Progress Toward the Elusive Pseudomonas aeruginosa Vaccine

Abstract

Background:

The gram-negative bacterial pathogen Pseudomonas aeruginosa causes a wide range of infections, mostly in hospitalized and immunocompromised patients, those with burns, surgical wounds, or combat-related wounds, and in people with cystic fibrosis. The increasing antibiotic resistance of P. aeruginosa confers a pressing need for vaccines, yet there are no P. aeruginosa vaccines approved for human use, and recent promising candidates have failed in large clinical trials.

Discussion:

In this review, we summarize recent clinical trials and pre-clinical studies of P. aeruginosa vaccines and provide a suggested framework for the makeup of a future successful vaccine. Murine models of infection suggest that antibodies, specifically opsonophagocytic killing antibodies (OPK), antitoxin antibodies, and anti-attachment antibodies, combined with T cell immunity, specifically T_H17 responses, are needed for broad and potent protection against P. aeruginosa infection. A better understanding of the human immune response to P. aeruginosa infections, and to vaccine candidates, will eventually pave the way to a successful vaccine for this wily pathogen.

P seudomonas aeruginosa is a gram-negative, motile, rod-shaped bacterium with approximately 6,000 genes and a complex gene regulation network comprising 690 genes with 1,020 described regulatory interactions among their products, all of which help orchestrate its formidable metabolic antibiotic resistance and pathogenic abilities [1,2]. This versatility allows it to adapt to many different environments, from water reservoirs to soil and from medical equipment to the human organism itself. Even though it is not able to cause serious infections in healthy individuals, the same is not true for patients with compromised immune systems and barriers. It causes a serious infection burden for patients in the intensive care unit (ICU), on mechanical ventilators, and those with burns, surgical site infections, and combat wounds [3]. Furthermore, patients with cystic fibrosis (CF) suffer from chronic lung infection with P. aeruginosa because of mucus accumulation and abnormal bacterial clearance in their lungs caused by the lack or malfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) channel. Versatility can also be found in the types of infections it causes. P. aeruginosa infections can be local (respiratory tract, urinary tract, gastrointestinal tract, eye, skin, ear, and joints) or systemic with bacteremia often related to central venous catheters. Regardless of the site, P. aeruginosa infections are often accompanied by clinical symptoms and signs of sepsis.

Data from the U.S. Centers for Disease Control and Prevention (CDC) [4], reports that P. aeruginosa was the most commonly isolated gram-negative pathogen in ventilator-associated pneumonia (VAP) in 2009–2010 and was the second most common pathogen overall, accounting for 11% of cases. In a more recent worldwide epidemiology study of VAP, P. aeruginosa was the most common pathogen, with a prevalence of 3%–5% in adults ventilated for longer than 48 hours [5]. P. aeruginosa infections are especially prevalent in the elderly. A Canadian population-based study [6] of P. aeruginosa blood stream infections reported an exponentially increasing risk after age 60, with an annual incidence (per 100,000) of 10 in those 60–69 years old, 20 in those 70–79 years old, and 35 in those over 80 years old, with overall mortality rate of 29%. P. aeruginosa is also a frequent cause of war trauma-associated infections. A review of the Joint Theater Trauma Registry for infections diagnosed in combat casualties in Iraq and Afghanistan noted that 5.5% of patients had one or more infection-related billing codes, 25% of which were Pseudomonas infection codes [7]. A more recent study reported that among personnel with combat-related injuries sustained in Afghanistan, P. aeruginosa was the most common gram-negative organism identified during infection, and 10% were multi-drug resistant [8]. P. aeruginosa continues to be a major pathogen in burn wound infections throughout the world, with attributable high mortality, high risk for outbreaks at burn centers [9,10], and increasing antibiotic resistance [11 –15]. Patients with cancer who are neutropenic after chemotherapy are also a high-risk group for P. aeruginosa infections, whether in the lungs, at surgical sites, or in the blood stream via translocation from the gastrointestinal tract or invasion from a central venous catheter [16 –18].

Treatment of P. aeruginosa infections can be extremely challenging. Multi-drug–resistant (MDR) P. aeruginosa strains exist mainly in the hospital environment and arise because of the resilient resistance mechanism armamentarium of the bacterium, which includes hypermutation leading to OprD loss (and carbapenem resistance), AmpC overproduction (leading to β-lactam and cephalosporin resistance), modifications of GyrA or type II topoisomerases (leading to fluoroquinolone resistance), and overexpression of MexAB-OprM (leading to efflux of multiple different antibiotic classes) [19]. For a recent review on P. aeruginosa antibiotic resistance, see Bassetti et al. [20].

