# Identification of multiple Acinetobacter baumannii protein antigens as targets for potential immunotherapies using a novel protein microarray screening approach

## TL;DR

A new protein microarray method identified multiple Acinetobacter baumannii antigens that could be used for vaccines or antibody therapies to treat drug-resistant infections.

## Contribution

A novel protein microarray approach was developed to rapidly identify multiple conserved A. baumannii antigens with immunotherapeutic potential.

## Key findings

- The microarray identified 66 A. baumannii proteins that elicited IgG responses in infected mice.
- Polyclonal antibodies to selected antigens enhanced immune clearance of multidrug-resistant A. baumannii in mice.
- Combination of antibodies to two antigens fully protected mice against sepsis, with protection dependent on complement and neutrophils.

## Abstract

The World Health Organisation has identified Acinetobacter baumannii as a critical priority antimicrobial resistant (AMR) pathogen for which new therapeutics are needed. Despite this, currently there are no antibody or vaccine candidates in advanced clinical development for A. baumannii. To help address this, we designed a protein microarray approach to identify multiple A. baumannii protein antigens for further investigation as potential targets for vaccination or an antibody therapy. An 868-protein microarray was constructed containing mainly highly conserved A. baumannii proteins, and was enriched for those predicted to be surface localised and for which the corresponding gene is highly expressed during culture in ex vivo human serum. Probing the protein microarray with sera obtained from mice after non-lethal infection with multiple different A. baumannii strains identified IgG responses to 66 proteins. Four proteins (three previously poorly described outer membrane proteins and BamA, a known protective vaccine antigen selected as a positive control) were selected for further investigation. Polyclonal rabbit IgG to all four protein antigens recognised multiple clinical AMR A. baumannii strains, and for selected strains promoted opsonisation with IgG and complement, improved neutrophil phagocytosis, and increased membrane attack complex formation. Passive immunisation with polyclonal IgG to each antigen partially protected mice against A. baumannii sepsis, and a combination of polyclonal to two antigens completely protected against A. baumannii murine sepsis. Repeating passive immunisation experiments in mice depleted of complement, neutrophils or tissue macrophages demonstrated protection against systemic infection was dependent on complement and neutrophils but not macrophages. Overall, the data demonstrate that our protein microarray is a novel approach that can rapidly identify multiple new protein antigens as potential antibody targets for preventing or treating AMR bacterial infections.

Antimicrobial-resistant (AMR) bacterial infections are predicted to cause 10 million deaths per year by 2050. One important AMR bacterium is Acinetobacter baumannii, which typically causes multidrug-resistant infections in hospitalised patients and is a particularly common problem in some lower- and middle- income countries. Vaccines or monoclonal antibody therapies could reduce the burden of infections caused by AMR A. baumannii, but need suitable target antigens that are conserved among clinical strains of A. baumannii and promote bacterial killing by the immune system. Here, we use a novel protein microarray technique to identify multiple novel target protein antigens that are present in the majority of clinical A. baumannii isolates and are expressed during infection. Further testing showed that polyclonal rabbit antibodies raised against selected antigenic proteins promoted immune recognition of clinical A. baumannii strains and increased clearance of multidrug-resistant A. baumannii infections in mice. Our study provides a novel approach for identifying protein antigen targets for bacterial pathogens that could lead to improved prevention or treatment of AMR infections.