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Respiratory infections caused by Pseudomonas aeruginosa are a major clinical problem globally, particularly for patients with chronic pulmonary disorders, such as those with cystic fibrosis (CF), non-CF bronchiectasis (nCFB) and severe chronic obstructive pulmonary disease (COPD). In addition, critically ill and immunocompromised patients are also at significant risk of P. aeruginosa infection. For almost half a century, research efforts have focused toward development of a vaccine against infections caused by P. aeruginosa, but a licensed vaccine is not yet available. Significant advances in identifying potential vaccine antigens have been made. Immunisations via both the mucosal and systemic routes have been trialled in animal models and their effectiveness in clearing acute infections demonstrated. The challenge for translation of this research to human applications remains, since P. aeruginosa infections in the human respiratory tract can present both as an acute or chronic infection. In addition, immunisation prior to infection may not be possible for many patients with CF, nCFB or COPD. Therefore, development of a therapeutic vaccine provides an alternative approach for treatment of chronic infection. Preliminary animal and human studies suggest that mucosal immunisation may be effective as a therapeutic vaccine against P. aeruginosa respiratory infections. Nevertheless, more research is needed to improve our understanding of the basic biology of P. aeruginosa and the mechanisms needed to upregulate the induction of host immune pathways to prevent infection. Recognition of variability in the host immune responses for a range of patient health conditions at risk from P. aeruginosa infection is also required to support development of a successful vaccine delivery strategy and vaccine. Activation of mucosal immune responses may provide improved efficacy of vaccination for P. aeruginosa during both acute exacerbations and chronic infection.

Pseudomonas aeruginosa is an important prognostic determinant in cystic fibrosis (CF). Little is known however, about P. aeruginosa induced local mucosal and systemic immune responses. Twenty CF children were categorized according to their P. aeruginosa status: (1) chronic lower respiratory tract infection (LRTI), (2) prior successfully treated initial LRTI, (3) isolated upper respiratory tract (URT) colonization, and (4) no known URT colonization or previous LRTI. Their antibody responses, and those of six non-CF disease controls, in serum and bronchoalveolar lavage (BAL) fluid to potential P. aeruginosa vaccine antigens outer membrane protein F (OprF), outer membrane protein H (OprH), catalase A (KatA) and a whole killed cell (WKC) extract were evaluated. Outer membrane protein G (OprG) responses were also measured in blood. Natural exposure, colonization and infection resulted in detectable antibody levels in BAL and serum in all CF groups. Both chronically infected and URT colonized CF children had substantially elevated immunoglobulin A antibody levels in the BAL fluid and sera toward the WKC extract and OprF antigen compared with the other groups of CF children and non-CF controls. The serum levels of specific P. aeruginosa antibodies involving immunoglobulin G and M isotypes increased with chronic LRTI, especially antibody levels to KatA, OprH and WKC extract, which were substantially greater in chronically infected children compared with all other groups. In conclusion, natural exposure, URT colonization and LRTI with P. aeruginosa all induce substantial mucosal and systemic antibody responses to potential vaccine antigens with chronically infected CF children having the highest levels.

Pseudomonas aeruginosa is a major cause of nosocomal and community acquired chronic infections in subjects with compromised respiratory function. The microbe is environmentally ubiquitious and has a high level of innate antimicrobial resistance. This has led researchers to investigate vaccine and immunotherapeutic approaches to prevent and treat P. aeruginosa infections. Seven cytosolic non-integral proteins were studied as vaccine candidates in an acute lung infection model in the rat. Five of these (amidase, amidopeptidase, KatE, KatE and Pa13 a novel 13kDa protein) enhanced bacterial clearance from the lung compared to control animals following challenge and are worthy of further study. Immune mechanisms stimulated by these proteins in response to both immunization and infection varied. The most pronounced degree of bacterial clearance from the lung was associated with antigens, which demonstrated greater surface exposure and induced an increase in phagocyte recruitment, in particular, an increased proportion of polymorphonuclear leukocytes. Lymphocytic proliferation and specific antibody responses in the absence of enhanced clearance were less informative as immune correlates.