Vaccination against P. aeruginosa would eliminate or reduce the need for antibiotic agents and overcome many of the problems associated with antibiotic resistance. P. aeruginosa vaccines have been in development since the 1970s, but there is no vaccine available for human use. This review focuses on some of the recent developments in vaccine development since 2014. For a review of developments prior to this refer to Priebe and Goldberg [21], Sharma et al. [3], and Worgall et al. [22]. There are several things to consider regarding the makeup of an effective P. aeruginosa vaccine. Many patients who are at risk of P. aeruginosa infection have impaired immune systems caused by other diseases or other treatments that might impair the immune response to active immunization. An effective vaccine should elicit an antibody response that can both mediate opsonophagocytic killing by phagocytic cells (predominantly neutrophils and macrophages) and neutralize P. aeruginosa virulence factors. The target antigen(s) should be present in and well-conserved in strains that cause infection. These antigens should also be expressed at concentrations high enough that they are adequately exposed to the immune system.

A P. aeruginosa vaccine should also elicit a T-cell response characterized by T_H17 immunity. T_H17 cells are helper T cells that secrete the cytokine interleukin (IL)-17, which in turn recruits and activates the phagocytic cells needed for bacterial killing. Growing evidence, including data from our laboratory [23,24] and others [25 –31], suggests that T_H17 cells and IL-17 play a critical role in innate and adaptive antibacterial host defense. The action of IL-17 centers on neutrophil recruitment via induction of CXC chemokine secretion (KC/MIP-2 in rodents, GRO/IL-8 in humans) along with granulopoietic factors (such as granulocyte colony-stimulating factor [G-CSF]) that lead to increased bone marrow production and/or prolonged survival of neutrophils [29,32,33]. T_H17 cells can also secrete granulocyte-macrophage colony stimulating factor (GM-CSF) [34 –36]. T_HGM-CSF cells elicited by a live-attenuated P. aeruginosa vaccine can recruit protective monocytes/macrophages to the murine airways in neutropenic pneumonia models [36]. Additional benefits of vaccines that use a T_H17-based mechanism are that they do not select for vaccine escape mutants and can potentially protect against mixed infections with other bacterial species.

In this review, we summarize recent clinical trials investigating both active and passive immunization strategies. We then review pre-clinical studies that are seeking to improve vaccines currently under clinical development. Vaccines comprising new antigens are discussed next and we close by describing some novel delivery mechanisms and antigen screening strategies.

Vaccines in Clinical Development

Passive immunization

Patients who are susceptible to infection by P. aeruginosa may not be able to produce a functional immune response to active vaccination or the immediate need for protection might not allow for the time needed to develop a protective immune response after active vaccination. Passive immunization, specifically with humanized monoclonal antibodies against one or more P. aeruginosa virulence factors, has been evaluated in clinical trials, but to date none of these immunotherapies have been approved for patient use. It should also be noted that by definition, passive immunoprophylaxis or immunotherapy strategies function in the absence of T-cell responses, and therefore, are not likely to be as effective as active vaccines. Monoclonal antibody approaches also suffer from ease of escape by point mutations. There are several ongoing clinical trials of passive immunization strategies that are discussed in more detail below.

MEDI3902

MEDI3902 is a multifunctional bispecific IgG1-type monoclonal antibody under development by MedImmune (Gaithersburg, MD). MEDI3902 targets two P. aeruginosa virulence factors: PcrV [37], a serotype-independent type III secretion system (T3SS) protein that is involved in host cell cytotoxicity and Psl [38], an exopolysaccharide implicated in immune evasion, epithelial attachment, and biofilm formation [39]. Pre-clinical data demonstrate that MEDI3902 is able to protect against pneumonia and thermal burn infections in multiple animal species [39,40]. MEDI3902 enhances neutrophil uptake of P. aeruginosa and also increases inhibition of T3SS function (compared with anti-PcrV alone), allowing for better phagosome acidification and bacterial killing [39 –42] One major benefit of this bispecific antibody is that both PcrV and Psl are serotype independent. In a recent study, it was reported that PcrV and Psl are well-conserved among different clinical isolates worldwide, with an intact psl operon present in 94% of isolates and pcrV gene present in 99% of isolates [43]. Notably, in the 46 PcrV variant sequences that were identified, the MEDI3902-PcrV contact residues were preserved [43]. In 2014, a phase 1 trial (NCT02255760) was conducted with the primary goal of evaluating the safety of MEDI3902. Additional goals included evaluating the pharmacokinetics, anti-drug antibody (ADA) response, opsonophagocytic killing (OPK), and ex vivo anti-cytotoxicity of this antibody. This study was conducted in healthy adults by a single intravenous administration in dose escalation. There were no serious treatment-related adverse events reported other than some minor infusion-related reactions. Anti-drug antibody response was only detected in one subject after administration of MEDI3902. The same subject showed lower serum MEDI3902 concentrations from day 43 to day 61 compared with the ADA-negative subjects, indicating that the ADA might impair therapeutic efficacy. MEDI3902 serum concentrations correlated with the OPK activity and the serum anti-cytotoxicity antibody concentrations [44]. Currently, a phase 2 clinical trial (NCT02696902) administrating MEDI3902 to mechanically ventilated subjects who are colonized with P. aeruginosa and at risk for P. aeruginosa pneumonia is recruiting and is expected to be completed in 2021.

