opsonophagocytic-killing-assay-to-assess-immunological-responses

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

University of Georgia

Most pathogenic bacteria express surface carbohydrates called capsular polysaccharides (CPSs). CPSs are important vaccine targets because they are easily accessible and recognizable by the immune system. However, CPS-specific adaptive humoral immune responses can only be achieved by the covalent conjugation of CPSs with carrier proteins to produce glycoconjugate vaccines. We previously described a mechanism by which a model glycoconjugate vaccine can activate the adaptive immune system and demonstrated that the mammalian CD4 T cell repertoire contains a population of carbohydrate-specific T cells. In this study, we use glycoconjugates of type 3  CPS (Pn3P) to assess whether the carbohydrate-specific adaptive immune response exemplified in our previous study can be applied to the conjugates of this lethal pathogen. In this article, we provide evidence for the functional roles of Pn3P-specific CD4 T cells utilizing mouse immunization schemes that induce Pn3P-specific IgG responses in a carbohydrate-specific T cell-dependent manner.

T Cell-Mediated Humoral Immune Responses to Type 3 Capsular Polysaccharide of .

Despite a century of investigation,  remains a major human pathogen, causing a number of diseases, such as pneumonia, meningitis, and otitis media. Like many encapsulated pathogens, the capsular polysaccharide (CPS) of  is a critical component for colonization and virulence in mammalian hosts. This study aimed to evaluate the protective role of a glycoside hydrolase, Pn3Pase, targeting the CPS of type 3 , which is one of the most virulent serotypes. We have assessed the ability of Pn3Pase to degrade the capsule on a live type 3 strain. Through  assays, we observed that Pn3Pase treatment increases the bacterium's susceptibility to phagocytosis by macrophages and complement-mediated killing by neutrophils. We have demonstrated that  Pn3Pase treatment reduces nasopharyngeal colonization and protects mice from sepsis caused by type 3  Due to the increasing shifts in serotype distribution, the rise in drug-resistant strains, and poor immune responses to vaccine-included serotypes, it is necessary to investigate approaches to combat pneumococcal infections. This study evaluates the interaction of pneumococcal CPS with the host at molecular, cellular, and systemic levels and offers an alternative therapeutic approach for diseases caused by  through enzymatic hydrolysis of the CPS.

Enzymatic Hydrolysis of Pneumococcal Capsular Polysaccharide Renders the Bacterium Vulnerable to Host Defense.

The glycan shield on the envelope glycoprotein gp120 of HIV-1 has drawn immense attention as a vulnerable site for broadly neutralizing antibodies and for its significant impact on host adaptive immune response to HIV-1. Glycosylation sites and glycan composition/structure at each site on gp120 along with the interactions of gp120 glycan shield with broadly neutralizing antibodies have been extensively studied. However, a method for directly and selectively tracking gp120 glycans has been lacking. Here, we integrate metabolic labeling and click chemistry technology with recombinant gp120 expression to demonstrate that gp120 glycans could be specifically labeled and directly detected. Selective labeling of gp120 by -azidoacetylmannosamine (ManNAz) and -azidoacetylgalactosamine (GalNAz) incorporation into the gp120 glycan shield was characterized by MS of tryptic glycopeptides. By using metabolically labeled gp120, we investigated the impact of gp120 glycosylation on its interaction with host cells and demonstrated that oligomannose enrichment and sialic acid deficiency drastically enhanced gp120 uptake by bone marrow-derived dendritic cells. Collectively, our data reveal an effective labeling and detection method for gp120, serving as a tool for functional characterization of the gp120 glycans and potentially other glycosylated proteins.

Fikri Y. Avci

Department of Biochemistry and Molecular Biology,
Center for Molecular Medicine and Complex Carbohydrate Research Center,
Department of Biochemistry and Molecular Biology, Center for Molecular Medicine and Complex Carbohydrate Research Center

University of Georgia

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens