High-Definition Mapping of Four Spatially Distinct Neutralizing Epitope Clusters on RiVax, a Candidate Ricin Toxin Subunit Vaccine

ABSTRACT RiVax is a promising recombinant ricin toxin A subunit (RTA) vaccine antigen that has been shown to be safe and immunogenic in humans and effective at protecting rhesus macaques against lethal-dose aerosolized toxin exposure. We previously used a panel of RTA-specific monoclonal antibodies (MAbs) to demonstrate, by competition enzyme-linked immunosorbent assay (ELISA), that RiVax elicits similar serum antibody profiles in humans and macaques. However, the MAb binding sites on RiVax have yet to be defined. In this study, we employed hydrogen exchange-mass spectrometry (HX-MS) to localize the epitopes on RiVax recognized by nine toxin-neutralizing MAbs and one nonneutralizing MAb. Based on strong protection from hydrogen exchange, the nine MAbs grouped into four spatially distinct epitope clusters (namely, clusters I to IV). Cluster I MAbs protected RiVax's α-helix B (residues 94 to 107), a protruding immunodominant secondary structure element known to be a target of potent toxin-neutralizing antibodies. Cluster II consisted of two subclusters located on the “back side” (relative to the active site pocket) of RiVax. One subcluster involved α-helix A (residues 14 to 24) and α-helices F-G (residues 184 to 207); the other encompassed β-strand d (residues 62 to 69) and parts of α-helices D-E (154 to 164) and the intervening loop. Cluster III involved α-helices C and G on the front side of RiVax, while cluster IV formed a sash from the front to back of RiVax, spanning strands b, c, and d (residues 35 to 59). Having a high-resolution B cell epitope map of RiVax will enable the development and optimization of competitive serum profiling assays to examine vaccine-induced antibody responses across species.

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Cloning, expression and immunogenic characterization of theapxIIAgene ofActinobacillus pleuropneumoniae

Chinese Journal of Agricultural Biotechnology ◽

10.1017/s1479236207001222 ◽

2007 ◽

Vol 4 (1) ◽

pp. 63-67

Author(s):

Yan Ke-Xia ◽

Liu Jian-Jie ◽

Wu Bin ◽

Tang Xi-Biao ◽

Cai Li-Jun ◽

...

Keyword(s):

Recombinant Protein ◽

Lethal Dose ◽

Serum Antibody ◽

Enzyme Linked Immunosorbent Assay ◽

Gene Encoding ◽

Lethal Challenge ◽

Sds Page ◽

Prokaryotic Expression Vector ◽

Protection Rate ◽

Protein Group

AbstractThe structural gene encoding ApxII toxin (apxIIA) was amplified from the genomic DNA ofActinobacillus pleuropneumoniae(APP) strain HB08 (serotype 2) and cloned into the prokaryotic expression vector pET-28a. SDS-PAGE and Western blot analysis showed that theapxIIAgene was expressed inEscherichia coliBL21 (DE3) and the expressed products could react with ApxII antibodies. The recombinant ApxIIA was purified from the inclusion bodies. Kunming mice were intraperitoneally vaccinated twice, with an interval of 2 weeks, using unfolded/refolded recombinant proteins, the native ApxII toxin extracted from the cultural supernatant of a strain of APP serotype 7 (APP-7) or phosphate-buffered saline (PBS). Serum antibody was examined by ApxIIA-specific enzyme-linked immunosorbent assay (ELISA) 2 weeks after every vaccination. Two weeks after the second vaccination, mice were challenged intraperitoneally with a lethal dose of APP-7 (1.08 × 108cfu per mouse). The protection rate reached 91.7% in the native ApxII group, 83.3% in the refolded recombinant protein group and 58.3% in the unfolded recombinant protein group, while all mice in the PBS group died within 36 h after challenge. Our data revealed that the refolded recombinant ApxIIA had excellent immunogenicity and could elicit protection against a lethal challenge of APP.