AR-105 (Aerucin^®)

A human IgG1 monoclonal antibody, the AR-105 (Aerucin^®, Aridis Pharmaceuticals, San Jose, CA) that targets P. aeruginosa alginate, is currently under development. A phase 1 trial (NCT02486770) was completed in 2015 in which the safety of Aerucin was evaluated in 16 healthy individuals after intravenous administration up to 20 mg/kg monitored for 84 days. There has not been a publication related to this trial, but the Food and Drug Administration (FDA) has granted it Fast-Track designation. Currently there is a placebo-controlled, double-blind, randomized phase 2 trial underway (NCT03027609) to investigate the efficacy of Aerucin in combination with standard antibiotic treatment in P. aeruginosa ventilator-associated pneumonia patients. The trial is expected to enroll 108 patients worldwide and conclude in June 2019.

Active immunization

An active immunization against P. aeruginosa infection could be administered to those at higher risk of P. aeruginosa infection. This population would include those at a higher risk of hospitalization for injury, patients who are admitted to ICUs, and potentially everyone over 60 years of age because this age group appears to be at increased risk of P. aeruginosa bacteremia in a population-based study from Canada [6], which reported an exponential increased risk after age 60.

IC43/VLA43

The IC43 vaccine was developed originally by Intercell AG (Vienna, Austria) but more recently has been under development by Valneva Austria GmbH (Vienna, Austria) and named VLA43. This vaccine consists of two outer membrane P. aeruginosa protein epitopes, the C-terminal of OprF and the entire protein OprI fused together as a single recombinant protein. The recombinant protein is expressed in Escherichia coli, purified, and combined with the adjuvant aluminum hydroxide (Alum)_. This OprF/I fusion protein vaccine (specifically OprF_190-342-OprI_21-83) has been shown in animal models (predominantly mouse models) to induce multiple immune effectors, including opsonophagocytic killing antibodies (OPK) [45], antibodies that inhibit interferon (IFN)-γ binding to P. aeruginosa [46] (thereby interfering with a virulence mechanism [47]), and via the OprF_329-342 epitope, IFN-γ⁺ T cell responses [48]. In the phase 1 trial (NCT00778388) four different doses of IC43 were administered intramuscularly to healthy adults, with two doses given seven days apart. There were no serious adverse events associated with vaccination. Specific OprF/I IgG antibodies were detected in all IC43 administered groups. From day 0 to day 14, a four-fold or more increase in the antibody titers were observed in more than 90% of subjects in all four treatment groups. At day 90, titers started to decline but nonetheless remained higher than the placebo groups for up to six months [45]. The phase 2 trial (NCT00876252) measured the immunogenicity of IC43 in patients admitted to the ICU with a predicted need for mechanical ventilation for more than 48 hours. Patients did not need to have a positive P. aeruginosa culture but needed to be deemed to be at risk for P. aeruginosa infection. Four hundred patients were given vaccine (100 mcg or 200 mcg IC43 with adjuvant, or 100 mcg IC43 without adjuvant, or placebo) intramuscularly at day 0 and 7 and evaluated up to 90 days. On day 14 there was a statistical increase in anti-OprF/I titers in all groups that were immunized with active vaccine, suggesting that rapid antibody responses could be achieved in this patient population. Interestingly, there was no statistically, significant difference in P. aeruginosa infection rates between patients vaccinated with IC43 and placebo, but there was lower mortality observed in patients immunized with IC43 compared with placebo. The study was underpowered to compare P. aeruginosa infection rates, and the authors noted that OprF/I-specific IgG immune response did not occur until 7–14 days post-immunization whereas most P. aeruginosa infections occurred before 14 days, suggesting these infections occurred too early, before vaccine-induced immune effectors could be mounted [49]. There was a follow-up phase 2/3 trial (NCT01563263) to confirm efficacy, immunogenicity, and safety of IC43. This study recruited approximately 800 adult patients in the ICU who were expected to be mechanically ventilated for at least 48 hours. Patients were given 100 mcg IC43 (without adjuvant) or placebo via an intramuscular injection at day 0 and again at day 7. According to a press release from Valneva, VAL43 (IC43) was unable to achieve its primary goal of reducing all-cause mortality, but was well tolerated and immunogenic [50]. The website of Valneva no longer lists VAL43, so it appears that its development has halted.

