Combination Antibody Therapy against Pseudomonas aeruginosa Infections: Comparison
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Despite advances in antimicrobial therapy and even the advent of some effective vaccines, Pseudomonas aeruginosa (P. aeruginosa) remains a significant cause of infectious disease, primarily due to antibiotic resistance. Although P. aeruginosa is commonly treatable with readily available therapeutics, these therapies are not always efficacious, particularly for certain classes of patients (e.g., cystic fibrosis (CF)) and for drug-resistant strains. Combinations of monoclonal antibodies against different targets and epitopes have demonstrated synergistic efficacy with each other as well as in combination with antimicrobial agents typically used to treat these infections. Such a strategy has reduced the ability of infectious agents to develop resistance. This entreviewy highlights potential targets secreted by  by P. aeruginosa that future polyclonal antibodies may directed against in order to develop more efficacious treatments against these infections. 

  • polyclonal antibodies
  • antibiotic resistance
  • antibiotics
  • Pseudomonas aeruginosa

1. Introduction

P. aeruginosa is a Gram-negative bacillus implicated in a wide variety of human infections. In acute infections, individual P. aeruginosa organisms exhibit swarming motility via a single flagellum and type 4 pili and express a wide variety of toxins, cell surface proteins, and other molecules that contribute to its immunogenicity and pathogenicity [1]. In order to establish chronic infection, P. aeruginosa transitions to a sessile, non-motile state marked by the formation of a mucoid biofilm, composed mainly of exo-polysaccharides, glycolipids, and mucin, which often poses a barrier to successful clinical treatment [2]. Regardless of if P. aeruginosa exists in an acute motile form or a chronic sessile biofilm, infection with P. aeruginosa is particularly perilous for immunosuppressed patients [1], ventilator-dependent patients, and cystic fibrosis patients.
Additionally, P. aeruginosa has been recognized as the causative organism in catheter-associated urinary tract infections, otitis externa, otitis media, contact lens keratitis, soft tissue infections in burn victims and AIDS patients, septic arthritis, folliculitis, meningitis, and sepsis. In fact, this broad array of associated disease states (Figure 1) has led P. aeruginosa to be recognized as the sixth leading cause of hospital-acquired infections, the second most common cause of ventilator-associated pneumonia and the most common multidrug-resistant Gram-negative cause of ventilator-associated pneumonia, the third most common cause of catheter-associated UTI, and the fifth most common cause of surgical site infections [1].
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Figure 1. Types of Acute P. Aeruginosa Infections [5][3]. P. aeruginosa is prevalent in skin and soft tissue infections (top right) including trauma, burns, and dermatitis. It also commonly causes swimmer’s’ ear (external otitis), hot tub folliculitis, and ocular infections, bacteremia and septicemia, especially in immunocompromised patients, and endocarditis associated with IV drug users and prosthetic heart valves (bottom right). P. aeruginosa can also cause central nervous system (CNS) infections such as meningitis and brain abscess (top left), bone and joint infections, including osteomyelitis and osteochondritis, respiratory tract infections, and hospital-acquired urinary tract infections (UTIs; bottom left). P. aeruginosa is also resistant to many common antibiotics [5][3].
The vast array of infectious complications that can arise from normal commensal and environmental strains of P. aeruginosa indicates that it is an opportunistic, adaptable, common environmental pathogen, making P. aeruginosa very robust and difficult to treat. Several antimicrobial agents possess the ability to treat P. aeruginosa infections [3][4]; however, successful clinical treatment regimens should include pre-treatment sensitivity testing, as different strains possess widely different antimicrobial resistances. Importantly, treatment is often dictated by the antibiogram of a specific hospital or region. P. aeruginosa is often susceptible to first-line agents, including beta-lactam antibiotics (e.g., piperacillin-tazobactam and ticarcillin-clavulanate), cephalosporins (e.g., ceftazidime, cefoperazone, and cefepime), and monobactams (e.g., Aztreonam). Carbapenems (e.g., meropenem and doripenem), which were historically seen as the “big gun”, last-ditch antimicrobials, can be used to treat highly resistant infections. However, as of 2019, the World Health Organization has listed carbapenem-resistant P. aeruginosa as one of three bacterial diseases in critical need of new treatment strategies, with up to 14% of P. aeruginosa isolates in the U.S. in 2019 expressing carbapenem resistance (Figure 2) [6][5]. 
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Figure 2. Treatment strategy for carbapenem-resistant P. aeruginosa isolates including future treatment options based on combinatorial antibody therapies [21][6]. In the ongoing battle between humans and the pathogenic microbes that cause disease, the CDC recognizes that the development of newer antimicrobial pharmacotherapeutics continues to be a pressing need, despite several current pharmaceutical agents that are reserved for the treatment of multidrug-resistant isolates [7]. In response to advancing antimicrobial pharmacotherapies, particularly bactericidal therapies that impose selective pressure, bacterial resistance mechanisms continue to evolve as opportunistic microbes adapt to an ever-changing therapeutic landscape. The evolution of multidrug-resistant P. aeruginosa can be considered as a case study based on its sophisticated quorum sensing communication system and phenotypic plasticity that has allowed it to adapt, survive, and thrive in a wide variety of environmental (e.g., aquatic and soil) and host conditions [11][8]. Among clinical isolates, a wide range of phenotypic variation has been identified including hyperpigmentation, small colony variant formation, autoaggregation, alginate overproduction, and autolysis [12,13,14,15,16][9][10][11][12][13]. These phenotypes change and adapt as an infection progresses, allowing for long-term survival in the differing conditions of the host [17][14]. 

