Structural characterization of a neutralizing mAb H16.001, a potent candidate for a common potency assay for various HPV16 VLPs

Abstract With more human papillomavirus (HPV) virus-like particle (VLP) vaccines to hit the market in future, a monoclonal antibody (mAb) with preferably comparable reactivity against vaccines from different expression systems and bioprocesses is urgently needed for the potency characterization. Among all mAbs against HPV16 collected, rabbit mAb H16.001 is potently neutralizing with the highest affinity, recognizes an immune-dominant epitope, and can comparably react with HPV16 vaccines from various sources. Cryo-electron microscopic (cryo-EM) structure demonstrated that 360 H16.001 Fabs could bind to HPV16 capsid in preferable binding manner without steric hindrance between neighboring Fabs, potentially supporting its identification for VLP structural integrity and utility in monitoring VLP structural probity. This structural analysis indicated that mAb H16.001 afforded unbiased potency characterization for various HPV16 vaccines and was potential for use in vaccine regulation practice. This study also showed a model process for selecting suitable mAbs for potency assays of other vaccines.

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Characterization of a Broadly Reactive Monoclonal Antibody against Norovirus Genogroups I and II: Recognition of a Novel Conformational Epitope

Journal of Virology ◽

10.1128/jvi.00891-07 ◽

2007 ◽

Vol 81 (22) ◽

pp. 12298-12306 ◽

Cited By ~ 44

Author(s):

Tomoyuki Shiota ◽

Michio Okame ◽

Sayaka Takanashi ◽

Pattara Khamrin ◽

Makiko Takagi ◽

...

Keyword(s):

Monoclonal Antibody ◽

Monoclonal Antibodies ◽

Conformational Epitope ◽

The Novel ◽

Antigen Specificity ◽

Virus Like Particle ◽

The Family ◽

Immunological Diagnosis ◽

Further Development

ABSTRACT Norovirus, which belongs to the family Caliciviridae, is one of the major causes of nonbacterial acute gastroenteritis in the world. The main human noroviruses are of genogroup I (GI) and genogroup II (GII), which were subdivided further into at least 15 and 18 genotypes (GI/1 to GI/15 and GII/1 to GII/18), respectively. The development of immunological diagnosis for norovirus had been hindered by the antigen specificity of the polyclonal antibody. Therefore, several laboratories have produced broadly reactive monoclonal antibodies, which recognize the linear GI and GII cross-reactive epitopes or the conformational GI-specific epitope. In this study, we characterized the novel monoclonal antibody 14-1 (MAb14-1) for further development of the rapid immunochromatography test. Our results demonstrated that MAb14-1 could recognize 15 recombinant virus-like particles (GI/1, 4, 8, and 11 and GII/1 to 7 and 12 to 15) and showed weak affinity to the virus-like particle of GI/3. This recognition range is the broadest of the existing monoclonal antibodies. The epitope for MAb14-1 was identified by fragment, sequence, structural, and mutational analyses. Both terminal antigenic regions (amino acid positions 418 to 426 and 526 to 534) on the C-terminal P1 domain formed the conformational epitope and were in the proximity of the insertion region (positions 427 to 525). These regions contained six amino acids responsible for antigenicity that were conserved among genogroup(s), genus, and Caliciviridae. This epitope mapping explained the broad reactivity and different titers among GI and GII. To our knowledge, we are the first group to identify the GI and GII cross-reactive monoclonal antibody, which recognizes the novel conformational epitope. From these data, MAb14-1 could be used further to develop immunochromatography.

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Production and characterization of virus-like particles of grapevine fanleaf virus presenting L2 epitope of human papillomavirus minor capsid protein

BMC Biotechnology ◽

10.1186/s12896-019-0566-y ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 4

Author(s):

Razieh Yazdani ◽

Masoud Shams-Bakhsh ◽

Afshin Hassani-Mehraban ◽

Seyed Shahriar Arab ◽

Nicolas Thelen ◽

...

