Neutralization of<i>Clostridium difficile</i>toxin A using antibody combinations

Demarest, Stephen J.; Hariharan, Mangala J.; Elia, Marikka; Salbato, Jared; Jin, Ping; Bird, C. C.; Short, Jay M.; Kimmel, Bruce E.; Dudley, Michael N.; Woodnutt, Gary; Hansen, Geneviève

doi:10.4161/mabs.2.2.11220

Cited by 30 publications

(24 citation statements)

References 51 publications

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“…While cooperativity and synergism of monoclonal antibody mixtures have been reported in the past (16)(17)(18)(19), emerging data have illustrated striking examples of situations wherein the activity of a monoclonal antibody can be drastically altered when assessed in the context of other antibodies (20,21). Indeed, the benefits of using polyclonal antibody preparations to treat disease in therapeutic settings are just beginning to be realized (22).…”

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confidence: 99%

Broadly Neutralizing Anti-Influenza Virus Antibodies: Enhancement of Neutralizing Potency in Polyclonal Mixtures and IgA Backbones

Mullarkey

Duty

et al. 2015

J Virol

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Current influenza virus vaccines rely upon the accurate prediction of circulating virus strains months in advance of the actual influenza season in order to allow time for vaccine manufacture. Unfortunately, mismatches occur frequently, and even when perfect matches are achieved, suboptimal vaccine efficacy leaves several high-risk populations vulnerable to infection. However, the recent discovery of broadly neutralizing antibodies that target the hemagglutinin (HA) stalk domain has renewed hope that the development of "universal" influenza virus vaccines may be within reach. Here, we examine the functions of influenza A virus hemagglutinin stalk-binding antibodies in an endogenous setting, i.e., as polyclonal preparations isolated from human sera. Relative to monoclonal antibodies that bind to the HA head domain, the neutralization potency of monoclonal stalk-binding antibodies was vastly inferior in vitro but was enhanced by several orders of magnitude in the polyclonal context. Furthermore, we demonstrated a surprising enhancement in IgA-mediated HA stalk neutralization relative to that achieved by antibodies of IgG isotypes. Mechanistically, this could be explained in two ways. Identical variable regions consistently neutralized virus more potently when in an IgA backbone compared to an IgG backbone. In addition, HA-specific memory B cells isolated from human peripheral blood were more likely to be stalk specific when secreting antibodies of IgA isotypes compared to those secreting IgG. Taken together, our data provide strong evidence that HA stalk-binding antibodies perform optimally when in a polyclonal context and that the targeted elicitation of HA stalk-specific IgA should be an important consideration during "universal" influenza virus vaccine design. IMPORTANCEInfluenza viruses remain one of the most worrisome global public health threats due to their capacity to cause pandemics. While seasonal vaccines fail to protect against the emergence of pandemic strains, a new class of broadly neutralizing antibodies has been recently discovered and may be the key to developing a "universal" influenza virus vaccine. While much has been learned about the biology of these antibodies, most studies have focused only on monoclonal antibodies of IgG subtypes. However, the study of monoclonal antibodies often fails to capture the complexity of antibody functions that occur during natural polyclonal responses. Here, we provide the first detailed analyses of the biological activity of these antibodies in polyclonal contexts, comparing both IgG and IgA isotypes isolated from human donors. The striking differences observed in the functional properties of broadly neutralizing antibodies in polyclonal contexts will be essential for guiding design of "universal" influenza virus vaccines and therapeutics. I nfluenza A viruses (IAVs) remain one of the most pressing global public health concerns due to their widespread distribution, rapid evolution, and potential for reassortment (1). These traits contribute to the abil...

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confidence: 99%

Broadly Neutralizing Anti-Influenza Virus Antibodies: Enhancement of Neutralizing Potency in Polyclonal Mixtures and IgA Backbones

Mullarkey

Duty

et al. 2015

J Virol

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“…Taken together, these data suggest that combining certain anti-toxin B MAbs offers a powerful synergistic benefit. The impact of a second antibody against the same toxin has been noted before (33), but the mechanism for this interaction remains unclear.…”

Section: Discussionmentioning

confidence: 99%

“…In contrast, a monoclonal antibody therapy offers control over timing and dose. For this reason, many have been actively working on the identification and development of toxin-specific monoclonal antibodies for treatment against recurrent CDI (29)(30)(31)(32)(33). Merck is currently developing a pair of monoclonal antibodies against toxins A and B.…”

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confidence: 99%

A Combination of Three Fully Human Toxin A- and Toxin B-Specific Monoclonal Antibodies Protects against Challenge with Highly Virulent Epidemic Strains of Clostridium difficile in the Hamster Model

Anosova¹,

Cole²,

Li³

et al. 2015

Clin. Vaccine Immunol.