Table 1 summarizes P. aeruginosa vaccine trials since 2008. This includes the trials described above and trials that were conducted between 2008 and 2014, which are discussed in detail in a prior review [21]. A brief summary of these trials follows. KB001, a humanized IgG Fab′ fragment against PcrV, showed promising results in safety and pharmacokinetics and even managed to reduce the incidence of pneumonia in P. aeruginosa-colonized patients on mechanical ventilation in a phase 1/2 trial (NCT00691587) [51]. In another phase 1/2 clinical trial (NCT00638365), there were no significant differences between KB0001 and the placebo group in colonization concentrations in patients with CF, but there was a trend of reduced lung inflammation [52]. In a follow-up study, also in patients with CF but using a PEGylated version of KB001 called KB001-A, no significant reduction in colonization or inflammation was observed between KB001-A and the placebo group. It was noted that this antibody may not be of benefit because of the reported low concentrations of T3SS (including PcrV) proteins in P. aeruginosa-colonized patients with CF [53]. The company developing these anti-PcrV monoclonal antibodies (Kalobios) is no longer in business, so the future of their development is uncertain and likely depends on the clinical trial results of MedImmune's bispecific anti-PcrV antibody.

Table 1.

Pseudomonas aeruginosa Vaccine Clinical Trials that Occurred between 2008 and 2018

Name/antigen/antibody/responsible party	Study design	Study population	Main findings	Reference number, registry number
Passive immunization
MEDI3902, an IgG1 mAb anti-Psl/PcrV; MedImmune LLC	Phase 1, randomized, double-blind, Placebo controlled, Intravenous administration, single dose, dose Escalation	Adults (≤60 y), healthy individuals	TEAEs were mild or moderate in severity; most common were infusion-related reactions Anti-cytotoxic activity and OPK activity correlated with MED3902 serum concentrations One subject tested for ADA at Day 61	[44], NCT02255760
MEDI3902, an IgG1 mAb Anti-Psl/PcrV; MedImmune LLC	Phase 2, randomized, double-blind, placebo controlled, intravenous administration, single dose	Adults, P. aeruginosa colonization, mechanical ventilation	Ongoing	NCT02696902
AR-105 (Aerucin^® Aridis Pharmaceuticals, San Jose, Ca), IgG1 mAb against Alginate	Phase 1, non-randomized, open label, not placebo-controlled study, intravenous administration, single dose, dose escalation	Adults (≤50 y), healthy individuals	Safety up to doses of 20 mg/kg	[50], NCT02486770
AR-105 (Aerucin), IgG1 mAb against Alginate	Phase 2, randomized, double-blind, placebo-controlled, intravenous administration, single dose	Adults (≥20 y in Tawain), mechanical ventilation, P. aeruginosa pneumonia, APACHE II score between 10 and 35	Ongoing	NCT03027609
KB001, PEGylated mAb against PcrV; KaloBios, Humanigen	Phase 1/2, randomized, double-blind, placebo-controlled, Intravenous administration, high and low dose	Adults, mechanical ventilation (at least 3 d) with P. aeruginosa colonization	Well tolerated at day 28 No anti-KB001 antibodies were detected	[51], NCT00691587
KB001, PEGylated mAb against PcrV; KaloBios, Humanigen	Phase 1/2, randomized, double-blind, placebo-controlled, Intravenous administration, high and low dose	Children (≥12 y), adults and older adults, CF	No deaths or life-threatening adverse events, two infusion-related AEs Mean serum half-life was 12 d PA T3SS expression detected in all subjects' sputum A trend of dose-dependent reduction in sputum myeloperoxidase, IL-1, and IL-8 detected at day 28 Sputum neutrophil elastase and neutrophil counts favoring the KB001 10 mg/kg group compared with placebo	[52], NCT00638365
KB001-A, PEGylated mAb against PcrV, differs from the preceding KB001 by a single amino acid Substitution; KaloBios, Humanigen	Phase 2, randomized, double-blind placebo-controlled, repeat-dose study	Children (≥12 yr and adults (≤50 y), CF	Week 16: Time to need for antibiotic agents did not differ between groups 3.2% increase in ppFEV₁ of treated group versus placebo Safe and well-tolerated	[53], NCT01695343
Avian polyclonal anti-P. aeruginosa antibodies (IgY); Mukoviszidose Institut gGmbH	Phase 3, randomized, double-blind, placebo-controlled, oral administration, every day dose up to 24 mo	Children (≥5 y), adults and older adults, CF	Not yet available	NCT01455675
Active immunization
IC43, a recombinant, OprF/I vaccine (also known as VLA43); Valneva Austria GmbH	Phase 1, randomized, double-blind, placebo-controlled, intra-muscular administration, 2 doses (0, 7 d) dose escalation	Adults, healthy individuals	Higher OprF/I IgG specific antibody titers detected at day 14 for all IC43 groups vs. placebo ≥Four-fold increase in OprF/I IgG titers from day 0 to day 14 On day 90, OprF/I-specific IgGs started to decline in all IC43 treatment groups but remained higher at 6 mo vs. placebo OPK followed a similar pattern OprF/I-specific IgG secreted by ASCs was detected in all IC43 groups after the second vaccination and up to 6 mo All vaccinations were well tolerated	[45], NCT00778388
IC43, a recombinant, OprF/I vaccine (also known as VLA43); Valneva Austria GmbH	Phase 2, randomized, partially blind, placebo- controlled, intramuscular administration, two doses (0, 7 days), with and without Alum adjuvant	Children (≥8 y), adults and older adults (≤80 y), ICU patients, mechanical ventilation (>48 h)	Higher OprF/I IgG specific antibody titers detected at day 14 for all IC43 groups vs. placebo ≥Four-fold increase in OprF/I IgG titers from day 0 to day 14 No significant differences in PA infection rates No deaths related to the vaccine treatment Less than 5% of patients showed local tolerability symptoms A low rate (3.1%– 10.6%) of treatment- related treatment- emergent adverse events was observed in the IC43 groups	[49], NCT00876252
IC43, a recombinant, OprF/I vaccine (also known as VLA43), Valneva Austria GmbH	Phase 2/3, randomized, double-blind, placebo- controlled, intramuscular administration, two doses (0, 7 days), single concentration, no adjuvant	Adults and older adults (≤80 y), ICU patients, mechanical ventilation (>48 h)	No clinically meaningful reduction of all- cause mortality in the vaccine group was detected compared with the placebo Overall survival did not differ between the treated group and the placebo	[86], NCT01563263