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Thermostable ricin vaccine protects rhesus macaques against aerosolized ricin: Epitope-specific neutralizing antibodies correlate with protection

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1502585112 ◽

2015 ◽

Vol 112 (12) ◽

pp. 3782-3787 ◽

Cited By ~ 40

Author(s):

Chad J. Roy ◽

Robert N. Brey ◽

Nicholas J. Mantis ◽

Kelly Mapes ◽

Iliodora V. Pop ◽

...

Keyword(s):

Castor Oil ◽

Mammalian Cells ◽

Neutralizing Antibodies ◽

Rhesus Macaques ◽

Lethal Dose ◽

Lung Damage ◽

Ricin Toxin ◽

A Cell ◽

A Chain ◽

Antibody Competition

Ricin toxin (RT) is the second most lethal toxin known; it has been designated by the CDC as a select agent. RT is made by the castor bean plant; an estimated 50,000 tons of RT are produced annually as a by-product of castor oil. RT has two subunits, a ribotoxic A chain (RTA) and galactose-binding B chain (RTB). RT binds to all mammalian cells and once internalized, a single RTA catalytically inactivates all of the ribosomes in a cell. Administered as an aerosol, RT causes rapid lung damage and fibrosis followed by death. There are no Food and Drug Administration-approved vaccines and treatments are only effective in the first few hours after exposure. We have developed a recombinant RTA vaccine that has two mutations V76M/Y80A (RiVax). The protein is expressed in Escherichia coli and is nontoxic and immunogenic in mice, rabbits, and humans. When vaccinated mice are challenged with injected, aerosolized, or orally administered (gavaged) RT, they are completely protected. We have now developed a thermostable, aluminum-adjuvant–containing formulation of RiVax and tested it in rhesus macaques. After three injections, the animals developed antibodies that completely protected them from a lethal dose of aerosolized RT. These antibodies neutralized RT and competed to varying degrees with a panel of neutralizing and nonneutralizing mouse monoclonal antibodies known to recognize specific epitopes on native RTA. The resulting antibody competition profile could represent an immunologic signature of protection. Importantly, the same signature was observed using sera from RiVax-immunized humans.

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Durable Immunity to Ricin Toxin Elicited by a Thermostable, Lyophilized Subunit Vaccine

10.1101/2021.09.08.459551 ◽

2021 ◽

Author(s):

Hayley Novak ◽

Jennifer Doering ◽

Dylan Ehrbar ◽

Oreola Donini ◽

Nicholas J. Mantis

Keyword(s):

Subunit Vaccine ◽

Phase 1 ◽

Lethal Dose ◽

Serum Antibody ◽

The United States ◽

Phase I Clinical Trials ◽

Complete Protection ◽

Ricin Toxin ◽

Immune Parameters ◽

One Year

ABSTRACTThe development of vaccines against biothreat toxins like ricin (RT) is considered an integral component of the United States national security efforts. RiVax® is a thermostable, lyophilized RT subunit vaccine adsorbed to aluminum salt adjuvant intended for use by military personnel and first responders. Phase 1 studies indicated that RiVax is safe and immunogenic, while a three dose, intramuscular vaccination regimen in non-human primates elicited protection against lethal dose RT challenge by aerosol. Here we investigated, in a mouse model, the durability of RiVax-induced antibody responses and corresponding immunity to lethal dose RT challenge. Groups of mice were subcutaneously administered 3 or 1 μg of RiVax on days 0 and 21 and challenged with 10 × LD50 RT by injection at six different intervals over the course of twelve months. Serum antibody titers and epitope-specific competition assays were determined prior to each challenge. We report that the two-dose, 3 μg regimen conferred near complete protection against RT challenge on day 35 and complete protection thereafter (challenge days 65, 95, 125, 245, and 365). The two-dose, 3 μg regimen was superior to the 1 μg regimen as revealed by slight differences in survival and morbidity scores (e.g., hypoglycemia, weight loss) on challenge days 35 and 365. In separate experiments, a single 3 μg RiVax vaccination proved only marginally effective at eliciting protective immunity to RT, underscoring the necessity of a prime-boost regimen to achieve full and long-lasting protection against RT.IMPORTANCERicin toxin (RT) is a notorious biothreat, as exposure to even trace amounts via injection or inhalation can induce organ failure and death within a matter of hours. In this study, we advance the preclinical testing of a candidate RT vaccine known as RiVax®. RiVax is a recombinant non-toxic derivative of RT’s enzymatic subunit that has been evaluated for safety in Phase I clinical trials and efficacy in a variety of animal models. We demonstrate that two doses of RiVax is sufficient to protect mice from lethal dose RT challenge for up to one year. We describe kinetics and other immune parameters of the antibody response to RiVax and discuss how these immune factors may translate to humans.

