A combination of RBD and NTD neutralizing antibodies limits the generation of SARS-CoV-2 spike neutralization-escape mutants

Most known SARS-CoV-2 neutralizing antibodies (nAbs), including those approved by the FDA for emergency use, inhibit viral infection by targeting the receptor-binding domain (RBD) of the spike (S) protein. Variants of concern (VOC) carrying mutations in the RBD or other regions of S reduce the effectiveness of many nAbs and vaccines by evading neutralization. Therefore, therapies that are less susceptible to resistance are urgently needed. Here, we characterized the memory B-cell repertoire of COVID-19 convalescent donors and analyzed their RBD and non-RBD nAbs. We found that many of the non-RBD-targeting nAbs were specific to the N-terminal domain (NTD). Using neutralization assays with authentic SARS-CoV-2 and a recombinant vesicular stomatitis virus carrying SARS-CoV-2 S protein (rVSV-SARS2), we defined a panel of potent RBD and NTD nAbs. Next, we used a combination of neutralization-escape rVSV-SARS2 mutants and a yeast display library of RBD mutants to map their epitopes. The most potent RBD nAb competed with hACE2 binding and targeted an epitope that includes residue F490. The most potent NTD nAb epitope included Y145, K150 and W152. As seen with some of the natural VOC, the neutralization potencies of COVID-19 convalescent sera were reduced by 4-16-fold against rVSV-SARS2 bearing Y145D, K150E or W152R spike mutations. Moreover, we found that combining RBD and NTD nAbs modestly enhanced their neutralization potential. Notably, the same combination of RBD and NTD nAbs limited the development of neutralization-escape mutants in vitro, suggesting such a strategy may have higher efficacy and utility for mitigating the emergence of VOC.

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Nucleic acid delivery of immune-focused SARS-CoV-2 nanoparticles drive rapid and potent immunogenicity capable of single-dose protection

10.1101/2021.04.28.441474 ◽

2021 ◽

Author(s):

Kylie M. Konrath ◽

Kevin Liaw ◽

Yuanhan Wu ◽

Xizhou Zhu ◽

Susanne N. Walker ◽

...

Keyword(s):

Single Dose ◽

Neutralizing Antibodies ◽

Dna Delivery ◽

Nucleic Acid Delivery ◽

Wild Type ◽

Cell Repertoire ◽

B Cell Repertoire ◽

Induced Immunity ◽

Human B Cell

AbstractAntibodies from SARS-CoV-2 vaccines may target epitopes which reduce durability or increase the potential for escape from vaccine-induced immunity. Using a novel synthetic vaccinology pipeline, we developed rationally immune focused SARS-CoV-2 Spike-based vaccines. N-linked glycans can be employed to alter antibody responses to infection and vaccines. Utilizing computational modeling and comprehensive in vitro screening, we incorporated glycans into the Spike Receptor-Binding Domain (RBD) and assessed antigenic profiles. We developed glycan coated RBD immunogens and engineered seven multivalent configurations. Advanced DNA delivery of engineered nanoparticle vaccines rapidly elicited potent neutralizing antibodies in guinea pigs, hamsters and multiple mouse models, including human ACE2 and human B cell repertoire transgenics. RBD nanoparticles encoding wild-type and the P.1 SARS-CoV-2 variant induced high levels of cross-neutralizing antibodies. Single, low dose immunization protected against a lethal SARS-CoV-2 challenge. Single-dose coronavirus vaccines via DNA-launched nanoparticles provide a platform for rapid clinical translation of novel, potent coronavirus vaccines.