The table is divided into passive and active immunization strategies. Clinical trials of the same vaccine are listed in chronological order [87].

mAb = monoclonal antibody; TEAEs = treatment-emergent adverse events; OPK = opsonophagocytic killing antibodies; ADA = anti-drug antibody; APACHE = Acute Physiologic Assessment and Chronic Health Evaluation; T3SS = type III secretion system; AEs = adverse effects; ppFEV = percent predicted forced expiratory volume in 1 second; LPS = lipopolysaccharide; ICU = intensive care unit.

Another passive immunization strategy, panobacumab (KBPA-101), which is an antibody against lipopolysaccharides (LPS) of serotype O11, was tested in a phase 1/2 trial (NCT00851435) that enrolled ICU patients who had pneumonia caused by P. aeruginosa serotype O11. This trial was open label and only had a treatment arm. There were no serious adverse events and although the study was not designed or powered to evaluate efficacy, the authors suggested an improvement in outcomes in patients who received KBPA-101 [54]. In a follow-up post hoc analysis of data from the trial in which the 13 patients who received KBPA-101 were compared with 14 patients who did not receive KBPA-101, survival and resolution of infection were substantially improved after KBPA-101 [55]. Another passive immunization strategy that has been evaluated in clinical trials is the avian antibody, IgY, purified from hen eggs immunized with P. aeruginosa. In a phase 1/2 trial (NCT00633191) in patients with CF who were colonized by P. aeruginosa, treatment showed prolonged time between colonization events compared with to historical controls but because of small numbers and lack of placebo, concrete conclusions could not be drawn [56]. A phase 3 trial (NCT01455675) with a higher number of participants and a control group was performed but results are not yet available [57].

Vaccines Under Development

In addition to the vaccines already in clinical trials, there are many vaccines in various stages of pre-clinical development. Many of these involve adding antigens and delivery systems to the vaccine antigens already in clinical trials. There also are several exciting novel antigens that are in the beginning stages of pre-clinical studies. A summary of the most extensively studied antigens is provided in Table 2.

Table 2.