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Validation of a Competitive Enzyme-Linked Immunosorbent Assay for Detection of Babesia bigemina Antibodies in Cattle

Clinical and Vaccine Immunology ◽

10.1128/cvi.00150-08 ◽

2008 ◽

Vol 15 (9) ◽

pp. 1316-1321 ◽

Cited By ~ 17

Author(s):

Will L. Goff ◽

Wendell C. Johnson ◽

John B. Molloy ◽

Wayne K. Jorgensen ◽

Susan J. Waldron ◽

...

Keyword(s):

Immunosorbent Assay ◽

Serum Antibody ◽

Cell Epitope ◽

Enzyme Linked Immunosorbent Assay ◽

Babesia Bigemina ◽

Characteristic Analysis ◽

Serum Samples ◽

Predictive Values ◽

Field Samples ◽

Species Specific

ABSTRACT A competitive enzyme-linked immunosorbent assay (cELISA) based on a broadly conserved, species-specific, B-cell epitope within the C terminus of Babesia bigemina rhoptry-associated protein 1a was validated for international use. Receiver operating characteristic analysis revealed 16% inhibition as the threshold for a negative result, with an associated specificity of 98.3% and sensitivity of 94.7%. Increasing the threshold to 21% increased the specificity to 100% but modestly decreased the sensitivity to 87.2%. By using 21% inhibition, the positive predictive values ranged from 90.7% (10% prevalence) to 100% (95% prevalence) and the negative predictive values ranged from 97.0% (10% prevalence) to 48.2% (95% prevalence). The assay was able to detect serum antibody as early as 7 days after intravenous inoculation. The cELISA was distributed to five different laboratories along with a reference set of 100 defined bovine serum samples, including known positive, known negative, and field samples. The pairwise concordance among the five laboratories ranged from 100% to 97%, and all kappa values were above 0.8, indicating a high degree of reliability. Overall, the cELISA appears to have the attributes necessary for international application.

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A Collection of Single-Domain Antibodies that Crowd Ricin Toxin’s Active Site

Antibodies ◽

10.3390/antib7040045 ◽

2018 ◽

Vol 7 (4) ◽

pp. 45 ◽

Cited By ~ 3

Author(s):

Siva Angalakurthi ◽

David Vance ◽

Yinghui Rong ◽

Chi Nguyen ◽

Michael Rudolph ◽

...

Keyword(s):

