Functional Characterization and Evaluation of In Vitro Protective Efficacy of Murine Monoclonal Antibodies BURK24 and BURK37 against Burkholderia pseudomallei

ABSTRACTOur laboratory has developed more than a hundred mouse monoclonal antibodies (MAbs) againstBurkholderia pseudomalleiandBurkholderia mallei. These antibodies have been categorized into different groups based on their specificities and the biochemical natures of their target antigens. The current study first examined the bactericidal activities of a number of these MAbs by anin vitroopsonic assay. Then, thein vivoprotective efficacy of selected MAbs was evaluated using BALB/c mice challenged intranasally with a lethal dose of the bacteria. The opsonic assay using dimethyl sulfoxide-treated human HL-60 cells as phagocytes revealed that 19 out of 47 tested MAbs (40%) have prominent bactericidal activities againstB. pseudomalleiand/orB. mallei. Interestingly, all MAbs with strong opsonic activities are those with specificity against either the capsular polysaccharides (PS) or the lipopolysaccharides (LPS) of the bacteria. On the other hand, none of the MAbs reacting to bacterial proteins or glycoproteins showed prominent bactericidal activity. Further study revealed that the antigenic epitopes on either the capsular PS or LPS molecules were readily available for binding in intact bacteria, while the epitopes on proteins/glycoproteins were less accessible to the MAbs. Ourin vivostudy showed that four MAbs reactive to either the capsular PS or LPS were highly effective in protecting mice against lethal bacterial challenge. The result is compatible with that of ourin vitrostudy. The MAbs with the highest protective efficacy are those reactive to either the capsular PS or LPS of theBurkholderiabacteria.

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In vitro proliferation of T-cells from patients treated with murine monoclonal antibodies (mAbs)

European Journal of Cancer and Clinical Oncology ◽

10.1016/0277-5379(90)90716-7 ◽

1990 ◽

Vol 26 (9) ◽

pp. 1019

Keyword(s):

T Cells ◽

Monoclonal Antibodies ◽

In Vitro Proliferation ◽

Murine Monoclonal Antibodies

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Murine monoclonal antibodies against RBD of SARS-CoV-2 neutralize authentic wild type SARS-CoV-2 as well as B.1.1.7 and B.1.351 viruses and protect in vivo in a mouse model in a neutralization dependent manner

10.1101/2021.04.05.438547 ◽

2021 ◽

Author(s):

Fatima Amanat ◽

Shirin Strohmeier ◽

Wen-Hsin Lee ◽

Sandhya Bangaru ◽

Andrew B Ward ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Severe Acute Respiratory Syndrome ◽

Mouse Model ◽

Neutralizing Antibodies ◽

Dependent Manner ◽

Receptor Binding Domain ◽

Wild Type ◽

Murine Monoclonal Antibodies

After first emerging in December 2019 in China, severe acute respiratory syndrome 2 (SARS-CoV-2) has since caused a pandemic leading to millions of infections and deaths worldwide. Vaccines have been developed and authorized but supply of these vaccines is currently limited. With new variants of the virus now emerging and spreading globally, it is essential to develop therapeutics that are broadly protective and bind conserved epitopes in the receptor binding domain (RBD) or the whole spike of SARS-CoV-2. In this study, we have generated mouse monoclonal antibodies (mAbs) against different epitopes on the RBD and assessed binding and neutralization against authentic SARS-CoV-2. We have demonstrated that antibodies with neutralizing activity, but not non-neutralizing antibodies, lower viral titers in the lungs when administered in a prophylactic setting in vivo in a mouse challenge model. In addition, most of the mAbs cross-neutralize the B.1.351 as well as the B.1.1.7 variants in vitro.