Candidate Pseudomonas aeruginosa Vaccine Antigens, their Bacterial Function, Role in Virulence, and Immune Responses after Vaccination

Antigen	Function in Pseudomonas aeruginosa	Role in virulence	Immune response in Mice	Reference
Live-attenuated vaccines	Bacteria missing the aroA gene		Opsonophagocytic antibodies T_H17 responses	[23]
Multi-valent live attenuated P. aeruginosa vaccines	Bacteria missing the aroA gene		Opsonophagocytic antibodies Local CD4 T cells in the lung T_H17 responses	[88]
O antigen	Polysaccharide repeating unit of LPS confers serogroup	Complement/serum resistance	Opsonophagocytic antibodies Antibody-dependent cellular cytotoxicity killing	[89 –91]
Alginate, also known as mucoid exopolysaccharide (MEP)	Secreted exopolysaccharide Confers mucoidy	Biofilm formation Antibiotic resistance Phagocyte resistance	Opsonophagocytic antibodies (variable)	[92 –95]
Flagella (FliC)	Motility	Attachment to mucin Mucosal colonization	Opsonophagocytic antibodies Adjuvant effect through TLR5 IgG, IgA, and secretory IgA antibody production PMN response	[96, 97]
PilA	Major pilin subunit of T4P (structural component)	T4P are responsible for twitching, adhesion, and invasion of tissue	Antibody-mediated immunity T_H2 polarized Immunity	[66,67,98]
PilQ	The outer membrane secretin channel protein of T4P	T4P are responsible for twitching, adhesion and invasion of tissue	T_H17-stimulating protein T_H2 polarized immunity when fused with PilA	[24,67,99]
PopB	T3SS translocator	Translocation of effector proteins leading to intoxication of eukaryotic cells	T_H17-stimulating protein	[24]
OprF	Outer membrane porin, contributes to cell structure maintenance, membrane permeability, and environmental sensing	Binds C3b Adhesion factor Biofilm formation Binds IFN-γ	Antibody-mediated immunity IFN-γ⁺ CD4 and CD8 responses	[47,100 –103,45]
OprI	Outer membrane protein		Opsonophagocytic antibodies	[104]
OprL	Outer membrane protein	Protection against H₂O₂	T_H17 response	[24,31,105]
PcrV	Surface-expressed, needle-tip protein component of the T3SS	Translocation of effector proteins leading to intoxication of eukaryotic cells	Blockage with an antibody: Reduces cytotoxicity and inflammationAllows internalization of bacteria by neutrophils	[42,106 –108]
Psl	Secreted exopolysaccharide	Biofilm formation Protection from opsonization Neutrophil and macrophage evasion	Blockage with an antibody: Allows phagocytosis by neutrophilsPrevents epithelial attachment	[38,42,109]

Ig = immunoglobulin; LPS = lipopolysaccharide; T3SS = type III secretion system;

T4P = subunit of type IV pili; IFN-γ = interferon-γ; PMN = polymorphonuclear neutrophils.

Modifications of and additions to vaccine antigens under clinical development

Several studies are investigating modifying vaccines that are under clinical development to improve their efficacy. One such study sought to improve the efficacy of the OprF and OprI antigens by adding the type B flagellin antigen. This trivalent vaccine was able to protect against both mucoid and non-mucoid P. aeruginosa strains after intra-nasal challenge. There was no improvement in bacterial clearance with the addition of flagellin, but there was a reduction in the level of inflammation observed by histology when flagellin was added [58]. PcrV, one of the targets of the MEDI3902 bispecific antibody, is being developed as an active vaccine antigen as part of a trivalent vaccine with OprI and Hcp1 (a central component of the type VI secretion system). Protection against burn wound infection and pneumonia was generated using this antigen as an active vaccine or to stimulate antibodies for passive immunization [59]. A recombinant attenuated Salmonella vaccine (RASV) that expresses a fusion protein containing domains of OprF and OprI has also been evaluated as a novel delivery mechanism of these well-tested vaccine antigens. The attenuated Salmonella was given orally or subcutaneously to mice and able to protect against a lethal intranasal challenge [60].

Several studies have used different approaches to deliver OprF antigens, including those investigating the use of DNA vaccines in which DNA encoding the antigen is administered and taken into cells that then produce the antigen, which in turn elicits an immune response. One study evaluated the immunogenicity of a DNA vaccine encoding OprF in combination with herpes simplex virus type 1 tegument protein, VP22 [61]. This vaccine was delivered intramuscularly to mice and it was found that the pVAX1-OprF-VP22 construct induced higher IgG serum titers and T-cell proliferation and better protected against P. aeruginosa IP infection in comparison to constructs containing only OprF or VP22. The immune response was characterized by a preferential increase of IgG2a and IFN-γ (T_H1 polarization) [61]. Another study used a DNA vaccine approach to vaccinate with the surface proteins OprF and OprL to protect against P. aeruginosa challenge in chickens [62]. PcrV has also been evaluated for use in a DNA vaccine along with exotoxin A [63]. Immunization with this construct generated both B- and T-cell responses and protected against intra-tracheal challenge of P. aeruginosa.