Active Site ◽

Hydrogen Exchange ◽

Single Domain ◽

Ricin Toxin ◽

Exchange Mass Spectrometry ◽

Health Authorities ◽

Public Health Authorities ◽

Hydrogen Exchange Mass Spectrometry ◽

Α Helix ◽

Upper Rim

In this report, we used hydrogen exchange-mass spectrometry (HX-MS) to identify the epitopes recognized by 21 single-domain camelid antibodies (VHHs) directed against the ribosome-inactivating subunit (RTA) of ricin toxin, a biothreat agent of concern to military and public health authorities. The VHHs, which derive from 11 different B-cell lineages, were binned together based on competition ELISAs with IB2, a monoclonal antibody that defines a toxin-neutralizing hotspot (“cluster 3”) located in close proximity to RTA’s active site. HX-MS analysis revealed that the 21 VHHs recognized four distinct epitope subclusters (3.1–3.4). Sixteen of the 21 VHHs grouped within subcluster 3.1 and engage RTA α-helices C and G. Three VHHs grouped within subcluster 3.2, encompassing α-helices C and G, plus α-helix B. The single VHH in subcluster 3.3 engaged RTA α-helices B and G, while the epitope of the sole VHH defining subcluster 3.4 encompassed α-helices C and E, and β-strand h. Modeling these epitopes on the surface of RTA predicts that the 20 VHHs within subclusters 3.1–3.3 physically occlude RTA’s active site cleft, while the single antibody in subcluster 3.4 associates on the active site’s upper rim.

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Recombinant Shiga Toxin B-Subunit-Keyhole Limpet Hemocyanin Conjugate Vaccine Protects Mice from Shigatoxemia

Infection and Immunity ◽

10.1128/iai.73.10.6523-6529.2005 ◽

2005 ◽

Vol 73 (10) ◽

pp. 6523-6529 ◽

Cited By ~ 33

Author(s):

Paola Marcato ◽

Thomas P. Griener ◽

George L. Mulvey ◽

Glen D. Armstrong

Keyword(s):

Shiga Toxin ◽

Lethal Dose ◽

Keyhole Limpet Hemocyanin ◽

Vaccine Antigen ◽

Enzyme Linked Immunosorbent Assay ◽

Toxin B ◽

B Subunit ◽

Adjuvant System ◽

Shiga Toxin 2 ◽

Uremic Syndrome

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) causes hemorrhagic colitis in humans and, in a subgroup of infected subjects, a more serious condition called hemolytic-uremic syndrome (HUS). These conditions arise because EHEC produces two antigenically distinct forms of Shiga toxin (Stx), called Stx1 and Stx2. Despite this, the production of Stx2 by virtually all EHEC serotypes and the documented role this toxin plays in HUS make it an attractive vaccine candidate. Previously, we assessed the potential of a purified recombinant Stx2 B-subunit preparation to prevent Shigatoxemia in rabbits. This study revealed that effective immunization could be achieved only if endotoxin was included with the vaccine antigen. Since the presence of endotoxin would be unacceptable in a human vaccine, the object of the studies described herein was to investigate ways to safely augment, in mice, the immunogenicity of the recombinant Stx2 B subunit containing <1 endotoxin unit per ml. The study revealed that sera from mice immunized with such a preparation, conjugated to keyhole limpet hemocyanin and administered with the Ribi adjuvant system, displayed the highest Shiga toxin 2 B-subunit-specific immunoglobulin G1 (IgG1) and IgG2a enzyme-linked immunosorbent assay titers and cytotoxicity-neutralizing activities in Ramos B cells. As well, 100% of the mice vaccinated with this preparation were subsequently protected from a lethal dose of Stx2 holotoxin. These results support further evaluation of a Stx2 B-subunit-based human EHEC vaccine.

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Immune Serum Produced by DNA Vaccination Protects Hamsters against Lethal Respiratory Challenge with Andes Virus

Journal of Virology ◽

10.1128/jvi.01822-07 ◽

2007 ◽

Vol 82 (3) ◽

pp. 1332-1338 ◽

Cited By ~ 51

Author(s):

Jay W. Hooper ◽

Anthony M. Ferro ◽

Victoria Wahl-Jensen

Keyword(s):