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Persistence of Viremia and Production of Neutralizing Antibodies Differentially Regulated by Polymorphic APOBEC3 and BAFF-R Loci in Friend Virus-Infected Mice

Journal of Virology ◽

10.1128/jvi.02516-09 ◽

2010 ◽

Vol 84 (12) ◽

pp. 6082-6095 ◽

Cited By ~ 27

Author(s):

Sachiyo Tsuji-Kawahara ◽

Tomomi Chikaishi ◽

Eri Takeda ◽

Maiko Kato ◽

Saori Kinoshita ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Early Stage ◽

Chromosome 15 ◽

Cell Repertoire ◽

Viral Loads ◽

Functional Polymorphisms ◽

B Cell Repertoire ◽

Retroviral Replication ◽

Different Strains ◽

Host Genes

ABSTRACT Several host genes control retroviral replication and pathogenesis through the regulation of immune responses to viral antigens. The Rfv3 gene influences the persistence of viremia and production of virus-neutralizing antibodies in mice infected with Friend mouse retrovirus complex (FV). This locus has been mapped within a narrow segment of mouse chromosome 15 harboring the APOBEC3 and BAFF-R loci, both of which show functional polymorphisms among different strains of mice. The exon 5-lacking product of the APOBEC3 allele expressed in FV-resistant C57BL/6 (B6) mice directly restricts viral replication, and mice lacking the B6-derived APOBEC3 exhibit exaggerated pathology and reduced production of neutralizing antibodies. However, the mechanisms by which the polymorphisms at the APOBEC3 locus affect the production of neutralizing antibodies remain unclear. Here we show that the APOBEC3 genotypes do not directly affect the B-cell repertoire, and mice lacking B6-derived APOBEC3 still produce FV-neutralizing antibodies in the presence of primed T helper cells. Instead, higher viral loads at a very early stage of FV infection caused by either a lack of the B6-derived APOBEC3 or a lack of the wild-type BAFF-R resulted in slower production of neutralizing antibodies. Indeed, B cells were hyperactivated soon after infection in the APOBEC3- or BAFF-R-deficient mice. In contrast to mice deficient in the B6-derived APOBEC3, which cleared viremia by 4 weeks after FV infection, mice lacking the functional BAFF-R allele exhibited sustained viremia, indicating that the polymorphisms at the BAFF-R locus may better explain the Rfv3-defining phenotype of persistent viremia.

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Comparison of Four SARS-CoV-2 Neutralization Assays

Vaccines ◽

10.3390/vaccines9010013 ◽

2020 ◽

Vol 9 (1) ◽

pp. 13

Author(s):

Lydia Riepler ◽

Annika Rössler ◽

Albert Falch ◽

André Volland ◽

Wegene Borena ◽

...

Keyword(s):

Vesicular Stomatitis Virus ◽

Neutralizing Antibodies ◽

The Other ◽

In Vitro Assays ◽

The Novel ◽

Effective Protection ◽

Vaccine Candidates ◽

Vesicular Stomatitis ◽

Novel Coronavirus

Neutralizing antibodies are a major correlate of protection for many viruses including the novel coronavirus SARS-CoV-2. Thus, vaccine candidates should potently induce neutralizing antibodies to render effective protection from infection. A variety of in vitro assays for the detection of SARS-CoV-2 neutralizing antibodies has been described. However, validation of the different assays against each other is important to allow comparison of different studies. Here, we compared four different SARS-CoV-2 neutralization assays using the same set of patient samples. Two assays used replication competent SARS-CoV-2, a focus forming assay and a TCID50-based assay, while the other two assays used replication defective lentiviral or vesicular stomatitis virus (VSV)-based particles pseudotyped with SARS-CoV-2 spike. All assays were robust and produced highly reproducible neutralization titers. Titers of neutralizing antibodies correlated well between the different assays and with the titers of SARS-CoV-2 S-protein binding antibodies detected in an ELISA. Our study showed that commonly used SARS-CoV-2 neutralization assays are robust and that results obtained with different assays are comparable.