Other Vaccine Antigens

Lipopolysaccharide and alginate in conjugate vaccines

Lipopolysaccharide (LPS) and alginate were among the first antigens pursued as vaccine antigens, but these antigens were limited by toxicity, pyrogenicity, the diversity in LPS serotypes, and lack of immunogenicity of alginate. One study revisited these antigens by evaluating these antigens by conjugating purified, detoxified, and depolymerized LPS or alginate to diphtheria toxoid (DT). Intra-peritoneal immunization of either vaccine resulted in antibody responses, but there were no challenge experiments reported, so it is unknown if this vaccine is protective [64]. An additional study evaluated alginate conjugated to the outer membrane vesicle (OMV) of Neisseria meningitidis (B serogroup). This conjugate vaccine was able to elicit opsonic antibodies and protect against an intra-nasal P. aeruginosa challenge with two different strains [65].

Multiple studies have evaluated components of the pilus and flagella for use as vaccine antigens. One study found that vaccination with recombinant PilA could protect against a lethal challenge in a murine burn model [66]. Protection in a murine burn model was also generated when mice were vaccinated with a fusion protein containing a domain of PilA along with a subunit of PilQ [67]. Vaccination with a mix of recombinant type a and b flagellin proteins were able to protect against a lethal challenge using the murine burn model [68] and a pneumonia model [69]. Bacterial flagella are potent stimulators of the immune system, and this protection was observed without the addition of adjuvant. Azad et al. [70] also showed protection against lethal challenge using a murine model using recombinant PilQ and type b-flagellin proteins. Lower protection using a murine burn model and lower OPK activity was noted when a heterologous strain was used compared with a homologous strain highlighting the importance of heterologous challenge experiments [71]. These results highlight a general feature of flagella-based vaccines in the lack of cross-protection against heterologous flagellin types, as previously delineated by Pier et al. [72]. A fusion protein of exotoxin A and flagellin domains was also found to be protective against intraperitoneal challenge using a single clinical strain [73]. Whether other flagella types are protected by this vaccine is unclear. Faezi et al. [74] found that immunization with flagella and pilin was able elicit similar levels of protection against a homologous strain and a non-typed, MDR clinical isolate in a murine burn model. Another study showed that immunization with pilin (PilA) and a novel adjuvant combination (alum and naloxone) could induce both antibody and T cell responses (including T_H17 responses) and achieve protection in a murine pneumonia model [75].

Several recent studies have evaluated novel vaccine antigens or approaches. A reverse vaccinology strategy identified a P. aeruginosa OmpA C-like protein (PA0833) as a potential vaccine, and intramuscular vaccination with a recombinant version of the protein partially protected against homologous challenge in murine sepsis and pneumonia models [76]. Zhang et al. [77] reported recently on the use of P. aeruginosa OMVs as intramuscular vaccines, showing the OMVs to be composed mostly of flagellin and other outer membrane proteins (specifically OprF and OprH/G), inducive of antibody and T-cell responses after intramuscular immunization with aluminum phosphate as adjuvant, and protective against a lethal intra-tracheal P. aeruginosa challenge using the strain from which the OMVs were prepared as well as three clinical isolates [77]. The role of LPS-induced antibodies in these studies is not clear and warrants further investigation.

New P. aeruginosa antigen delivery systems

Several research groups are pursuing novel antigen delivery systems that could potentially increase the efficacy of these vaccines. These novel approaches include the DNA vaccines and attenuated Salmonella strains listed above. A new and innovative delivery mechanism is to use polymeric intra-cellular polyhydroxyalkanoate (PHA) inclusions. These spheres are produced by many bacteria, including P. aeruginosa, and are used as energy and carbon storage. In one study, P. aeruginosa PHA inclusions were constructed to be decorated on their surface with OprI, OprF, and AlgE peptide antigens. Mice vaccinated with the PHA inclusions produced a T_H1-type immune response characterized by antigen-specific production of IFN-γ and IgG2c isotype antibodies with OPK activity [78]. The protective efficacy of these vaccines is currently unknown because protection studies have not been published. Another novel delivery mechanism is a live-attenuated Francisella tularensis strain called LVS [79]. LVS was engineered to express three P. aeruginosa antigens, PilA, OprF, and FliC (flagellin), separately. Mice immunized with LVS expressing FliC, but not PilA or OprF, showed high titers of specific antibodies, but there were no challenge experiments or other measures of protection reported in this study [80]. Another approach using a Salmonella enterica serovar typhimurium live-attenuated strain (wecA mutant) expressing P. aeruginosa LPS O antigen found that mice immunized intra-peritoneally elicited specific opsonophagocytic antibodies and were protected from a lethal challenge in a murine pneumonia model [81].