Dna Vaccine ◽

Neutralizing Antibodies ◽

Lethal Dose ◽

Case Fatality ◽

Immune Serum ◽

Human Infection ◽

M Gene ◽

Hamster Model ◽

Highly Pathogenic ◽

Andes Virus

ABSTRACT Hantavirus pulmonary syndrome (HPS) is a highly pathogenic disease (40% case fatality rate) carried by rodents chronically infected with certain viruses within the genus Hantavirus of the family Bunyaviridae. The primary mode of transmission to humans is thought to be inhalation of excreta from infected rodents; however, ingestion of contaminated material and rodent bites are also possible modes of transmission. Person-to-person transmission of HPS caused by one species of hantavirus, Andes virus (ANDV), has been reported. Previously, we reported that ANDV injected intramuscularly causes a disease in Syrian hamsters that closely resembles HPS in humans. Here we tested whether ANDV was lethal in hamsters when it was administered by routes that more accurately model the most common routes of human infection, i.e., the subcutaneous, intranasal, and intragastric routes. We discovered that ANDV was lethal by all three routes. Remarkably, even at very low doses, ANDV was highly pathogenic when it was introduced by the mucosal routes (50% lethal dose [LD50], ∼100 PFU). We performed passive transfer experiments to test the capacity of neutralizing antibodies to protect against lethal intranasal challenge. The neutralizing antibodies used in these experiments were produced in rabbits vaccinated by electroporation with a previously described ANDV M gene-based DNA vaccine, pWRG/AND-M. Hamsters that were administered immune serum on days −1 and +5 relative to challenge were protected against intranasal challenge (21 LD50). These findings demonstrate the utility of using the ANDV hamster model to study transmission across mucosal barriers and provide evidence that neutralizing antibodies produced by DNA vaccine technology can be used to protect against challenge by the respiratory route.

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Immunization with Epstein–Barr Virus Core Fusion Machinery Envelope Proteins Elicit High Titers of Neutralizing Activities and Protect Humanized Mice from Lethal Dose EBV Challenge

Vaccines ◽

10.3390/vaccines9030285 ◽

2021 ◽

Vol 9 (3) ◽

pp. 285

Author(s):

Xinle Cui ◽

Zhouhong Cao ◽

Yuriko Ishikawa ◽

Sara Cui ◽

Ken-Ichi Imadome ◽

...

Keyword(s):

Epithelial Cells ◽

B Lymphocytes ◽

Neutralizing Antibodies ◽

Epstein Barr Virus ◽

Lethal Dose ◽

Envelope Proteins ◽

Humanized Mice ◽

Target Cells ◽

Barr Virus ◽

Epstein Barr

Epstein–Barr virus (EBV) is the primary cause of infectious mononucleosis and is strongly implicated in the etiology of multiple lymphoid and epithelial cancers. EBV core fusion machinery envelope proteins gH/gL and gB coordinately mediate EBV fusion and entry into its target cells, B lymphocytes and epithelial cells, suggesting these proteins could induce antibodies that prevent EBV infection. We previously reported that the immunization of rabbits with recombinant EBV gH/gL or trimeric gB each induced markedly higher serum EBV-neutralizing titers for B lymphocytes than that of the leading EBV vaccine candidate gp350. In this study, we demonstrated that immunization of rabbits with EBV core fusion machinery proteins induced high titer EBV neutralizing antibodies for both B lymphocytes and epithelial cells, and EBV gH/gL in combination with EBV trimeric gB elicited strong synergistic EBV neutralizing activities. Furthermore, the immune sera from rabbits immunized with EBV gH/gL or trimeric gB demonstrated strong passive immune protection of humanized mice from lethal dose EBV challenge, partially or completely prevented death respectively, and markedly decreased the EBV load in peripheral blood of humanized mice. These data strongly suggest the combination of EBV core fusion machinery envelope proteins gH/gL and trimeric gB is a promising EBV prophylactic vaccine.