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Mouse APOBEC3 affects the production of virus-neutralizing antibodies by restricting early retroviral replication, not by altering the B-cell repertoire

Retrovirology ◽

10.1186/1742-4690-6-s2-o9 ◽

2009 ◽

Vol 6 (S2) ◽

Author(s):

Masaaki Miyazawa ◽

Sachiyo Tsuji-Kawahara ◽

Tomomi Chikaishi ◽

Maiko Kato ◽

Shiki Takamura

Keyword(s):

B Cell ◽

Neutralizing Antibodies ◽

Cell Repertoire ◽

B Cell Repertoire ◽

Retroviral Replication

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Long antibody HCDR3s from HIV-naïve donors presented on a PG9 neutralizing antibody background mediate HIV neutralization

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1518405113 ◽

2016 ◽

Vol 113 (16) ◽

pp. 4446-4451 ◽

Cited By ~ 13

Author(s):

Jordan R. Willis ◽

Jessica A. Finn ◽

Bryan Briney ◽

Gopal Sapparapu ◽

Vidisha Singh ◽

...

Keyword(s):

Amino Acid ◽

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Computational Design ◽

Cell Repertoire ◽

B Cell Repertoire ◽

Amino Acid Length ◽

Massive Scale ◽

Amino Acid Mutations ◽

Hiv 1

Development of broadly neutralizing antibodies (bnAbs) against HIV-1 usually requires prolonged infection and induction of Abs with unusual features, such as long heavy-chain complementarity-determining region 3 (HCDR3) loops. Here we sought to determine whether the repertoires of HIV-1–naïve individuals contain Abs with long HCDR3 loops that could mediate HIV-1 neutralization. We interrogated at massive scale the structural properties of long Ab HCDR3 loops in HIV-1–naïve donors, searching for structured HCDR3s similar to those of the HIV-1 bnAb PG9. We determined the nucleotide sequences encoding 2.3 × 107unique HCDR3 amino acid regions from 70 different HIV-1–naïve donors. Of the 26,917 HCDR3 loops with 30-amino acid length identified, we tested 30 for further study that were predicted to have PG9-like structure when chimerized onto PG9. Three of these 30 PG9 chimeras bound to the HIV-1 gp120 monomer, and two were neutralizing. In addition, we found 14 naturally occurring HCDR3 sequences that acquired the ability to bind to the HIV-1 gp120 monomer when adding 2- to 7-amino acid mutations via computational design. Of those 14 designed Abs, 8 neutralized HIV-1, with IC50values ranging from 0.7 to 98 µg/mL. These data suggest that the repertoire of HIV-1–naïve individuals contains rare B cells that encode HCDR3 loops that bind or neutralize HIV-1 when presented on a PG9 background with relatively few or no additional mutations. Long HCDR3 sequences are present in the HIV-naïve B-cell repertoire, suggesting that this class of bnAbs is a favorable target for rationally designed preventative vaccine efforts.

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Incorporation of apolipoprotein E into HBV–HCV subviral envelope particles to improve the hepatitis vaccine strategy

Scientific Reports ◽

10.1038/s41598-021-01428-7 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Elsa Gomez-Escobar ◽

Julien Burlaud-Gaillard ◽

Clara Visdeloup ◽

Adeline Ribeiro E. Silva ◽

Pauline Coutant ◽

...

Keyword(s):

