Structurally Mapping Antigenic Epitopes of Adeno-Associated Virus 9: Development of Antibody Escape Variants

Adeno-associated viruses (AAV) serve as vectors for therapeutic gene delivery. AAV9 vectors have been FDA approved, as Zolgensma®, for the treatment of spinal muscular atrophy and is being evaluated in clinical trials for the treatment of neurotropic and musculotropic diseases. A major hurdle for AAV-mediated gene delivery is the presence of pre-existing neutralizing antibodies in 40 to 80% of the general population. These pre-existing antibodies can reduce therapeutic efficacy through viral neutralization, and the size of the patient cohort eligible for treatment. In this study, cryo-electron microscopy and image reconstruction was used to define the epitopes of five anti-AAV9 monoclonal antibodies (MAbs); ADK9, HL2368, HL2370, HL2372, and HL2374, on the capsid surface. Three of these, ADK9, HL2370, and HL2374, bound on or near the icosahedral 3-fold axes, HL2368 to the 2/5-fold wall, and HL2372 to the region surrounding the 5-fold axes. Pseudo-atomic modeling enabled the mapping and identification of antibody contact amino acids on the capsid, including S454 and P659. These epitopes overlap with previously defined parvovirus antigenic sites. Capsid amino acids critical for the interactions were confirmed by mutagenesis followed by biochemical assays testing recombinant AAV9 (rAAV9) variants capable of escaping recognition and neutralization by the parental MAbs. These variants retained parental tropism and had similar or improved transduction efficiency compared to AAV9. These engineered rAAV9 variants could expand the patient cohort eligible for AAV9-mediated gene delivery by avoiding pre-existing circulating neutralizing antibodies. IMPORTANCE The use of recombinant AAVs (rAAVs) as delivery vectors for therapeutic genes is becoming increasingly popular, especially following the FDA approval of Luxturna® and Zolgensma®, based on serotypes AAV2 and AAV9, respectively. However, high titer anti-AAV neutralizing antibodies in the general population, exempts patients from treatment. The goal of this study is to circumvent this issue by creating AAV variant vectors not recognized by pre-existing neutralizing antibodies. The mapping of the antigenic epitopes of five different monoclonal antibodies (MAbs) on AAV9, to recapitulate a polyclonal response, enabled the rational design of escape variants with minimal disruption to cell tropism and gene expression. This study, which included four newly developed and now commercially available MAbs, provides a platform for the engineering of rAAV9 vectors that can be used to deliver genes to patients with pre-exiting AAV antibodies.

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Elucidation of the Binding Regions of PAI-1 Neutralizing Antibodies Using Chimeric Variants of Human and Rat PAI-1

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615761 ◽

2001 ◽

Vol 85 (05) ◽

pp. 866-874 ◽

Cited By ~ 16

Author(s):

A. P. Bijnens ◽

T. H. Ngo ◽

A. Gils ◽

J. Dewaele ◽

I. Knockaert ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Plasminogen Activator ◽

Neutralizing Antibodies ◽

Rational Design ◽

Biochemical Characterization ◽

Tissue Type ◽

Binding Region ◽

Plasminogen Activator Inhibitor 1 ◽

Inhibitory Antibodies ◽

Pai 1

SummaryIncreased levels of plasminogen activator inhibitor-1 (PAI-1), the main physiological inhibitor of tissue-type plasminogen activator (t-PA) in plasma, are a known risk factor for thromboembolic and cardiovascular diseases. The elucidation of the binding site of inhibitory monoclonal antibodies may contribute to the rational design of PAI-1 modulating therapeutics. In this study, homolog-scanning mutagenesis was used to identify the binding region of a variety of human PAI-1 inhibitory antibodies, lacking cross-reactivity with rat PAI-1. Therefore, eight chimeric human/rat PAI-1 variants, containing rat PAI-1 substitutions at the N-terminal or C-terminal end with splicing sites at positions 26, 81, 187, 277 or 327, were generated and purified. Biochemical characterization revealed that all chimeras were folded properly. Subsequently, surface plasmon resonance was used to determine the affinity of various monoclonal antibodies for these chimera. Comparative analysis of the affinity and ELISA data allowed the identification of the major binding region of the inhibitory antibodies MA-8H9D4, MA-33B8F7, MA-44E4, MA-42A2F6 and MA-56A7C10. Thus, three segments in human PAI-1 containing each at least one site involved in the neutralization of PAI-1 activity could be identified, i.e. (1) the segment from residue 81 to residue 187 (comprising -helices hD, hE and hF, -strands s4C, s3A, s2A and s1A and the loops connecting these elements), (2) the segment between residues 277 and 327 (hI, thIs5A, s5A and s6A) and (3) the region C-terminal from amino acid 327, including the reactive site loop. The current data, together with previous data, indicate that PAI-1 contains at least four different regions that could be considered as putative targets to modulate its activity.