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Novel Cross-Reactive Monoclonal Antibodies against Ebolavirus Glycoproteins Show Protection in a Murine Challenge Model

Journal of Virology ◽

10.1128/jvi.00652-17 ◽

2017 ◽

Vol 91 (16) ◽

Cited By ~ 18

Author(s):

James Duehr ◽

Teddy John Wohlbold ◽

Lisa Oestereich ◽

Veronika Chromikova ◽

Fatima Amanat ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Vesicular Stomatitis Virus ◽

Viral Vector ◽

Virus Disease ◽

Protective Efficacy ◽

Influenza Viruses ◽

Content Type ◽

Zaire Ebolavirus ◽

Vesicular Stomatitis

ABSTRACT Out of an estimated 31,100 cases since their discovery in 1976, ebolaviruses have caused approximately 13,000 deaths. The vast majority (∼11,000) of these occurred during the 2013-2016 West African epidemic. Three out of five species in the genus are known to cause Ebola Virus Disease in humans. Several monoclonal antibodies against the ebolavirus glycoprotein are currently in development as therapeutics. However, there is still a paucity of monoclonal antibodies that can cross-react between the glycoproteins of different ebolavirus species, and the mechanism of these monoclonal antibody therapeutics is still not understood in detail. Here, we generated a panel of eight murine monoclonal antibodies (MAbs) utilizing a prime-boost vaccination regimen with a Zaire ebolavirus glycoprotein expression plasmid followed by infection with a vesicular stomatitis virus expressing the Zaire ebolavirus glycoprotein. We tested the binding breadth of the resulting monoclonal antibodies using a set of recombinant surface glycoproteins from Reston, Taï Forest, Bundibugyo, Zaire, Sudan, and Marburg viruses and found two antibodies that showed pan-ebolavirus binding. An in vivo Stat2 −/− mouse model was utilized to test the ability of these MAbs to protect from infection with a vesicular stomatitis virus expressing the Zaire ebolavirus glycoprotein. Several of our antibodies, including the broadly binding ones, protected mice from mortality despite lacking neutralization capability in vitro, suggesting their protection may be mediated by Fc-FcR interactions. Indeed, three antibodies displayed cellular phagocytosis and/or antibody-dependent cell-mediated cytotoxicity in vitro. Our antibodies, specifically the two identified cross-reactive monoclonal antibodies (KL-2E5 and KL-2H7), might add to the understanding of anti-ebolavirus humoral immunity. IMPORTANCE This study describes the generation of a panel of novel anti-ebolavirus glycoprotein monoclonal antibodies, including two antibodies with broad cross-reactivity to all known ebolavirus species. The antibodies were raised using a heterologous DNA-viral vector prime-boost regimen, resulting in a high proportion of cross-reactive antibodies (25%). Similar vaccination regimens have been used successfully to induce broad protection against influenza viruses in humans, and our limited data indicate that this might be a useful strategy for filovirus vaccines as well. Several of our antibodies showed protective efficacy when tested in a novel murine challenge model and may be developed into future therapeutics.

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Development of Murine Monoclonal Antibodies for the Immunohistochemical Diagnosis of Systemic Bovine Aspergillosis

Journal of Veterinary Diagnostic Investigation ◽

10.1177/104063879600800111 ◽

1996 ◽

Vol 8 (1) ◽

pp. 68-75 ◽

Cited By ~ 31

Author(s):

H. E. Jensen ◽

B. Aalbaek ◽

P. Lind ◽

H. V. Krogh ◽

P. L. Frandsen

Keyword(s):