Apolipoprotein E ◽

Neutralizing Antibodies ◽

Envelope Proteins ◽

Viral Envelope ◽

Vaccine Strategy ◽

S Protein ◽

Hepatitis Vaccine ◽

Protein Immunization ◽

Neutralizing Potential

AbstractHepatitis C is a major threat to public health for which an effective treatment is available, but a prophylactic vaccine is still needed to control this disease. We designed a vaccine based on chimeric HBV–HCV envelope proteins forming subviral particles (SVPs) that induce neutralizing antibodies against HCV in vitro. Here, we aimed to increase the neutralizing potential of those antibodies, by using HBV–HCV SVPs bearing apolipoprotein E (apoE). These particles were produced by cultured stable mammalian cell clones, purified and characterized. We found that apoE was able to interact with both chimeric HBV–HCV (E1-S and E2-S) proteins, and with the wild-type HBV S protein. ApoE was also detected on the surface of purified SVPs and improved the folding of HCV envelope proteins, but its presence lowered the incorporation of E2-S protein. Immunization of New Zealand rabbits resulted in similar anti-S responses for all rabbits, whereas anti-E1/-E2 antibody titers varied according to the presence or absence of apoE. Regarding the neutralizing potential of these anti-E1/-E2 antibodies, it was higher in rabbits immunized with apoE-bearing particles. In conclusion, the association of apoE with HCV envelope proteins may be a good strategy for improving HCV vaccines based on viral envelope proteins.

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Shared HIV envelope-specific B cell clonotypes induced by a pox-protein vaccine regimen

10.1101/2021.08.23.21262508 ◽

2021 ◽

Author(s):

Kristen W. Cohen ◽

Lamar Ballweber-Fleming ◽

Michael Duff ◽

Rachael E. Whaley ◽

Aaron Seese ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Neutralizing Antibodies ◽

Rational Design ◽

Critical Role ◽

Memory B Cells ◽

Protein Vaccine ◽

Cell Repertoire ◽

B Cell Repertoire ◽

Specific Igg

An effective HIV-1 vaccine will likely induce potent, broad neutralizing antibodies. No candidate vaccines have elicited these responses presumably because they fail to activate human B cell precursors that can affinity mature to generate broad neutralizing antibodies. To identify the B cell clonotypes that are elicited, we conducted in-depth analyses of the envelope-specific B cell repertoire in recipients of ALVAC-HIV vector (vCP2438) and bivalent subtype C gp120 protein (HVTN100). We observed high frequencies of envelope-specific IgG+ memory B cells with restricted immunogenetic diversity, relative to non-vaccine induced memory B cells, with preferential expansions of distinct variable genes but limited accumulation of mutations. Many envelope-specific clonotypes were shared across vaccinees, but did not overlap with the envelope-negative memory repertoire, within and across subjects. Single-cell sequencing of envelope-specific IgG+ memory B cells often revealed VH1-2*02 and VK3-20 sequence co-expression and in one case, contained a 5 amino acid CDRL3, the canonical signature of VRC01-class antibodies, confirming that these B cells are extremely rare but detectable. Our study provides evidence that immunogens play a critical role in selecting and restricting the responding B cell repertoire and supports the rational design of HIV vaccines targeting specific B cell lineages for induction of broadly-reactive neutralizing antibodies.

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Landscape analysis of escape variants identifies SARS-CoV-2 spike mutations that attenuate monoclonal and serum antibody neutralization

10.1101/2020.11.06.372037 ◽

2020 ◽

Cited By ~ 3

Author(s):

Zhuoming Liu ◽

Laura A. VanBlargan ◽

Paul W. Rothlauf ◽

Louis-Marie Bloyet ◽

Rita E. Chen ◽

...

Keyword(s):

Immune Responses ◽

Neutralizing Antibodies ◽

Serum Antibody ◽

Single Amino Acid ◽

Cross Resistance ◽

S Protein ◽

Mutational Landscape ◽

Immune Mediated ◽

Human Sera ◽

Escape Mutants

ABSTRACTAlthough neutralizing antibodies against the SARS-CoV-2 spike (S) protein are a goal of most COVID-19 vaccines and being developed as therapeutics, escape mutations could compromise such countermeasures. To define the immune-mediated mutational landscape in S protein, we used a VSV-eGFP-SARS-CoV-2-S chimeric virus and 19 neutralizing monoclonal antibodies (mAbs) against the receptor binding domain (RBD) to generate 48 escape mutants. These variants were mapped onto the RBD structure and evaluated for cross-resistance by convalescent human plasma. Although each mAb had unique resistance profiles, many shared residues within an epitope, as several variants were resistant to multiple mAbs. Remarkably, we identified mutants that escaped neutralization by convalescent human sera, suggesting that some humans induce a narrow repertoire of neutralizing antibodies. By comparing the antibody-mediated mutational landscape in S protein with sequence variation in circulating SARS-CoV-2 strains, we identified single amino acid substitutions that could attenuate neutralizing immune responses in some humans.