2. Host Immune Response

P. aeruginosa infection commonly induces a robust humoral response including IgG antibodies towards lipopolysaccharide (LPS), alginate, alkaline protease, elastase, exotoxin A, and many other surface antigens and proteins of P. aeruginosa, which are often upregulated as virulence factors during various stages of biofilm development [36,37][15][16]. Unfortunately, the host antibodies produced typically have low affinity for their respective targets and are not effective at eliminating the infection [25][17]. As an aside, host opsonizing antibodies also cannot eliminate these mucoid microorganisms [38][18]. Nevertheless, anti-P. aeruginosa IgG binds to its antigen and immune complexes are formed, activating complement and recruiting macrophages. As macrophages and immune cells bind the anti-P. aeruginosa IgG, they create reactive oxygen species (ROS), consuming oxygen, making the biofilm environment more anaerobic and thus more favorable for the organism. The anaerobic environment is unsuitable for host macrophages, neutrophils, and other immune cells. Phagocytosis may occur, but without sufficient oxygen, ROS cannot be produced to eliminate bacteria. This creates an inflammatory environment causing tissue damage without efficient disruption of P. aeruginosa biofilms [25][17]. The ongoing inflammatory state in chronic infections is thus not linked to immunogenicity of the bacterial organisms themselves, but rather the secreted products that leave the biofilm and induce immune responses in the airway epithelium [23,24][19][20]; hence, the humoral responses produced by many people in response to P. aeruginosa infection are not effective in eliminating the infection.

3. Description of Targets

Taking inspiration from the immune response and in the context of P. aeruginosa’s life cycle and its antibiotic resistance mechanisms, several potential targets secreted by P. aeruginosa were identified. These targets, outlined in Table 1, produce a wide variety of effects in hosts and the bacteria, contributing to the pathogenesis of the entire spectrum of infections caused by this organism.
Table 1.
 Potential Therapeutic Antibody Targets.
Location or Class Examples Activity/Effects on Host
Cell surface Alginate Antiphagocytic, resists opsonic killing
Lipopolysaccharide Endotoxic, antiphagocytic, avoids preformed antibody to previously encountered O antigens
Pili (produced by type IV secretion) Twitching motility, biofilm formation, adherence to host tissues
Flagella Motility, biofilm formation, adherence to host tissues and mucin components
Injection of type III secretion factors PcrG, PcrV, PcrH, PopB, and PopD proteins form injection bridge for type III effectors
Outer membrane Siderophore receptors Provides iron for microbial growth and survival
  Efflux pumps Remove antibiotics
Secretion systems    
  • Type II
 Elastase, lipase, phospholipases, chitin-binding protein, exotoxin A, and others Variety of proteolytic, lipolytic, and toxic factors; degrade host immune effectors
  • Type III
 ExoS, ExoT, ExoU, ExoY Intoxicates cells (ExoS, ExoT); cytotoxic (ExoU); disrupts actin cytoskeleton
  • Type VI
 Cytoplasmic and membrane-associated proteins, ATPases, lipoproteins, Hcp1 protein Poorly characterized but found in animal studies to be needed for optimal virulence, particularly in chronic infection
Iron acquisition Pyoverdin, pyochelin, HasAP Scavenge iron from the host for bacterial use
Secreted toxins Hemolysins, rhamnolipid phospholipases Kill leukocytes, hemolysis of red cells, degrade host cell surface glycolipids
Secreted oxidative factors Pyocyanin, ferric pyochelin, HCN Produce reactive oxygen species: H2O2, O2−

Inflammatory, disrupts epithelial cell function
Quorum sensing LasR/LasI, RhlR/RhlI, PQS Biofilm formation, regulation of virulence factor secretion
ATPases = adenosine triphosphatases; PQS = Pseudomonas quinolone signal.

4. Antibodies as Therapeutics

Therapeutic antibodies work by activating and modulating our own host effector mechanisms including direct neutralization of toxins and pathogens, activation of the complement pathway, activation of neutrophil and macrophage opsonophagocytosis, activation of natural killer cells, enhancement of antigen presentation from dendritic cells to T cells and follicular dendritic cells to B cells, and degranulation of mast cells, eosinophils, and basophils. Additionally, Fc receptors that correspond to different classes of antibodies can activate the complement pathway, induce innate immune responses, and enhance natural adaptive immunity, providing a multi-faceted strategy to address disease [186][21]. Therapeutic monoclonal antibodies (mAbs) have emerged as a tour de force in the drug market and are projected to capture a rising percentage of the worldwide drug market—a trend that reflects increased prescription use of existing therapeutic mAbs and a large number (~50) of new therapeutic mAb drugs approved for use [187][22]. Currently, mAbs have been developed to treat a broad array of clinical conditions including cancer, autoimmune diseases, transplants, infectious disease, and toxin neutralization [188][23]. In contrast to mAbs, polyclonal antibodies (pAbs) have multiple epitope binding sites, thereby allowing greater coverage of neutralizing antigens. Much like our native immune system, pAbs are produced as a collection of antibodies from multiple B cell lineages, thus producing many different sites and affinities for the same antigen. Having multiple epitope binding sites allows for greater neutralization options and less opportunities for pathogens to develop escape mutants [189][24].

Combinatorial Therapy

Recently, it has been shown that antibodies and antibiotics can act concomitantly or even synergistically against pathogens. Several studies have shown that exposure to antibiotic therapy alters the expression of secreted factors, cell surface proteins, and other cellular products that may be viable immunotargets [190][25]. These altered levels of expression can change and potentially enhance the innate and humoral immune responses to pathogens—namely through increased antigen exposure, increased protein secretion, and potentially decreased virulence of pathogens [190][25]. Inspired by these same principles, combination or conjugate antibiotic/antibody therapeutic strategies, particularly those based on polyclonal antibodies, could be designed to capitalize on these same altered expression levels to enhance therapeutic efficacy and combat resistance.

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