversed myelin loss in the corpus callosum and midbrain of newborn mice, and prevented the impaired cognition observed in mice with NEC-induced brain injury. In seeking to define the mechanisms involved, 2'-FL or 6'-SL administration resulted in a restoration of the blood-brain barrier in newborn mice and also had a direct anti-inflammatory effect on the brain as revealed through the study of brain organoids. Metabolites of 2'-FL were detected in the infant mouse brain by nuclear magnetic resonance (NMR), whereas intact 2'-FL was not. Strikingly, the beneficial effects of 2'-FL or 6'-SL against NEC-induced brain injury required the release of the neurotrophic factor brain-derived neurotrophic factor (BDNF), as mice lacking BDNF were not protected by these HMOs from the development of NEC-induced brain injury. Taken in aggregate, these findings reveal that the HMOs 2'-FL and 6'-SL interrupt the gut-brain inflammatory axis and reduce the risk of NEC-induced brain injury.
This study reveals that the administration of human milk oligosaccharides, which are present in human breast milk, can interfere with the proinflammatory gut-brain axis and prevent neuroinflammation in the setting of necrotizing enterocolitis, a major intestinal disorder seen in premature infants.
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