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Directed Evolution of Immune-Escaping Adeno-Associated Virus Vectors

Blood ◽

10.1182/blood.v112.11.3532.3532 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3532-3532

Author(s):

Stephan Maersch ◽

Anke Huber ◽

Michael Hallek ◽

Hildegard Buening ◽

Luca Perabo

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Gene Transfer ◽

Directed Evolution ◽

Neutralizing Antibodies ◽

Rational Design ◽

Multiple Point ◽

Repeated Treatment ◽

Therapeutic Gene ◽

Adeno Associated Virus

Abstract Efficiency of therapeutic gene transfer by adeno-associated virus of serotype 2 (AAV-2) vectors is hampered in patients with pre-existing immunity against the natural virus. Genetic engineering by rational design or directed evolution has been employed in the last 3 years to generate capsids that escape antibody neutralization and has led to identify several amino acid residues of the capsid proteins that can be mutated in order to decrease antibody recognition (Perabo et al., 2006; Maheshri et al, 2006; Lochrie et al., 2006). In this novel study, we aimed to exploit the comprehensive knowledge gathered so far by generating novel capsid variants that carried multiple point mutations at these previously identified sites. Capsid libraries were generated by codon randomization of several immunogenic residues and screened to isolate mutants that most efficiently infected human cells despite the presence of anti-AAV2 neutralizing antibodies. Besides testing novel combinations of concomitant mutations at these sites, this approach allowed for the first time an exhaustive scanning of combinations of all 20 natural amino acids at each position. We identified several novel capsid mutants that remain highly infectious even when incubated with serum concentrations that completely neutralize wild type AAV2. Our results demonstrate that combining mutations at several sites it is possible to improve the immune-escaping ability of the capsid. In addition, we show that escaping ability and other biological characteristics of these mutants are strongly dependent on the type of amino acid substituted, demonstrating that an exact choice of substituted amino acids is essential to maximize stealth properties and minimize loss of packaging ability, particle stability and transduction efficacy. These vectors can be used for therapeutic gene transfer to patients with pre-existing immunity, or for repeated treatment after antibodies are generated upon first application.

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Examining the cross-reactivity and neutralization mechanisms of a panel of mAbs against adeno-associated virus serotypes 1 and 5

Journal of General Virology ◽

10.1099/vir.0.035113-0 ◽

2012 ◽

Vol 93 (2) ◽

pp. 347-355 ◽

Cited By ~ 30

Author(s):

Carole E. Harbison ◽

Wendy S. Weichert ◽

Brittney L. Gurda ◽

John A. Chiorini ◽

Mavis Agbandje-McKenna ◽

...

Keyword(s):

Clinical Trials ◽

Gene Delivery ◽

Receptor Binding ◽

Neutralizing Antibodies ◽

Viral Infections ◽

Cross Reactivity ◽

Adeno Associated Virus ◽

Viral Capsids ◽

Serotype 1 ◽

Antigenic Epitopes

Neutralizing antibodies play a central role in the prevention and clearance of viral infections, but can be detrimental to the use of viral capsids for gene delivery. Antibodies present a major hurdle for ongoing clinical trials using adeno-associated viruses (AAVs); however, relatively little is known about the antigenic epitopes of most AAV serotypes or the mechanism(s) of antibody-mediated neutralization. We developed panels of AAV mAbs by repeatedly immunizing mice with AAV serotype 1 (AAV1) capsids, or by sequentially immunizing with AAV1 followed by AAV5 capsids, in order to examine the efficiency and mechanisms of antibody-mediated neutralization. The antibodies were not cross-reactive between heterologous AAV serotypes except for a low level of recognition of AAV1 capsids by the AAV5 antibodies, probably due to the initial immunization with AAV1. The neutralization efficiency of different IgGs varied and Fab fragments derived from these antibodies were generally poorly neutralizing. The antibodies appeared to display various alternative mechanisms of neutralization, which included inhibition of receptor-binding and interference with a post-attachment step.

