Discovery of antibodies that boost complement activation against K. pneumoniae

New study uncovers how antibody combinations synergistically boost complement activation to combat drug-resistant Klebsiella pneumoniae infections.

K. pneumoniae infection

K. pneumoniae is frequently the source of hospital-acquired infections and, due to the widespread use of antibiotics in these settings, has also acquired numerous resistance genes. In fact, over 700,000 antibiotic resistance-related deaths were attributed to K. pneumoniae infections in 2019 alone.

The lipopolysaccharide (LPS) outer membrane of K. pneumoniae consists of a membrane-anchored lipid A that is connected to a core oligosaccharide region. An O-antigen polysaccharide chain can be found on the surface of this oligosaccharide region and consists of repeating sugar moieties.

K. pneumoniae infections are often classified based on the expression of different O-antigen types, with O1, O2, and O3 O-types responsible for about 80% of clinical infections. Most antibiotic and hypervirulent strains of K. pneumoniae express either the O1 or O2 antigen.

In addition to the O-type, the type of polysaccharide capsule present on the surface of K. pneumoniae can also provide important insights into the immune-activating properties of this bacteria. To date, over 70 different capsule types have been identified.

Although most patients infected with K. pneumoniae are immunocompromised, they typically retain a functioning complement system, thus suggesting the potential utility of complement-enhancing antibodies for treating this type of infection. Nevertheless, it remains unclear which bacterial antigens should be targeted to trigger a potent complement response.

Identifying antibodies against K. pneumoniae

Memory B-cells (mBCs) express membrane-bound antibodies as B-cell receptors (BCRs) and are the preferred source for antibody discovery. To identify human monoclonal antibodies (mAbs) against bacteria, most research has investigated antibodies that target a single antigen. However, it is important to obtain antibodies against different antigens on the same bacterial strain to study differences in antibody-regulated complement activation and better assess the efficacy of combining mAbs.

Fluorescent microscopy analysis revealed that the double-positive B-cell population was strongly enriched for CD27⁺ mBCs. Many bacteria attached to the double-positive sorted B-cells, whereas single-positive sorted B-cells did not exhibit significant bacterial attachment.

Antibody-dependent complement activation on the surface target

Although antibody binding to the target surface is considered to be the first step of the complement cascade, the researchers of the current study observed that antibody binding K. pneumoniae did not induce the complement system and, rather, that this activation of the complement system is dependent on the target antigen.

A total of 12 antibodies specifically targeted the O2-antigen, whereas three targeted the capsule (KL110). Although all anti-O2-antigen antibodies enabled activation of the complement system, anti-capsule antibodies were less effective in their stimulation and ability to to form hexameric IgG clusters.

Four antibodies including UKpn72, UKpn77, UKpn69, and UKpn76 effectively recognized the conserved epitope within the O1-antigen and cross-reacted with all tested O1 strains. These antibodies also successfully detectd the O1-antigen outside the bacterium.

Antibody combinations synergistically activate the complement system

A combination of two anti-capsule antibody clones caused synergistic binding and complement activation. Experiments using F(ab’)2 fragments revealed that cooperative binding between anti-capsular antibodies is independent of the Fc tails. However, the subsequent complement activation is mediated by the Fc tail. The two antibody clones mediating synergy were derived from the same donor, thus highlighting the possibility that these antibodies could act together in vivo.

Conclusions

Journal reference:

• van der Lans, S. P. A., Bardoel, B. W., Ruyken, M., et al. (2024) Agnostic B cell selection approach identifies antibodies against K. Pneumoniae that synergistically drive complement activation. Nature Communications 15(1); 1-17. doi:10.1038/s41467-024-52372-9