Keyword(s):

Electron Microscopy ◽

Human Papillomavirus ◽

Capsid Protein ◽

Surface Display ◽

Grapevine Fanleaf Virus ◽

E Coli ◽

Virus Like Particle ◽

Epitope Sequence ◽

Lower Production

Abstract Background Virus-like particle (VLP) platform represents a promising approach for the generation of efficient and immunogenic subunit vaccines. Here, the feasibility of using grapevine fanleaf virus (GFLV) VLPs as a new carrier for the presentation of human papillomavirus (HPV) L2 epitope was studied. To achieve this goal, a model of the HPV L2 epitope secondary structure was predicted and its insertion within 5 external loops in the GFLV capsid protein (CP) was evaluated. Results The epitope sequence was genetically inserted in the αB-αB” domain C of the GFLV CP, which was then over-expressed in Pichia pastoris and Escherichia coli. The highest expression yield was obtained in E. coli. Using this system, VLP formation requires a denaturation-refolding step, whereas VLPs with lower production yield were directly formed using P. pastoris, as confirmed by electron microscopy and immunostaining electron microscopy. Since the GFLV L2 VLPs were found to interact with the HPV L2 antibody under native conditions in capillary electrophoresis and in ELISA, it can be assumed that the inserted epitope is located at the VLP surface with its proper ternary structure. Conclusions The results demonstrate that GFLV VLPs constitute a potential scaffold for surface display of the epitope of interest.

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Characterization of Monoclonal Antibody Glycosylation: Comparison of Expression Systems and Identification of Terminal α-Linked Galactose

Analytical Biochemistry ◽

10.1006/abio.1997.2036 ◽

1997 ◽

Vol 247 (1) ◽

pp. 102-110 ◽

Cited By ~ 106

Author(s):

Douglas M. Sheeley ◽

Barbara M. Merrill ◽

Lester C.E. Taylor

Keyword(s):

Monoclonal Antibody ◽

Expression Systems

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Fuc(α1→3)GalNAc-: the major antigenic motif of Schistosoma mansoni glycolipids implicated in infection sera and keyhole-limpet haemocyanin cross-reactivity

Biochemical Journal ◽

10.1042/bj20011678 ◽

2002 ◽

Vol 366 (1) ◽

pp. 217-223 ◽

Cited By ~ 43

Author(s):

Sven R. KANTELHARDT ◽

Manfred WUHRER ◽

Roger D. DENNIS ◽

Michael J. DOENHOFF ◽

Quentin BICKLE ◽

...

Keyword(s):

Monoclonal Antibody ◽

Schistosoma Mansoni ◽

High Performance ◽

Cross Reactivity ◽

Keyhole Limpet Haemocyanin ◽

The Common ◽

Dominant Epitope ◽

Fucose Residue ◽

Structural Determinant

The aim of the present study was the characterization of the dominant epitope present on Schistosoma mansoni glycolipids, which causes cross-reactivity of S. mansoni and S. haematobium infection sera with keyhole-limpet haemocyanin (KLH). To this end, the monoclonal antibody M2D3H was chosen for its similar behaviour in high-performance TLC immunostaining and inhibition-ELISA to infection sera. Individual, structurally defined oligosaccharides derived from S. mansoni egg glycolipids were tested for their binding to this monoclonal antibody by immunoaffinity chromatography. A terminal fucose residue linked in the (α1→3) position to N-acetylgalactosamine was found to be the common structural determinant of the four oligosaccharides binding to M2D3H. The Fuc(α1→3)GalNAc-motif also appeared to be the basis for the cross-reactivity with KLH, a phenomenon used in the serodiagnosis of S. mansoni, S. haematobium and S. japonicum infections.