Monoclonal Antibodies ◽

Polyclonal Antibodies ◽

Fungal Species ◽

Cross Reactivity ◽

Water Soluble ◽

Enzyme Linked Immunosorbent Assay ◽

Immunohistochemical Techniques ◽

Immunohistochemical Diagnosis ◽

Murine Monoclonal Antibodies

Murine monoclonal antibodies (MAbs) against water-soluble somatic antigens (WSSA) and the wall fraction (WF) from Aspergillus fumigatus were produced by fusion of splenocytes from immunized BALB/c mice with mouse myeloma X63-Ag 8.653 cells. The supernatants of in vitro cultured hybridomas were initially screened for reactivity with the WSSA and the WF from A. fumigatus and WSSA of other fungi in an enzyme-linked immunosorbent assay (ELISA). Supernatants reacting only with A. fumigatus antigens were subsequently screened for homologous and heterologous reactivity with immunohistochemical techniques using formalin-fixed, paraffin-embedded tissues from experimentally infected mice. Because of a high immunohistochemical reactivity with homologous fungi, 4 MAbs raised against A. fumigatus WSSA and WF were selected for a further evaluation of cross-reactivity (diagnostic specificity) in immunohistochemical and immunoblotting assays. In immunohistochemical assays, all MAbs raised against WSSA cross-reacted heavily with a number of other fungal species. All 4 MAbs (MAb-WF-AF-1-4) raised against the WF reacted strongly with hyphae of Aspergillus spp.; hyphae of Scedosporium apiospermum were also strongly labeled by MAb-WF-AF-3 and-4. The 2 specifically reacting MAbs (MAb-WF-AF-1 and-2) were of the IgM biotype and were precipitating, and in immunoblotting experiments both bound to a 106-kD antigen of the WF, whereas they did not bind to WSSA of A. fumigatus. One of the 2 aspergillosis-specific MAbs (MAb-WF-AF-1) was used to screen 145 mycotic lesions of cattle. The diagnoses on bovine lesions obtained by MAb-WF-AF-1 were compared with results based on reactivity with heterologously absorbed polyclonal antibodies and, for some lesions, to culture results. In the vast majority of lesions ( n = 133), the MAb-WF-AF-1 and the polyclonal anti-Aspergillus antibodies reacted in a similar pattern, i.e., positively in 41 aspergillosis lesions and negatively in 92 zygomycotic lesions. Hyphae in 3 of 12 lesions that were not stained by the polyclonal antibodies reacted with the specific MAb-WF-AF-1; i.e., aspergillosis was diagnosed. The characteristics of the 2 MAbs (MAb-WF-AF-1 and-2) raised against the WF of A. fumigatus in ELISA and immunoblotting and immunohistochemical assays justify their application for the in situ diagnosis of systemic aspergillosis of cattle.

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The proliferative response of B cell chronic lymphocytic leukemia to interleukin 2: functional characterization of the interleukin 2 membrane receptors

Blood ◽

10.1182/blood.v69.6.1667.1667 ◽

1987 ◽

Vol 69 (6) ◽

pp. 1667-1673 ◽

Cited By ~ 30

Author(s):

I Touw ◽

L Dorssers ◽

B Lowenberg

Keyword(s):

Monoclonal Antibodies ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Functional Characterization ◽

Interleukin 2 ◽

Lymphocytic Leukemia ◽

Membrane Receptors ◽

Thymidine Uptake ◽

Cell Chronic Lymphocytic Leukemia

Abstract To determine the growth properties of B cell chronic lymphocytic leukemia (B CLL) and to identify possible abnormalities thereof, we examined the in vitro action of interleukin 2 (IL2) in four patients. Using radiolabeled IL2 and monoclonal antibodies reactive with IL2 membrane receptors we show that CLL cells, after their activation in vitro, express IL2 receptors of a high- as well as a low-affinity type, exactly as has been reported for normal T and B blasts. In three of the four reported cases, CLL proliferation (measured with 3H-thymidine incorporation) depended on the addition of phytohemagglutinin (PHA) to activate the cells and IL2 (optimal concentration, 10 to 100 U IL2/mL). In contrast, the cells of the fourth case of CLL (CLL-4) proliferated in an autonomous fashion, ie, without a need for PHA and IL2 in culture. Specific blocking of the IL2-binding sites with anti-IL2 receptor monoclonal antibodies almost completely inhibited the proliferation of these cells, which indicated that functional IL2 receptors were required for the autonomous proliferation. The demonstration of low concentrations of IL2 activity in the culture medium conditioned by the cells suggests that endogenous IL2 had been responsible for the spontaneous 3H-thymidine uptake by the CLL cells of patient 4. However, we were unable to extract IL2 mRNA from the cells (neither fresh nor after various in vitro incubations) in quantities detectable by Northern blot analysis that would prove that the CLL cells of patient 4 were actively synthesizing IL2 during culture. Thus, individual cases of B CLL are subject to variable growth regulation involving functional IL2 receptors on the cell surface: after activation with PHA the cells respond to exogenous IL2 in a fashion similar to normal B lymphocytes, or the cells are stimulated by endogenous IL2 (or an IL2-like activity) and do not require activation with PHA.

