High genetic variability of Schmallenberg virus M-segment leads to efficient immune escape from neutralizing antibodies

Schmallenberg virus (SBV) is the cause of severe fetal malformations when immunologically naïve pregnant ruminants are infected. In those malformed fetuses, a “hot-spot”-region of high genetic variability within the N-terminal region of the viral envelope protein Gc has been observed previously, and this region co-localizes with a known key immunogenic domain. We studied a series of M-segments of those SBV variants from malformed fetuses with point mutations, insertions or large in-frame deletions of up to 612 nucleotides. Furthermore, a unique cell-culture isolate from a malformed fetus with large in-frame deletions within the M-segment was analyzed. Each Gc-protein with amino acid deletions within the “hot spot” of mutations failed to react with any neutralizing anti-SBV monoclonal antibodies or a domain specific antiserum. In addition, in vitro virus replication of the natural deletion variant could not be markedly reduced by neutralizing monoclonal antibodies or antisera from the field. The large-deletion variant of SBV that could be isolated in cell culture was highly attenuated with an impaired in vivo replication following the inoculation of sheep. In conclusion, the observed amino acid sequence mutations within the N-terminal main immunogenic domain of glycoprotein Gc result in an efficient immune evasion from neutralizing antibodies in the special environment of a developing fetus. These SBV-variants were never detected as circulating viruses, and therefore should be considered to be dead-end virus variants, which are not able to spread further. The observations described here may be transferred to other orthobunyaviruses, particularly those of the Simbu serogroup that have been shown to infect fetuses. Importantly, such mutant strains should not be included in attempts to trace the spatial-temporal evolution of orthobunyaviruses in molecular-epidemiolocal approaches during outbreak investigations.

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Assorted Mutations in the Envelope Gene of Simian Immunodeficiency Virus Lead to Loss of Neutralization Resistance against Antibodies Representing a Broad Spectrum of Specificities

Journal of Virology ◽

10.1128/jvi.77.18.9993-10003.2003 ◽

2003 ◽

Vol 77 (18) ◽

pp. 9993-10003 ◽

Cited By ~ 94

Author(s):

Welkin E. Johnson ◽

Hannah Sanford ◽

Linda Schwall ◽

Dennis R. Burton ◽

Paul W. H. I. Parren ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Amino Acid ◽

Simian Immunodeficiency Virus ◽

Relative Sensitivity ◽

Viral Envelope ◽

Envelope Gene ◽

Attachment Sites ◽

Immunodeficiency Virus ◽

Increased Sensitivity

ABSTRACT Simian immunodeficiency virus (SIV) of macaques isolate SIVmac239 is highly resistant to neutralization by polyclonal antisera or monoclonal antibodies, a property that it shares with most primary isolates of human immunodeficiency virus type 1 (HIV-1). This resistance is important for the ability of the virus to persist at high levels in vivo. To explore the physical features of the viral envelope complex that contribute to the neutralization-resistant phenotype, we examined a panel of SIVmac239 derivatives for sensitivity to neutralization by a large collection of monoclonal antibodies (MAbs). These MAbs recognize both linear and conformational epitopes throughout the viral envelope proteins. The variant viruses included three derivatives of SIVmac239 with substitutions in specific N-linked glycosylation sites of gp120 and a fourth variant that lacked the100 amino acids that encompass the V1 and V2 loops. Also included in this study was SIVmac316, a variant of SIVmac239 with distributed mutations in env that confer significantly increased replicative capacity in tissue macrophages. These viruses were chosen to represent a broad range of neutralization sensitivities based on susceptibility to pooled, SIV-positive plasma. All three of these very different kinds of mutations (amino acid substitutions, elimination of N-glycan attachment sites, and a 100-amino-acid deletion spanning variable loops V1 and V2) dramatically increased sensitivity to neutralization by MAbs from multiple competition groups. Thus, the mutations did not simply expose localized epitopes but rather conferred global increases in neutralization sensitivity. The removal of specific N-glycan attachment sites from V1 and V2 led to increased sensitivity to neutralization by antibodies recognizing epitopes from both within and outside of the V1-V2 sequence. Surprisingly, while most of the mutations that gave rise to increased sensitivity were located in the N-terminal half of gp120 (surface subunit [SU]), the greatest increases in sensitivity were to MAbs recognizing the C-terminal half of gp120 or the ectodomain of gp41 (transmembrane subunit [TM]). This reagent set and information should now be useful for defining the physical, structural, thermodynamic, and kinetic factors that influence relative sensitivity to antibody-mediated neutralization.