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Exploring antibody repurposing for COVID-19: beyond presumed roles of therapeutic antibodies

Scientific Reports ◽

10.1038/s41598-021-89621-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Puneet Rawat ◽

Divya Sharma ◽

Ambuj Srivastava ◽

Vani Janakiraman ◽

M. Michael Gromiha

Keyword(s):

Clinical Trials ◽

Monoclonal Antibodies ◽

Neutralizing Antibodies ◽

Effective Vaccine ◽

Therapeutic Antibodies ◽

Viral Neutralization ◽

Investigational Drugs ◽

Therapeutic Monoclonal Antibodies ◽

Interaction Surface ◽

Limited Choice

AbstractThe urgent need for a treatment of COVID-19 has left researchers with limited choice of either developing an effective vaccine or identifying approved/investigational drugs developed for other medical conditions for potential repurposing, thus bypassing long clinical trials. In this work, we compared the sequences of experimentally verified SARS-CoV-2 neutralizing antibodies and sequentially/structurally similar commercialized therapeutic monoclonal antibodies. We have identified three therapeutic antibodies, Tremelimumab, Ipilimumab and Afasevikumab. Interestingly, these antibodies target CTLA4 and IL17A, levels of which have been shown to be elevated during severe SARS-CoV-2 infection. The candidate antibodies were evaluated further for epitope restriction, interaction energy and interaction surface to gauge their repurposability to tackle SARS-CoV-2 infection. Our work provides candidate antibody scaffolds with dual activities of plausible viral neutralization and immunosuppression. Further, these candidate antibodies can also be explored in diagnostic test kits for SARS-CoV-2 infection. We opine that this in silico workflow to screen and analyze antibodies for repurposing would have widespread applications.

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Distinct Mechanisms of Neutralization by Monoclonal Antibodies Specific for Sites in the N-Terminal or C-Terminal Domain of Murine Leukemia Virus SU

Journal of Virology ◽

10.1128/jvi.77.7.3993-4003.2003 ◽

2003 ◽

Vol 77 (7) ◽

pp. 3993-4003 ◽

Cited By ~ 15

Author(s):

Michael Dominic Burkhart ◽

Samuel C. Kayman ◽

Yuxian He ◽

Abraham Pinter

Keyword(s):

Monoclonal Antibodies ◽

Receptor Binding ◽

Murine Leukemia Virus ◽

Neutralizing Antibodies ◽

Leukemia Virus ◽

Binding Pocket ◽

Protein Subunit ◽

Viral Neutralization ◽

Terminal Domain ◽

Murine Leukemia

ABSTRACT The epitope specificities and functional activities of monoclonal antibodies (MAbs) specific for the murine leukemia virus (MuLV) SU envelope protein subunit were determined. Neutralizing antibodies were directed towards two distinct sites in MuLV SU: one overlapping the major receptor-binding pocket in the N-terminal domain and the other involving a region that includes the most C-terminal disulfide-bonded loop. Two other groups of MAbs, reactive with distinct sites in the N-terminal domain or in the proline-rich region (PRR), did not neutralize MuLV infectivity. Only the neutralizing MAbs specific for the receptor-binding pocket were able to block binding of purified SU and MuLV virions to cells expressing the ecotropic MuLV receptor, mCAT-1. Whereas the neutralizing MAbs specific for the C-terminal domain did not interfere with the SU-mCAT-1 interaction, they efficiently inhibited cell-to-cell fusion mediated by MuLV Env, indicating that they interfered with a postattachment event necessary for fusion. The C-terminal domain MAbs displayed the highest neutralization titers and binding activities. However, the nonneutralizing PRR-specific MAbs bound to intact virions with affinities similar to those of the neutralizing receptor-binding pocket-specific MAbs, indicating that epitope exposure, while necessary, is not sufficient for viral neutralization by MAbs. These results identify two separate neutralization domains in MuLV SU and suggest a role for the C-terminal domain in a postattachment step necessary for viral fusion.