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Robust neutralization assay based on SARS-CoV-2 S-bearing vesicular stomatitis virus (VSV) pseudovirus and ACE2-overexpressed BHK21 cells

10.1101/2020.04.08.026948 ◽

2020 ◽

Cited By ~ 13

Author(s):

Hua-Long Xiong ◽

Yang-Tao Wu ◽

Jia-Li Cao ◽

Ren Yang ◽

Jian Ma ◽

...

Keyword(s):

Vesicular Stomatitis Virus ◽

Neutralizing Antibodies ◽

Neutralization Assay ◽

Cell Number ◽

New Drugs ◽

Human Society ◽

Angiotensin Converting Enzyme 2 ◽

Global Pandemic ◽

Vesicular Stomatitis

AbstractThe global pandemic of Coronavirus disease 2019 (COVID-19) is a disaster for human society. A convenient and reliable in vitro neutralization assay is very important for the development of neutralizing antibodies, vaccines and other inhibitors. In this study, G protein-deficient vesicular stomatitis virus (VSVdG) bearing full-length and truncated spike (S) protein of SARS-CoV-2 were evaluated. The virus packaging efficiency of VSV-SARS-CoV-2-Sdel18 (S with C-terminal 18 amino acid truncation) is much higher than VSV-SARS-CoV-2-S. A neutralization assay for antibody screening and serum neutralizing titer quantification was established based on VSV-SARS-CoV-2-Sdel18 pseudovirus and human angiotensin-converting enzyme 2 (ACE2) overexpressed BHK21 cell (BHK21-hACE2). The experimental results can be obtained by automatically counting EGFP positive cell number at 12 hours after infection, making the assay convenient and high-throughput. The serum neutralizing titer of COVID-19 convalescent patients measured by VSV-SARS-CoV-2-Sdel18 pseudovirus assay has a good correlation with live SARS-CoV-2 assay. Seven neutralizing monoclonal antibodies targeting receptor binding domain (RBD) of SARS-CoV-2-S were obtained. This efficient and reliable pseudovirus assay model could facilitate the development of new drugs and vaccines.

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Broad sarbecovirus neutralizing antibodies define a key site of vulnerability on the SARS-CoV-2 spike protein

10.1101/2020.05.15.096511 ◽

2020 ◽

Cited By ~ 9

Author(s):

Anna Z. Wec ◽

Daniel Wrapp ◽

Andrew S. Herbert ◽

Daniel Maurer ◽

Denise Haslwanter ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Rational Design ◽

Somatic Hypermutation ◽

Protein A ◽

Neutralizing Antibody ◽

Memory B Cells ◽

Antibody Responses ◽

Cell Repertoire ◽

Human Coronavirus ◽

B Cell Repertoire

Broadly protective vaccines against known and pre-emergent coronaviruses are urgently needed. Critical to their development is a deeper understanding of cross-neutralizing antibody responses induced by natural human coronavirus (HCoV) infections. Here, we mined the memory B cell repertoire of a convalescent SARS donor and identified 200 SARS-CoV-2 binding antibodies that target multiple conserved sites on the spike (S) protein. A large proportion of the antibodies display high levels of somatic hypermutation and cross-react with circulating HCoVs, suggesting recall of pre-existing memory B cells (MBCs) elicited by prior HCoV infections. Several antibodies potently cross-neutralize SARS-CoV, SARS-CoV-2, and the bat SARS-like virus WIV1 by blocking receptor attachment and inducing S1 shedding. These antibodies represent promising candidates for therapeutic intervention and reveal a new target for the rational design of pan-sarbecovirus vaccines.

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