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Characterization of Chimpanzee/Human Monoclonal Antibodies to Vaccinia Virus A33 Glycoprotein and Its Variola Virus Homolog In Vitro and in a Vaccinia Virus Mouse Protection Model

Journal of Virology ◽

10.1128/jvi.00906-07 ◽

2007 ◽

Vol 81 (17) ◽

pp. 8989-8995 ◽

Cited By ~ 34

Author(s):

Zhaochun Chen ◽

Patricia Earl ◽

Jeffrey Americo ◽

Inger Damon ◽

Scott K. Smith ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Vaccinia Virus ◽

Protective Efficacy ◽

Variola Virus ◽

Binding Affinities ◽

Amino Acid Residues ◽

C Terminus ◽

20 Nm

ABSTRACT Three distinct chimpanzee Fabs against the A33 envelope glycoprotein of vaccinia virus were isolated and converted into complete monoclonal antibodies (MAbs) with human γ1 heavy-chain constant regions. The three MAbs (6C, 12C, and 12F) displayed high binding affinities to A33 (Kd of 0.14 nM to 20 nM) and may recognize the same epitope, which was determined to be conformational and located within amino acid residues 99 to 185 at the C terminus of A33. One or more of the MAbs were shown to reduce the spread of vaccinia virus as well as variola virus (the causative agent of smallpox) in vitro and to more effectively protect mice when administered before or 2 days after intranasal challenge with virulent vaccinia virus than a previously isolated mouse anti-A33 MAb (1G10) or vaccinia virus immunoglobulin. The protective efficacy afforded by anti-A33 MAb was comparable to that of a previously isolated chimpanzee/human anti-B5 MAb. The combination of anti-A33 MAb and anti-B5 MAb did not synergize the protective efficacy. These chimpanzee/human anti-A33 MAbs may be useful in the prevention and treatment of vaccinia virus-induced complications of vaccination against smallpox and may also be effective in the immunoprophylaxis and immunotherapy of smallpox and other orthopoxvirus diseases.

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Manufacturing of hybridomas, producing monoclonal antibodies against Burkholderia mallei and Burkholderia pseudomallei antigens

Journal of microbiology epidemiology immunobiology ◽

10.36233/0372-9311-2019-4-78-82 ◽

2019 ◽

pp. 78-82

Author(s):

G. V. Kuklina ◽

G. D. Elagin ◽

D. V. Pechenkin ◽

O. O. Fomenkov ◽

A. V. Eremkin ◽

...

Keyword(s):

Monoclonal Antibodies ◽

B Lymphocytes ◽

Burkholderia Pseudomallei ◽

Specific Activity ◽

Burkholderia Mallei ◽

Causative Agents ◽

Biological Tests ◽

Diagnostic Capabilities

Aim. Obtaining hybridomas, stable producing specific monoclonal antibodies against Burkholderia mallei and Burkholderia pseudomallei antigens. Materials and methods. The microbial cultures from State Collection of Microorganisms from the Branch of 48 CSRI of the Defense Ministry of Russian Federation (Kirov) and BALB/c mouse were used in research. Hybridization of B lymphocytes with SP2/0-Ag14 myeloma cells was performed by G.Kohler and C.Milstein procedure in De St. Fazekas and D.Scheidegger modification. The specific activity of immune sera, hybridoma supernatants, ascites and evaluating the diagnostic capabilities of monoclonal antibodies was studied by ELISA. Results. Hybridomas, producing monoclonal antibodies against causative agents of glanders and melioidosis antigens, were obtained and characterized. Obtained hybridomas are active and stable antibody producers after repeated in vitro and in vivo passaging. Immunoglobulins from obtained ascites were isolated. Antibodies provided the greatest sensitivity and specificity were selected. Conclusion. Monoclonal antibodies, producing by obtained hybridomas may be used for creating of immune biological tests.