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Mutations in SARS-CoV-2 Variants Modulate the Microscopic Dynamics of Neutralizing Antibodies

10.1101/2021.08.13.456317 ◽

2021 ◽

Author(s):

Dhiman Ray ◽

Riley Nicolas Quijano ◽

Ioan Andricioaei

Keyword(s):

Monoclonal Antibodies ◽

Neutralizing Antibodies ◽

Rational Design ◽

Graph Connectivity ◽

World Population ◽

The Novel ◽

Long Distance ◽

Correlated Motion ◽

Novel Coronavirus ◽

Microscopic Dynamics

Monoclonal antibodies have emerged as viable treatment for the COVID-19 disease caused by the SARS-CoV-2 virus. But the new viral variants can reduce the efficacy of the currently available antibodies, as well as diminish the vaccine induced immunity. Here, we demonstrate how the microscopic dynamics of the SARS-CoV-2 neutralizing monoclonal antibodies, can be modulated by the mutations present in the spike proteins of the variants currently circulating in the world population. We show that the dynamical perturbation in the antibody structure can be diverse, depending both on the nature of the antibody and on the location of the mutation. The correlated motion between the antibody and the receptor binding domain (RBD) can also be changed, altering the binding affinity. By constructing a protein graph connectivity network, we could delineate the mutant induced modifications in the allosteric information flow pathway through the antibody, and observed the presence of both localized and long distance effects. We identified a loop consisting of residues 470-490 in the RBD which works like an anchor preventing the detachment of the antibodies, and individual mutations in that region can significantly affect the antibody binding propensity. Our study provides fundamental and atomistically detailed insight on how virus neutralization by monoclonal antibody can be impacted by the mutations in the epitope, and can potentially facilitate the rational design of monoclonal antibodies, effective against the new variants of the novel coronavirus.

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Functional Analysis of Neutralizing Antibodies against Clostridium perfringens Epsilon-Toxin

Infection and Immunity ◽

10.1128/iai.01643-06 ◽

2007 ◽

Vol 75 (4) ◽

pp. 1785-1793 ◽

Cited By ~ 31

Author(s):

Mark S. McClain ◽

Timothy L. Cover

Keyword(s):

Amino Acids ◽

Monoclonal Antibodies ◽

Clostridium Perfringens ◽

Neutralizing Antibodies ◽

Plasma Membranes ◽

Mdck Cells ◽

Enzyme Linked Immunosorbent Assay ◽

Neutralizing Monoclonal Antibody ◽

Epsilon Toxin ◽

Antibody Competition

ABSTRACT The Clostridium perfringens epsilon-toxin causes a severe, often fatal illness (enterotoxemia) characterized by cardiac, pulmonary, kidney, and brain edema. In this study, we examined the activities of two neutralizing monoclonal antibodies against the C. perfringens epsilon-toxin. Both antibodies inhibited epsilon-toxin cytotoxicity towards cultured MDCK cells and inhibited the ability of the toxin to form pores in the plasma membranes of cells, as shown by staining cells with the membrane-impermeant dye 7-aminoactinomycin D. Using an antibody competition enzyme-linked immunosorbent assay (ELISA), a peptide array, and analysis of mutant toxins, we mapped the epitope recognized by one of the neutralizing monoclonal antibodies to amino acids 134 to 145. The antibody competition ELISA and analysis of mutant toxins suggest that the second neutralizing monoclonal antibody also recognizes an epitope in close proximity to this region. The region comprised of amino acids 134 to 145 overlaps an amphipathic loop corresponding to the putative membrane insertion domain of the toxin. Identifying the epitopes recognized by these neutralizing antibodies constitutes an important first step in the development of therapeutic agents that could be used to counter the effects of the epsilon-toxin.

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Landscape of Monoclonal Antibodies Targeting Zika and Dengue: Therapeutic Solutions and Critical Insights for Vaccine Development

Frontiers in Immunology ◽

10.3389/fimmu.2020.621043 ◽

2021 ◽

Vol 11 ◽

Author(s):

Vincent Dussupt ◽

Kayvon Modjarrad ◽

Shelly J. Krebs

Keyword(s):