Engineering monoclonal antibodies for enhanced airway delivery

In addition to the better delivery systems for active immunization being investigated, work to improve passive immunization is also ongoing. Commonly, IgG antibodies are used for therapy because they are now easy to manufacture and have a relatively long serum half-life. One limitation is that IgG molecules are typically not as abundant as IgA and IgM classes in mucosal sites, such as the lung airways, which are important in P. aeruginosa infections. One recent study modified the Cam-003 mAb (an IgG1 antibody against the P. aeruginosa biofilm polysaccharide Psl) to carry polymeric immunoglobulin receptor (pIgR) binding peptides, promoting transcytosis of the antibody from the mucosal tissue to the lumen. This modified monoclonal antibody was found to have increased localization in the bronchoalveolar space in comparison with the non-engineered version, and it improved survival in an acute pneumonia model [82].

Screens for novel vaccine candidates

In addition to the vaccine antigens described above, there is ongoing work to identify novel vaccine antigens. Reverse vaccinology was first used 20 years ago for identification of vaccine antigens by analyzing the genome of a pathogen to identify antigens that are predicted to make desirable vaccine antigens [83]. In a recent study, nine P. aeruginosa vaccine antigens were identified by screening the proteome for proteins with subcellular localization and a for potential immunogenic surface exposed epitopes. The proteins identified included efflux pumps, a penicillin binding protein, chaperone–usher pathway components, an extracellular component of T3SS and three uncharacterized secretory proteins. The study did not evaluate the antigens further but did determine that these antigens are well conserved across P. aeruginosa strains [84]. One recent study measured the concentrations of transcription of 158 P. aeruginosa predicted outer membrane and secreted proteins during a murine acute pneumonia murine model and found that OprF and three iron-uptake proteins (FpvA, FoxA, and HasR) were highly expressed. When mice were immunized with the four antigens separately or in combination, only immunization with OprF resulted in protective efficacy against acute pneumonia in mice [85].

Conclusion

Despite more than four decades of study and clinical trials, no vaccine for P. aeruginosa has been licensed, suggesting that our fundamental approaches have been flawed. None of the recent human clinical trials of active vaccines has evaluated T_H17 responses, and there is an overall dearth of translational studies of the human immune response to P. aeruginosa infection. It is very likely that multiple immune effectors will be needed to control this versatile pathogen. A major challenge lies in the fact that different immune effectors might be needed for different sites and types of infection (e.g., lung vs. burn wound vs. surgical wound). Thus, different vaccine antigens and adjuvants might need to be utilized in different patient populations. For example, a vaccine that induces GM-CSF–secreting T cells is likely to be more effective in patients with neutropenic cancer compared with one that does not. In the future, new technologies such as dual-RNA-seq (where the transcriptomic response of both the pathogen and host during infection are delineated) will help define which bacterial virulence factors are most important for each type of infection. A better understanding of the impact of the host microbiome on vaccine-induced immune responses will also likely be needed. Despite these challenges, new formulations and delivery systems of known antigens show great promise in murine models and await testing in humans.

Footnotes

Acknowledgments

This work was supported in part by the Cystic Fibrosis Foundation (grant number PRIEBE17G0, to G.P.P.); by the Richard A. and Susan F. Smith President's Innovation Award (to G.P.P.); by the Technology and Innovation Development Office (TIDO) at Boston Children's Hospital (to G.P.P.); and by funds from the Translational Research for Infection Prevention in Pediatric Anesthesia and Critical Care (TRIPPACC) Program of the Department of Anesthesiology, Critical Care and Pain Medicine at Boston Children's Hospital (to G.P.P.).

Author Disclosure Statement

The authors have nothing to disclose.

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Progress Toward the Elusive Pseudomonas aeruginosa Vaccine

Abstract

Abstract

Background:

Discussion:

Vaccines in Clinical Development

Passive immunization

MEDI3902

AR-105 (Aerucin®)

Active immunization

IC43/VLA43

Vaccines Under Development

Modifications of and additions to vaccine antigens under clinical development

Other Vaccine Antigens

Lipopolysaccharide and alginate in conjugate vaccines

New P. aeruginosa antigen delivery systems

Engineering monoclonal antibodies for enhanced airway delivery

Screens for novel vaccine candidates

Conclusion

Footnotes

Acknowledgments

Author Disclosure Statement

References

AR-105 (Aerucin^®)