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Electron Microscopic Detection and Characterization of Nonhuman Primate Enteric Viruses

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100054145 ◽

1982 ◽

Vol 40 ◽

pp. 306-307

Author(s):

G. C. Smith ◽

R. L. Heberling ◽

S. S. Kalter

Keyword(s):

Electron Microscopy ◽

Animal Model ◽

Nonhuman Primate ◽

Clinical Condition ◽

Intestinal Tract ◽

Enteric Viruses ◽

Electron Microscopic ◽

Microscopic Detection

A number of viral agents are recognized as and suspected of causing the clinical condition “gastroenteritis.” In our attempts to establish an animal model for studies of this entity, we have been examining the nonhuman primate to ascertain what viruses may be found in the intestinal tract of “normal” animals as well as animals with diarrhea. Several virus types including coronavirus, adenovirus, herpesvirus, and picornavirus (Table I) were detected in our colony; however, rotavirus, astrovirus, and calicivirus have not yet been observed. Fecal specimens were prepared for electron microscopy by procedures reported previously.

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The Immuno-Electron Microscopic Localization of the Mo-Fe Protein Component of Nitrogenase in Cells of Azotobacter Vinelandii

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100073313 ◽

1973 ◽

Vol 31 ◽

pp. 574-575

Author(s):

J. T. Stasny ◽

R. C. Burns ◽

R. W. F. Hardy

Keyword(s):

Cellular Localization ◽

Direct Evidence ◽

Azotobacter Vinelandii ◽

Intracellular Localization ◽

Protein Component ◽

Electron Microscopic ◽

Thin Sections ◽

Fe Protein ◽

Intracytoplasmic Membranes

Structure-functlon studies of biological N2-fixation have correlated the presence of the enzyme nitrogenase with increased numbers of intracytoplasmic membranes in Azotobacter. However no direct evidence has been provided for the internal cellular localization of any nitrogenase. Recent advances concerned with the crystallizatiorTand the electron microscopic characterization of the Mo-Fe protein component of Azotobacter nitrogenase, prompted the use of this purified protein to obtain antibodies (Ab) to be conjugated to electron dense markers for the intracellular localization of the protein by electron microscopy. The present study describes the use of ferritin conjugated to goat antitMo-Fe protein immunoglobulin (IgG) and the observations following its topical application to thin sections of N2-grown Azotobacter.

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High-resolution transmission electron microscopic study of highly oxygen doped silicon layer

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100155438 ◽

1989 ◽

Vol 47 ◽

pp. 692-693

Author(s):

H. Takaoka ◽

M. Tomita ◽

T. Hayashi

Keyword(s):

High Resolution ◽

Oxygen Concentration ◽

Silicon Layer ◽

Detailed Structure ◽

Electron Microscopic ◽

Cross Sectional ◽

Transmission Electron ◽

Doped Silicon ◽

Argon Ion

High resolution transmission electron microscopy (HRTEM) is the effective technique for characterization of detailed structure of semiconductor materials. Oxygen is one of the important impurities in semiconductors. Detailed structure of highly oxygen doped silicon has not clearly investigated yet. This report describes detailed structure of highly oxygen doped silicon observed by HRTEM. Both samples prepared by Molecular beam epitaxy (MBE) and ion implantation were observed to investigate effects of oxygen concentration and doping methods to the crystal structure.The observed oxygen doped samples were prepared by MBE method in oxygen environment on (111) substrates. Oxygen concentration was about 1021 atoms/cm3. Another sample was silicon of (100) orientation implanted with oxygen ions at an energy of 180 keV. Oxygen concentration of this sample was about 1020 atoms/cm3 Cross-sectional specimens of (011) orientation were prepared by argon ion thinning and were observed by TEM at an accelerating voltage of 400 kV.

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Electron microscopic characterization of diamond thin films and substrates for diamond heteroepitaxial growth

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100154937 ◽

1989 ◽

Vol 47 ◽

pp. 592-593

Author(s):

J.B. Posthill ◽

R.P. Burns ◽

R.A. Rudder ◽

Y.H. Lee ◽

R.J. Markunas ◽

...