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Aptamer BC 007’s Affinity to Specific and Less-Specific Anti-SARS-CoV-2 Neutralizing Antibodies

Viruses ◽

10.3390/v13050932 ◽

2021 ◽

Vol 13 (5) ◽

pp. 932

Author(s):

Annekathrin Haberland ◽

Oxana Krylova ◽

Heike Nikolenko ◽

Peter Göttel ◽

Andre Dallmann ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Cross Reactivity ◽

Enzyme Linked Immunosorbent Assay ◽

Resonance Spectroscopy ◽

Plastic Plate ◽

Calorimetric Titration ◽

Background Signal ◽

High Background ◽

Specific Virus ◽

Binding Sequence

COVID-19 is a pandemic respiratory disease that is caused by the highly infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Anti-SARS-CoV-2 antibodies are essential weapons that a patient with COVID-19 has to combat the disease. When now repurposing a drug, namely an aptamer that interacts with SARS-CoV-2 proteins for COVID-19 treatment (BC 007), which is, however, a neutralizer of pathogenic autoantibodies in its original indication, the possibility of also binding and neutralizing anti-SARS-CoV-2 antibodies must be considered. Here, the highly specific virus-neutralizing antibodies have to be distinguished from the ones that also show cross-reactivity to tissues. The last-mentioned could be the origin of the widely reported SARS-CoV-2-induced autoimmunity, which should also become a target of therapy. We, therefore, used enzyme-linked immunosorbent assay (ELISA) technology to assess the binding of well-characterized publicly accessible anti-SARS-CoV-2 antibodies (CV07-209 and CV07-270) with BC 007. Nuclear magnetic resonance spectroscopy, isothermal calorimetric titration, and circular dichroism spectroscopy were additionally used to test the binding of BC 007 to DNA-binding sequence segments of these antibodies. BC 007 did not bind to the highly specific neutralizing anti-SARS-CoV-2 antibody but did bind to the less specific one. This, however, was a lot less compared to an autoantibody of its original indication (14.2%, range 11.0–21.5%). It was also interesting to see that the less-specific anti-SARS-CoV-2 antibody also showed a high background signal in the ELISA (binding on NeutrAvidin-coated or activated but noncoated plastic plate). These initial experiments suggest that the risk of binding and neutralizing highly specific anti-SARS CoV-2 antibodies by BC 007 should be low.

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Elicitation of Broadly Neutralizing Antibodies against B.1.1.7, B.1.351, and B.1.617.1 SARS-CoV-2 Variants by Three Prototype Strain-Derived Recombinant Protein Vaccines

Viruses ◽

10.3390/v13081421 ◽

2021 ◽

Vol 13 (8) ◽

pp. 1421

Author(s):

Yong Yang ◽

Jinkai Zang ◽

Shiqi Xu ◽

Xueyang Zhang ◽

Sule Yuan ◽

...

Keyword(s):

Recombinant Protein ◽

Neutralizing Antibodies ◽

Wild Type Strain ◽

Immune Escape ◽

High Titer ◽

Prototype Strain ◽

Vaccine Antigen ◽

Broadly Neutralizing Antibodies ◽

Continued Use ◽

Further Development

The ongoing coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Most of the currently approved SARS-CoV-2 vaccines use the prototype strain-derived spike (S) protein or its receptor-binding domain (RBD) as the vaccine antigen. The emergence of several novel SARS-CoV-2 variants has raised concerns about potential immune escape. In this study, we performed an immunogenicity comparison of prototype strain-derived RBD, S1, and S ectodomain trimer (S-trimer) antigens and evaluated their induction of neutralizing antibodies against three circulating SARS-CoV-2 variants, including B.1.1.7, B.1.351, and B.1.617.1. We found that, at the same antigen dose, the RBD and S-trimer vaccines were more potent than the S1 vaccine in eliciting long-lasting, high-titer broadly neutralizing antibodies in mice. The RBD immune sera remained highly effective against the B.1.1.7, B.1.351, and B.1.617.1 variants despite the corresponding neutralizing titers decreasing by 1.2-, 2.8-, and 3.5-fold relative to that against the wild-type strain. Significantly, the S-trimer immune sera exhibited comparable neutralization potency (less than twofold variation in neutralizing GMTs) towards the prototype strain and all three variants tested. These findings provide valuable information for further development of recombinant protein-based SARS-CoV-2 vaccines and support the continued use of currently approved SARS-CoV-2 vaccines in the regions/countries where variant viruses circulate.

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