Monoclonal Antibodies ◽

Neutralizing Antibodies ◽

Rational Design ◽

Vaccine Development ◽

Therapeutic Potential ◽

Cell Responses ◽

Therapeutic Value ◽

Global Concern ◽

Species Specific ◽

Rapid Deployment

The unprecedented 2015–2016 Zika outbreak in the Americas sparked global concern and drove the rapid deployment of vaccine and therapeutic countermeasures against this re-emerging pathogen. Alongside vaccine development, a number of potent neutralizing antibodies against Zika and related flaviviruses have been identified in recent years. High-throughput antibody isolation approaches have contributed to a better understanding of the B cell responses elicited following infection and/or vaccination. Structure-based approaches have illuminated species-specific and cross-protective epitopes of therapeutic value. This review will highlight previously described monoclonal antibodies with the best therapeutic potential against ZIKV and related flaviviruses, and discuss their implications for the rational design of better vaccine strategies.

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AAV-mediated delivery of optogenetic constructs to the macaque brain triggers humoral immune responses

Journal of Neurophysiology ◽

10.1152/jn.00780.2016 ◽

2017 ◽

Vol 117 (5) ◽

pp. 2004-2013 ◽

Cited By ~ 14

Author(s):

Skyler D. Mendoza ◽

Yasmine El-Shamayleh ◽

Gregory D. Horwitz

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Gene Delivery ◽

Immune Responses ◽

Neutralizing Antibodies ◽

Viral Vector ◽

Transduction Efficiency ◽

Vitro Assay ◽

Humoral Immune ◽

Vector Transduction

Gene delivery to the primate central nervous system via recombinant adeno-associated viral vectors (AAV) allows neurophysiologists to control and observe neural activity precisely. A current limitation of this approach is variability in vector transduction efficiency. Low levels of transduction can foil experimental manipulations, prompting vector readministration. The ability to make multiple vector injections into the same animal, even in cases where successful vector transduction has already been achieved, is also desirable. However, vector readministration has consequences for humoral immunity and gene delivery that depend on vector dosage and route of administration in complex ways. As part of optogenetic experiments in rhesus monkeys, we analyzed blood sera collected before and after AAV injections into the brain and quantified neutralizing antibodies to AAV using an in vitro assay. We found that injections of AAV1 and AAV9 vectors elevated neutralizing antibody titers consistently. These immune responses were specific to the serotype injected and were long lasting. These results demonstrate that optogenetic manipulations in monkeys trigger immune responses to AAV capsids, suggesting that vector readministration may have a higher likelihood of success by avoiding serotypes injected previously.NEW & NOTEWORTHY Adeno-associated viral vector (AAV)-mediated gene delivery is a valuable tool for neurophysiology, but variability in transduction efficiency remains a bottleneck for experimental success. Repeated vector injections can help overcome this limitation but affect humoral immune state and transgene expression in ways that are poorly understood. We show that AAV vector injections into the primate central nervous system trigger long-lasting and serotype-specific immune responses, raising the possibility that switching serotypes may promote successful vector readministration.

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An Exposed Domain in the Severe Acute Respiratory Syndrome Coronavirus Spike Protein Induces Neutralizing Antibodies

Journal of Virology ◽

10.1128/jvi.78.13.7217-7226.2004 ◽

2004 ◽

Vol 78 (13) ◽

pp. 7217-7226 ◽

Cited By ~ 59

Author(s):

Tong Zhou ◽

Hong Wang ◽

Danlin Luo ◽

Thomas Rowe ◽

Zheng Wang ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Monoclonal Antibodies ◽

Severe Acute Respiratory Syndrome ◽

Neutralizing Antibodies ◽

Vaccine Development ◽

Vero Cells ◽

Spike Protein ◽

Protein Fragment

ABSTRACT Exposed epitopes of the spike protein may be recognized by neutralizing antibodies against severe acute respiratory syndrome (SARS) coronavirus (CoV). A protein fragment (S-II) containing predicted epitopes of the spike protein was expressed in Escherichia coli. The properly refolded protein fragment specifically bound to the surface of Vero cells. Monoclonal antibodies raised against this fragment recognized the native spike protein of SARS CoV in both monomeric and trimeric forms. These monoclonal antibodies were capable of blocking S-II attachment to Vero cells and exhibited in vitro antiviral activity. These neutralizing antibodies mapped to epitopes in two peptides, each comprising 20 amino acids. Thus, this region of the spike protein might be a target for generation of therapeutic neutralizing antibodies against SARS CoV and for vaccine development to elicit protective humoral immunity.

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