Antibody and B cell responses in Staphylococcus aureus, Klebsiella pneumoniae and Escherichia coli infection

We will study the antibody and B cell response in patients with S. aureus, K. pneumoniae, and E. coli bacteraemia. From the blood we will isolate B cells and purify antibodies (Immunoglobulins). We have multiple approaches to identify interesting antibodies. From isolated B cells, we can: 1) select bacterium-specific B cells based on flow cytometry; 2) isolate RNA and generate yeast libraries that display the antibodies; 3) select all B cells and perform single-cell sequencing to select cells that respond to the infection. For method 1) and 2) sorted B cells will be lysed and RNA will be converted to cDNA by RT-PCR. The cDNA will be used to either amplify the variable heavy- and light-chain regions (VH/VL) of the BCR, which will be used for sequencing and cloning into antibody expression vectors or yeast display vectors. Alternatively, bulk isolated cells will be analyzed with 10x Genomics to obtain single cell immune profile data with paired BCR sequences. These sequences will be synthesized and cloned into antibody expression vectors. Cloning will be done in human embryonic kidney 293 (HEK-293) suspension cell lines to produce full-length antibodies. The supernatants, containing antibodies, will be tested for their capability to bind to bacteria and trigger complement activation and bacterial killing (either directly by complement or after phagocytosis by human neutrophils). This way we can select the B cells producing functionally relevant antibodies. The best antibodies will be produced at a larger scale and further developed towards a clinical therapeutic. A potent antibody is defined as: 1 *g/ml of purified antibody should trigger >80% phagocytosis or complement-dependent killing of bacteria. From isolate antibodies (present in serum/plasma), we will isolate specific antibodies by pull-down experiments, characterize the antibodies by mass spectrometry including de novo sequencing of the variable domains (mass spectrometry).

The serum/plasma of the patients will also be used for an initial screening

test to determine which patient has the best antibodies. In a later stage of

the project, the serum/plasma will be used to study whether the antibodies

identified in this project are better than the antibodies already present.

Also, we will isolate neutrophils (from the waste of the PBMC isolation) to

study whether the antibodies can work together with the patients* own

neutrophils.



Subject age, gender and days between bacteraemia and collection of blood for

the study will be recorded. Data will be collected regarding medical history,

use of medication (especially immunosuppressive therapy), baseline

characteristics (among which source of infection, e.g. catheter associated

sepsis, endocarditis, osteomyelitis, urinary, abdominal, other), antimicrobial

therapy, laboratory, radiology, cultures (any material), morbidity, mortality

within 90 days after the day of bacteraemia.





Other study parameters (if applicable)

Subject age, gender and days between bacteraemia and collection of blood for

the study will be recorded. Data will be collected regarding medical history,

use of medication (especially immunosuppressive therapy), baseline

characteristics (among which source of infection, e.g.: catheter associated

sepsis, endocarditis, osteomyelitis, urinary, abdominal, other), antimicrobial

therapy, laboratory, radiology, cultures (any material), morbidity, mortality

within 90 days after the day of bacteraemia.