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Structural and functional characterization of the mouse von Willebrand factor receptor GPIb-IX with novel monoclonal antibodies

Blood ◽

10.1182/blood.v95.3.886.003k45_886_893 ◽

2000 ◽

Vol 95 (3) ◽

pp. 886-893 ◽

Cited By ~ 105

Author(s):

Wolfgang Bergmeier ◽

Kirsten Rackebrandt ◽

Werner Schröder ◽

Hubert Zirngibl ◽

Bernhard Nieswandt

Keyword(s):

Monoclonal Antibodies ◽

Von Willebrand Factor ◽

Functional Characterization ◽

Receptor Internalization ◽

Inflammatory Conditions ◽

Structural And Functional Properties ◽

Von Willebrand ◽

Willebrand Factor

Five novel monoclonal antibodies (mAbs; p0p 1-5) were used to characterize the structural and functional properties and the in vivo expression of the murine GPIb-IX complex (von Willebrand factor receptor). The molecular weights of the subunits are similar to the human homologs: GPIb (150 kd), GPIbβ (25 kd), and GPIX (25 kd). Activation of platelets with thrombin or PMA predominantly induced shedding of glycocalicin (GC; 130 kd) but only low levels of receptor internalization. The GC concentration in normal mouse plasma was found to be at least 10 times higher than that described for human plasma (approximately 25 μg/mL versus 1-2 μg/mL). Two additional cleavage sites for unidentified platelet-derived proteases were found on GPIb, as demonstrated by the generation of 3 N-terminal fragments during in vitro incubation of washed platelets (GC, 60 kd, 45 kd). Occupancy of GPIb with p0p mAbs or F(ab)2-fragments resulted in aggregate formation in vitro and rapid irreversible thrombocytopenia in vivo, irrespective of the exact binding epitopes of the individual antibodies. GPIb-IX was not detectable immunohistochemically on endothelial cells in the major organs under normal or inflammatory conditions. The authors conclude that the mouse system might become an interesting model for studies on GPIb-IX function and regulation.

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The Effect of Human Serum on the Binding Activity of Radiolabelled Monoclonal Antibodies

Tumori Journal ◽

10.1177/030089168707300602 ◽

1987 ◽

Vol 73 (6) ◽

pp. 547-554

Author(s):

Silvia Camagni ◽

Silvana Canevari ◽

Marina Ripamonti ◽

Delia Mezzanzanica ◽

Rosaria Orlandi ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Human Blood ◽

Solid Phase ◽

Binding Capacity ◽

Binding Activity ◽

Ovarian Carcinomas ◽

Radiolabelled Monoclonal Antibodies ◽

In Vitro Stability ◽

Murine Monoclonal Antibodies

Three murine monoclonal antibodies (MoAbs), MBrl and MOv2 of IgM isotype and MOv8 of IgG isotype, with restricted reactivity for breast or ovarian carcinomas, were labelled with 125I in the perspective of obtaining specific and stable radioimmunopharmaceutical reagents. The radiolabeled MoAbs were analyzed for their « in vitro » stability in human blood. They were incubated at 37 °C for various lengths of time in human or, as a control, in murine blood and their binding capacity was evaluated by solid-phase RIA and compared with that obtained after incubation with buffer. In human blood, serum and plasma, but not with other components such as erythrocytes, leukocytes, HSA and IgG, the MoAbs revealed a loss of binding reactivity which was marked and constant for the IgM MoAbs, and only occasional for the IgG MoAb. In murine serum the decrease was not so rapid. The same change in the binding capacity was observed when the MoAbs were labelled with 3H or 35S, excluding the involvement of dehalogenating mechanisms. In the perspective of using MoAbs for intracavity therapy the effect of ascitic or pleural fluids on their binding activity was also evaluated. The inhibition of the binding reactivity was not as evident and was not related to the MoAb isotype.

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