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C lostridium difficile infection (CDI) is a leading cause of pseudomembranous colitis and diarrhea (C. difficile-associated diarrhea [CDAD]) causally related to a perturbation of the intestinal microbiota due to antibiotic use. Although the transmission of CDI is primarily associated with health care and long-term care facilities, C. difficile is a ubiquitous microorganism that has been found in the environment. There are documented cases of community-acquired CDI; in fact, the community-acquired C. difficile infection rates in the United States have been reported to be 7.7 cases per 100,000 person-years, of which 35% were not associated with antibiotics (1). However, the rates associated with health care and long-term care facilities are much higher, possibly due to the colocalization of a reservoir of the pathogen and a high number of susceptible individuals housed in those environments (2). As the eradication of C. difficile spores is very difficult, spore reservoirs can persist within the health care and long-term care environment for long periods (3-6). In recent years, CDI has increased in severity and incidence, and part of this increase is due to the spread of epidemic antibiotic-resistant strains (7,8). Treatment options remain limited and even appear to be losing efficacy, as evidenced by the continued spread of the epidemic strain and increasing numbers of patients who experience relapses and recurrent disease (9).Clostridial species are normal members of the human gut flora, usually as a small fraction of the microbiome and mostly nontoxigenic species (10). C. difficile pathogenesis in humans is associated with the disruption of the normal enteric flora and colonization with a toxigenic C. difficile strain. This is followed by overgrowth of vegetative cells and production of toxins that damage the cells of the colon through enzymatic activity of a glucosyltransferase, which glucosylates cytoskeletal regulators, such as Ras and Rac (11). Toxigenic C. difficile strains produce at least one of the two major exotoxins, toxin A or toxin B, and most produce both. Only toxigenic strains have been shown to cause intestinal inflammatory and diarrheal disease (12, 13); therefore, toxins A and B are believed to be major virulence factors of CDI, although other lessstudied virulence components of the bacterium can contribute to the disease. For example, the presence of a third toxin known as binary toxin has been associated with a marked increase in disease severity and risk of death. This increase was seen in all strains carrying the gene for the binary toxin, not just the C. difficile NAP1/027 strain associated with recent virulent outbreaks (14), but it remains unclear whether the binary toxin itself causes in-

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“…Therefore, the observed difference in TcdA sensitivity between C-PGC2 and CHO-K1 cells may be explained by the greater density in C-PGC2 cells of cell surface receptors binding the CROP region of TcdA. Furthermore, the crucial step for pathogenicity of the toxins is the translocation of the catalytic domain into the cytosol of target cells, a process for which TcdA CROPs are necessary (48,50).…”

Section: Discussionmentioning

confidence: 99%

Recombinant Mucin-Type Fusion Proteins with a Galα1,3Gal Substitution as Clostridium difficile Toxin A Inhibitors

et al. 2016

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The capability of a recombinant mucin-like fusion protein, P-selectin glycoprotein ligand-1/mouse IgG2b (PSGL-1/mIgG2b), carrying Gal␣1,3Gal␤1,4GlcNAc determinants to bind and inhibit Clostridium difficile toxin A (TcdA) was investigated. The fusion protein, produced by a glyco-engineered stable CHO-K1 cell line and designated C-PGC2, was purified by affinity and gel filtration chromatography from large-scale cultures. Liquid chromatography-mass spectrometry was used to characterize Oglycans released by reductive ␤-elimination, and new diagnostic ions to distinguish Gal␣1,3Gal-from Gal␣1,4Gal-terminated O-glycans were identified. The C-PGC2 cell line, which was 20-fold more sensitive to TcdA than the wild-type CHO-K1, is proposed as a novel cell-based model for TcdA cytotoxicity and neutralization assays. The C-PGC2-produced fusion protein could competitively inhibit TcdA binding to rabbit erythrocytes, making it a high-efficiency inhibitor of the hemagglutination property of TcdA. The fusion protein also exhibited a moderate capability for neutralization of TcdA cytotoxicity in both C-PGC2 and CHO-K1 cells, the former with and the latter without cell surface Gal␣1,3Gal␤1,4GlcNAc sequences. Future studies in animal models of C. difficile infection will reveal its TcdA-inhibitory effect and therapeutic potential in C. difficile-associated diseases.C lostridium difficile is an opportunistic Gram-positive pathogenic bacterium responsible for antibiotic-associated diarrhea and other gastrointestinal diseases. C. difficile infections, collectively known as C. difficile-associated disease (CDAD), range from mild cases of diarrhea to fatal pseudomembranous colitis (1, 2). Various treatment options for CDAD include antibiotics, fecal transplantation therapy, and, potentially, toxin-specific antibodies, vaccines, and replenishment of the patient microflora with oral probiotic therapy (3, 4). However, the emergence of strains with reduced susceptibility to antibiotics such as metronidazole and vancomycin and high rates of recurrent infection have limited the treatment options, necessitating more effective therapeutics that target the pathogenic mechanism of C. difficile (5-7).Toxin A (TcdA) and toxin B (TcdB), with molecular masses of 308 kDa and 270 kDa, respectively, are the two primary virulence factors secreted by C. difficile, and can inactivate the Rho/Ras superfamily of GTPases by glucosylation (8-10). As Rho GTPases regulate a number of vital cellular processes, their functional inactivation results in the inhibition of cell migration, morphogenesis, division, and membrane trafficking. Glucosylation of Rho family GTPases causes breakdown of the actin cytoskeleton and activation of caspase-3, leading to apoptosis of intoxicated cells. Diarrhea and inflammation ensue, and ultimately, the control of intestinal epithelial barrier function is lost (11-15). Much of the pathology seen during a C. difficile infection is believed to be due to toxin effects. TcdA exhibits many pathobiological functions, such as cell roundin...

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Neutralization ofClostridium difficiletoxin A using antibody combinations

Cited by 30 publications

References 51 publications

Broadly Neutralizing Anti-Influenza Virus Antibodies: Enhancement of Neutralizing Potency in Polyclonal Mixtures and IgA Backbones

Broadly Neutralizing Anti-Influenza Virus Antibodies: Enhancement of Neutralizing Potency in Polyclonal Mixtures and IgA Backbones

A Combination of Three Fully Human Toxin A- and Toxin B-Specific Monoclonal Antibodies Protects against Challenge with Highly Virulent Epidemic Strains of Clostridium difficile in the Hamster Model

Recombinant Mucin-Type Fusion Proteins with a Galα1,3Gal Substitution as Clostridium difficile Toxin A Inhibitors

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