Keyword(s):

Thin Films ◽

Wide Band ◽

Deposition Process ◽

Heteroepitaxial Growth ◽

Electron Microscopic ◽

Wide Band Gap ◽

Lattice Matching ◽

Different Types ◽

Diamond Thin Films

Because of diamond’s wide band gap, high thermal conductivity, high breakdown voltage and high radiation resistance, there is a growing interest in developing diamond-based devices for several new and demanding electronic applications. In developing this technology, there are several new challenges to be overcome. Much of our effort has been directed at developing a diamond deposition process that will permit controlled, epitaxial growth. Also, because of cost and size considerations, it is mandatory that a non-native substrate be developed for heteroepitaxial nucleation and growth of diamond thin films. To this end, we are currently investigating the use of Ni single crystals on which different types of epitaxial metals are grown by molecular beam epitaxy (MBE) for lattice matching to diamond as well as surface chemistry modification. This contribution reports briefly on our microscopic observations that are integral to these endeavors.

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Functional Characterization of PM6/13, a β3-specific (GPIIIa/CD61) Monoclonal Antibody that Shows Preferential Inhibition of Fibrinogen Binding over Fibronectin Binding to Activated Human Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614247 ◽

1998 ◽

Vol 79 (01) ◽

pp. 177-185 ◽

Cited By ~ 10

Author(s):

Ashia Siddiqua ◽

Michael Wilkinson ◽

Vijay Kakkar ◽

Yatin Patel ◽

Salman Rahman ◽

...

Keyword(s):

Monoclonal Antibody ◽

Platelet Aggregation ◽

Platelet Rich Plasma ◽

Functional Characterization ◽

Human Platelets ◽

Western Blots ◽

Activated Platelets ◽

Reducing Conditions ◽

Fibronectin Binding

SummaryWe report the characterization of a monoclonal antibody (MAb) PM6/13 which recognises glycoprotein IIIa (GPIIIa) on platelet membranes and in functional studies inhibits platelet aggregation induced by all agonists examined. In platelet-rich plasma, inhibition of aggregation induced by ADP or low concentrations of collagen was accompanied by inhibition of 5-hydroxytryptamine secretion. EC50 values were 10 and 9 [H9262]g/ml antibody against ADP and collagen induced responses respectively. In washed platelets treated with the cyclooxygenase inhibitor, indomethacin, PM6/13 inhibited platelet aggregation induced by thrombin (0.2 U/ml), collagen (10 [H9262]g/ml) and U46619 (3 [H9262]M) with EC50 = 4, 8 and 4 [H9262]g/ml respectively, without affecting [14C]5-hydroxytryptamine secretion or [3H]arachidonate release in appropriately labelled cells. Studies in Fura 2-labelled platelets revealed that elevation of intracellular calcium by ADP, thrombin or U46619 was unaffected by PM6/13 suggesting that the epitope recognised by the antibody did not influence Ca2+ regulation. In agreement with the results from the platelet aggregation studies, PM6/13 was found to potently inhibit binding of 125I-fibrinogen to ADP activated platelets. Binding of this ligand was also inhibited by two other MAbs tested, namely SZ-21 (also to GPIIIa) and PM6/248 (to the GPIIb-IIIa complex). However when tested against binding of 125I-fibronectin to thrombin stimulated platelets, PM6/13 was ineffective in contrast with SZ-21 and PM6/248, that were both potent inhibitors. This suggested that the epitopes recognised by PM6/13 and SZ-21 on GPIIIa were distinct. Studies employing proteolytic dissection of 125I-labelled GPIIIa by trypsin followed by immunoprecipitation with PM6/13 and analysis by SDS-PAGE, revealed the presence of four fragments at 70, 55, 30 and 28 kDa. PM6/13 did not recognize any protein bands on Western blots performed under reducing conditions. However Western blotting analysis with PM6/13 under non-reducing conditions revealed strong detection of the parent GP IIIa molecule, of trypsin treated samples revealed recognition of an 80 kDa fragment at 1 min, faint recognition of a 60 kDa fragment at 60 min and no recognition of any product at 18 h treatment. Under similar conditions, SZ-21 recognized fragments at 80, 75 and 55 kDa with the 55kDa species persisting even after 18 h trypsin treatment. These studies confirm the epitopes recognised by PM6/13 and SZ-21 to be distinct and that PM6/13 represents a useful tool to differentiate the characteristics of fibrinogen and fibronectin binding to the GPIIb-IIIa complex on activated platelets.

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