Antibodies that boost complement activation vs. K. pneumoniae

Recent research uncovers how antibody combinations synergistically enhance complement activation to combat drug-resistant Klebsiella pneumoniae infections.

Test: Agnostic B cell selection method identifies K. Pneumoniae antibodies that synergistically drive complement activation. Photo source: Kateryna Kon / Shutterstock.com

In recent Nature communication In the study, researchers examine antibody-dependent complement activation during Klebsiella pneumoniae infection.

K. pneumonia infection

K. pneumonia is often a source of hospital infections, and due to the widespread exploit of antibiotics in these facilities, it has also acquired numerous resistance genes. In fact, over 700,000 deaths have been attributed to antibiotic resistance K. pneumonia infections in 2019 alone

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

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

In addition to the O-type, a type of polysaccharide capsule present on the surface K. pneumonia may also provide significant information about the immune system-activating properties of this bacterium. To date, over 70 different types of capsules have been identified.

Although most patients infected K. pneumonia are immunocompromised, usually retain a functioning complement system, suggesting the potential utility of complement-enhancing antibodies in the treatment of this type of infection. Nevertheless, it is unclear which bacterial antigens should be targeted to induce a robust complement response.

Identification of antibodies against K. pneumonia

Memory B cells (mBC) express membrane-bound antibodies as B cell receptors (BCR) and are the preferred source of antibody discovery. To identify human monoclonal antibodies (mAbs) against bacteria, most studies have examined antibodies targeting a single antigen. However, it is significant to obtain antibodies against different antigens of the same bacterial strain to study differences in antibody-regulated complement activation and to better assess the effectiveness of mAb combinations.

In the current study, human antibodies are targeted K. pneumonia determined using B cells that can identify fluorescently labeled cells K. pneumonia based on their BCR. The fluorescent dyes ATTO-488 and Cy5 were labeled with LPS, which is a conserved component of Gram-negative bacteria. Flow cytometry and direct stochastic optical reconstruction microscopy (dSTORM) techniques were used to detect effective surface labeling. K. pneumonia.

B cells from fit people were used to isolate those they were directed against K. pneumonia antigens. B cells that tested positive for ATTO-488 or Cy5 labeling K. pneumonia have been identified. However, approximately 0.05% of B cells were positive for both ATTO-488 and Cy5-labeled bacteria.

Fluorescence microscopy analysis revealed that the double-positive B cell population was highly enriched in CD27⁺ mBC. Many bacteria attached to double-positive sorted B cells, whereas single-positive sorted B cells showed no significant bacterial attachment.

B cells were selected from two clinical cells K. pneumonia isolates KpnO2 ii KpnO1, both of which are dominantly multidrug resistant K. pneumonia strains. Initially, 368 double-positive B cells were isolated KpnO2 by single cell sorting. After screening, 17 and 12 mAbs were bound to the unique CDR3 KpnO1 i KpnO2 identified accordingly.

Antibody-dependent complement activation on the target surface

Although antibody binding to the target surface is thought to be the first step in the complement cascade, investigators in the current study observed that antibody binding K. pneumonia 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, while three targeted the capsule (KL110). Although all anti-O2 antibodies enabled activation of the complement system, anti-capsular antibodies were less effective in their stimulation and ability to form hexameric IgG clusters.

The introduction of clustered enhancer mutations led to complement activation by anticapsular antibodies in both cases K. pneumonia AND S. pneumonia. Further research is required to better understand the relative mobility, surface concentrations and clustering of capsular polysaccharides and O2 glycans on K. pneumonia.

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

With all complement-activating antibodies, C3b is deposited on the surface K. pneumonia caused activation of complement-mediated phagocytosis and elimination by human neutrophils.

Combinations of antibodies synergistically activate the complement system

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

Conclusions

The current study discovered potential treatable antibodies K. pneumonia infection and revealed that combinations of different antibodies can potently and synergistically enhance complement activation K. pneumonia.