Autotransporter-Mediated Display of Complement Receptor Ligands by Gram-Negative Bacteria Increases Antibody Responses and Limits Disease Severity

Abstract

The targeting of immunogens/vaccines to specific immune cells is a promising approach for amplifying immune responses in the absence of exogenous adjuvants. However, the targeting approaches reported thus far require novel, labor-intensive reagents for each vaccine and have primarily been shown as proof-of-concept with isolated proteins and/or inactivated bacteria. We have engineered a plasmid-based, complement receptor-targeting platform that is readily applicable to live forms of multiple gram-negative bacteria, including, but not limited to, Escherichia coli, Klebsiella pneumoniae, and Francisella tularensis. Using F. tularensis as a model, we find that targeted bacteria show increased binding and uptake by macrophages, which coincides with increased p38 and p65 phosphorylation. Mice vaccinated with targeted bacteria produce higher titers of specific antibody that recognizes a greater diversity of bacterial antigens. Following challenge with homologous or heterologous isolates, these mice exhibited less weight loss and/or accelerated weight recovery as compared to counterparts vaccinated with non-targeted immunogens. Collectively, these findings provide proof-of-concept for plasmid-based, complement receptor-targeting of live gram-negative bacteria.

Francisella and Antibodies

“…A second one consists of a generation of pathogen-specific IgM antibody clones produced by B1a B cells, which have been shown to induce protection while assuming the mutual interaction of cellular and humoral immune mechanisms [155], or in preparing antibody clones based on the knowledge of B-cell-activating epitopes on F. tularensis proteins [156]. Another, more sophisticated, approach is to construct a combined bacterial protein or whole bacterium with targeting (homing) components of eukaryotic molecules, such as Fc fragments of antibodies or the C3 component of complement, to provide directed opsonophagocytosis of tularemic antigens or of whole microbes and initiate the effective protective response (see, for example, Holland-Tummillo et al [157]. However, we should once more repeat that, in relation to effective immunoprophylaxis of tularemia, we still have substantial gaps in our knowledge regarding the effective immune mechanisms, their collaboration and precise timing during innate and adaptive phases of immune response, and the bacterial molecular mechanisms interfering with induced immune responses.…”

The O-Ag Antibody Response to Francisella Is Distinct in Rodents and Higher Animals and Can Serve as a Correlate of Protection

“…The RML isolate of LVS was provided by Catharine Bosio. Plasmid-modification of Ft was by electroporation [51]. WT Ft S4 was used within the CDC-certified A/BSL-3 facilities of Albany Medical College or the University of Pittsburgh.…”

“…With three exceptions detailed below (Rt α-LVS, Rt α-S4∆clpB, and Rb α-S4∆clpB), the generation/provision of all sera has been previously described. Mice: 2 groups of LVS primed C57BL/6 [51]; BalbC 1×, 3×; B6 3× and 3 × LVS/3 × S4 [29]; LVS∆sodB vaccinated C57BL/6 and Swiss Webster [34]. Rabbits: LVS [13], S4∆aroD and S4∆guaBA [14].…”

Co-Opting Host Receptors for Targeted Delivery of Bioconjugates—From Drugs to Bugs

“…Therefore, transformation of bacteria with the plasmid resulted in expression of OM-localized, surface-displayed C3d. This promotes targeting of the entire bacterial immunogen to CRs on immune cells and enhances protective responses; we have demonstrated this utilizing murine pulmonary Francisella tularensis infection as a model [ 51 ]. This technique is diagramed in Figure 1 f.…”

“…( f ) A plasmid encoding an OMP-anchored conjugate (lacking a cleavage site) is expressed in bacteria, effectively targeting the whole bacterial cell to immune cells when administered as a vaccine. Modeled after targeting Ft to CRs [ 51 ].…”