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Rapid determination of neutralizing antibodies to Semliki Forest virus in serum by enzyme immunoassay in cell culture with virus-specific monoclonal antibodies.

Journal of Clinical Microbiology ◽

10.1128/jcm.24.4.665-668.1986 ◽

1986 ◽

Vol 24 (4) ◽

pp. 665-668 ◽

Cited By ~ 7

Author(s):

F H van Tiel ◽

T Harmsen ◽

M Wagenaar ◽

W A Boere ◽

C A Kraaijeveld ◽

...

Keyword(s):

Cell Culture ◽

Monoclonal Antibodies ◽

Enzyme Immunoassay ◽

Neutralizing Antibodies ◽

Semliki Forest Virus ◽

Rapid Determination

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Application of immunoassay of encephalomyocarditis virus in cell culture with enzyme-labeled virus-specific monoclonal antibodies for rapid detection of virus, neutralizing antibodies, and interferon.

Journal of Clinical Microbiology ◽

10.1128/jcm.26.12.2593-2597.1988 ◽

1988 ◽

Vol 26 (12) ◽

pp. 2593-2597 ◽

Cited By ~ 5

Author(s):

F Vlaspolder ◽

T Harmsen ◽

D van Veenendaal ◽

C A Kraaijeveld ◽

H Snippe

Keyword(s):

Cell Culture ◽

Monoclonal Antibodies ◽

Rapid Detection ◽

Neutralizing Antibodies ◽

Encephalomyocarditis Virus

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A New Quaternary Structure Epitope on Dengue Virus Serotype 2 Is the Target of Durable Type-Specific Neutralizing Antibodies

mBio ◽

10.1128/mbio.01461-15 ◽

2015 ◽

Vol 6 (5) ◽

Cited By ~ 55

Author(s):

E. N. Gallichotte ◽

D. G. Widman ◽

B. L. Yount ◽

W. M. Wahala ◽

A. Durbin ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Dengue Virus ◽

Neutralizing Antibodies ◽

Quaternary Structure ◽

Natural Infection ◽

Viral Envelope ◽

Protective Response ◽

Denv Serotypes ◽

Virus Serotype ◽

Dengue Virus Serotype 2

ABSTRACT Dengue virus serotype 2 (DENV2) is widespread and responsible for severe epidemics. While primary DENV2 infections stimulate serotype-specific protective responses, a leading vaccine failed to induce a similar protective response. Using human monoclonal antibodies (hMAbs) isolated from dengue cases and structure-guided design of a chimeric DENV, here we describe the major site on the DENV2 envelope (E) protein targeted by neutralizing antibodies. DENV2-specific neutralizing hMAb 2D22 binds to a quaternary structure epitope. We engineered and recovered a recombinant DENV4 that displayed the 2D22 epitope. DENV2 neutralizing antibodies in people exposed to infection or a live vaccine tracked with the 2D22 epitope on the DENV4/2 chimera. The chimera remained sensitive to DENV4 antibodies, indicating that the major neutralizing epitopes on DENV2 and -4 are at different sites. The ability to transplant a complex epitope between DENV serotypes demonstrates a hitherto underappreciated structural flexibility in flaviviruses, which could be harnessed to develop new vaccines and diagnostics. IMPORTANCE Dengue virus causes fever and dengue hemorrhagic fever. Dengue serotype 2 (DENV2) is widespread and frequently responsible for severe epidemics. Natural DENV2 infections stimulate serotype-specific neutralizing antibodies, but a leading DENV vaccine did not induce a similar protective response. While groups have identified epitopes of single monoclonal antibodies (MAbs), the molecular basis of DENV2 neutralization by polyclonal human immune sera is unknown. Using a recombinant DENV displaying serotype 2 epitopes, here we map the main target of DENV2 polyclonal neutralizing antibodies induced by natural infection and a live DENV2 vaccine candidate. Proper display of the epitope required the assembly of viral envelope proteins into higher-order structures present on intact virions. Despite the complexity of the epitope, it was possible to transplant the epitope between DENV serotypes. Our findings have immediate implications for evaluating dengue vaccines in the pipeline as well as designing next-generation vaccines.

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Experimental estimation of the effects of all amino-acid mutations to HIV Env

10.1101/067470 ◽

2016 ◽

Author(s):

Hugh K. Haddox ◽

Adam S. Dingens ◽

Jesse D. Bloom

Keyword(s):

Cell Culture ◽

Amino Acid ◽

Neutralizing Antibodies ◽

Purifying Selection ◽

Sequence Evolution ◽

Broadly Neutralizing Antibodies ◽

Naturally Occurring ◽

Rapid Sequence ◽

Amino Acid Mutations

AbstractHIV is notorious for its capacity to evade immunity and anti-viral drugs through rapid sequence evolution. Knowledge of the functional effects of mutations to HIV is critical for understanding this evolution. HIV’s most rapidly evolving protein is its envelope (Env). Here we use deep mutational scanning to experimentally estimate the effects of all amino-acid mutations to Env on viral replication in cell culture. Most mutations are under purifying selection in our experiments, although a few sites experience strong selection for mutations that enhance HIV’s growth in cell culture. We compare our experimental measurements of each site’s preference for each amino acid to the actual frequencies of these amino acids in naturally occurring HIV sequences. Our measured amino-acid preferences correlate with amino-acid frequencies in natural sequences for most sites. However, our measured preferences are less concordant with natural amino-acid frequencies at surface-exposed sites that are subject to pressures absent from our experiments such as antibody selection. We show that some regions of Env have a high inherent tolerance to mutation, whereas other regions (such as epitopes of broadly neutralizing antibodies) have a significantly reduced capacity to tolerate mutations. Overall, our results help disentangle the role of inherent functional constraints and external selection pressures in shaping Env’s evolution.

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Complete mapping of viral escape from neutralizing antibodies

10.1101/086611 ◽

2016 ◽

Cited By ~ 1

Author(s):

Michael B. Doud ◽

Scott E. Hensley ◽

Jesse D. Bloom

Keyword(s):

Monoclonal Antibodies ◽

Amino Acid ◽

Neutralizing Antibodies ◽

Viral Evolution ◽

The Other ◽

Viral Escape ◽

Amino Acid Mutation ◽

Influenza Hemagglutinin ◽

Complete Mapping ◽

Protein Variants

AbstractIdentifying viral mutations that confer escape from antibodies is crucial for understanding the interplay between immunity and viral evolution. Here we quantify how every amino-acid mutation to influenza hemagglutinin affects neutralization by monoclonal antibodies targeting several antigenic regions. Our approach involves creating all replication-competent protein variants of the virus, selecting these variants with antibody, and using deep sequencing to identify enriched mutations. These high-throughput measurements are predictive of the effects of individual mutations in traditional neutralization assays. At many residues, only some of the possible mutations escape from an antibody. For instance, at a single residue targeted by two different antibodies, we identify some mutations that escape both antibodies and other mutations that escape only one or the other. Therefore, our approach maps how viruses can escape antibodies with mutation-level sensitivity, and shows that only some mutations at antigenic residues actually alter antigenicity.

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The Impact on Infectivity and Neutralization Efficiency of SARS-CoV-2 Lineage B.1.351 Pseudovirus

Viruses ◽

10.3390/v13040633 ◽

2021 ◽

Vol 13 (4) ◽

pp. 633

Author(s):

Yeong Jun Kim ◽

Ui Soon Jang ◽

Sandrine M. Soh ◽

Joo-Youn Lee ◽

Hye-Ra Lee

Keyword(s):

South Africa ◽

Monoclonal Antibodies ◽

Cell Fusion ◽

Receptor Binding ◽

Neutralizing Antibodies ◽

Receptor Binding Domain ◽

Viral Spread ◽

New Variant ◽

Global Concern ◽

The Impact

A new variant of SARS-CoV-2 B.1.351 lineage (first found in South Africa) has been raising global concern due to its harboring of multiple mutations in the spike that potentially increase transmissibility and yield resistance to neutralizing antibodies. We here tested infectivity and neutralization efficiency of SARS-CoV-2 spike pseudoviruses bearing particular mutations of the receptor-binding domain (RBD) derived either from the Wuhan strains (referred to as D614G or with other sites) or the B.1.351 lineage (referred to as N501Y, K417N, and E484K). The three different pseudoviruses B.1.351 lineage related significantly increased infectivity compared with other mutants that indicated Wuhan strains. Interestingly, K417N and E484K mutations dramatically enhanced cell–cell fusion than N501Y even though their infectivity were similar, suggesting that K417N and E484K mutations harboring SARS-CoV-2 variant might be more transmissible than N501Y mutation containing SARS-CoV-2 variant. We also investigated the efficacy of two different monoclonal antibodies, Casirivimab and Imdevimab that neutralized SARS-CoV-2, against several kinds of pseudoviruses which indicated Wuhan or B.1.351 lineage. Remarkably, Imdevimab effectively neutralized B.1.351 lineage pseudoviruses containing N501Y, K417N, and E484K mutations, while Casirivimab partially affected them. Overall, our results underscore the importance of B.1.351 lineage SARS-CoV-2 in the viral spread and its implication for antibody efficacy.

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