This study examines the safety and immunogenicity of an oral, whole-cell Pseudomonas aeruginosa vaccine administered to healthy volunteers. Thirty subjects received an oral dose of Pseudostat in two timed, measured doses with serological follow-up to 56 days postvaccination. Following vaccination, several individuals were identified as antibody responders for all three immunoglobulin (Ig) isotypes tested, specifically against whole-cell P. aeruginosa extract and outer membrane proteins F and I. The mean pooled lipopolysaccharide antigen-specific IgA showed the most significant and constant increases in titer postdose, with a similar increase in titer for whole-cell P. aeruginosa extract-specific IgA. The results demonstrated an increased phagocytic ability of the selected macrophage cell line in post vaccination sera. Furthermore a significant increase in intracellular macrophage killing of opsonized P. aeruginosa was also demonstrated (82% on day 14 postdose) in the presence of the postvaccination sera. The safety component of the study did not show any vaccine-attributable adverse effects in any of the subjects, as documented by clinical evidence, hematology, and biochemistry profiles. We conclude that Pseudostat is safe and immunogenic in humans at this dose and that further studies to determine the appropriate dosage and efficacy are needed. In our study, we have shown that the most significant and sustained responses to oral vaccination in human adult volunteers were serum IgA levels and that pooled sera collected postimmunization have an increased capacity to promote opsonophagocytotic killing of P. aeruginosa.

Pseudomonas aeruginosa remains a serious pathogen for specific cohorts of patients where chronic infection is a poor prognostic indicator, such as those with cystic fibrosis, burn wounds or those who are immunocompromised. Significant disease burden is associated with a diverse spectrum of both nosocomial and community-acquired infections. To date, vaccines against P. aeruginosa have shown limited and often conflicting efficacy data, especially against heterologous strains, which are increasingly identified as co-colonisers of biofilms. While few studies have gone beyond Phase II clinical trials, a particular concern is the ability of P. aeruginosa to evade the immune system while provoking an immune response that contributes to the destructive nature of infection. Therefore, vaccine development needs to focus on preventing attachment and colonisation, as well as preventing conversion to a mucoid phenotype that is characteristic of the chronic condition that promotes pathology.

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen, which causes serious debilitating infections in patients with compromised lung function. The mechanism by which P. aeruginosa is cleared from the lung is not fully defined, although our previous studies have established a role for cellular immunity in protection against P. aeruginosa infections. This study aimed to evaluate the role of P. aeruginosa-specific IgG in protection against P. aeruginosa in a rat model of acute pulmonary infection. Immunoaffinity chromatography was used to purify total rat IgG from rat immune serum (rats immunised with P. aeruginosa) and non-immune serum. Untreated recipient rats were injected intravenously with different concentrations of pure IgG prepared from serum of unimmunised rats (non-immune IgG) or from rats immunised intestinally with killed P. aeruginosa (immune IgG) and infected intratracheally with P. aeruginosa 18 h later. The protective capability of the purified IgG against P. aeruginosa was assessed by measurement of reduction in P. aeruginosa infection in the lung 4 h after instillation of bacteria. Enhanced bacterial clearance induced by IgG was determined to be dose-dependent with a 1 mg dose failing to enhance clearance, whereas 5 mg of immune IgG enhanced clearance from the airways and the lung tissue. Measurement of the IgG1, IgG2a and IgG2b isotypes in serum and the lung lavage following transfer of P. aeruginosa-specific IgG found that all three were present. These results demonstrate that anti-P. aeruginosa IgG can enhance bacterial clearance from the airways in an acute infection and identify an important role for IgG in acute respiratory infections caused by P. aeruginosa.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that can cause fatal acute lung infections in critically ill individuals. Lung damage due to chronic infections in cystic fibrosis sufferers is the major cause of morbidity and mortality in this group. The bacterium produces various immunomodulatory products that enable it to survive in the lung. Innate and increasing resistance to antibiotic therapy shown by this organism heightens the need for development of a vaccine. This study reports the identification of six non-integral protein antigens; Pa13, azurin, acyl carrier protein (ACP), amidase, aminopeptidase and KatE, purified from a mucoid strain of P. aeruginosa. N-terminal amino acid sequencing was used to identify these proteins and, based on their ascribed functions, determined that their normal cellular location was cytosolic. A rat model of acute pulmonary infection was used to investigate the ability of these protein antigens to enhance pulmonary clearance of a live P. aeruginosa challenge. Mucosal immunisation with four of the six antigens significantly enhanced bacterial clearance from both the lavage fluid and lung tissue. The greatest level of clearance was demonstrated for the antigens; KatE, aminopeptidase and amidase. Enhanced bacterial clearance was maintained when the antigens amidase and aminopeptidase were produced in recombinant form. When delivered parenterally, aminopeptidase demonstrated its continued efficacy as a vaccine candidate. This study has demonstrated that non-integral outer membrane proteins are antigenic and protective and warrant further investigation as potential components of a vaccine.

Pseudomonas aeruginosa is a common cause of infection in immunocompromised patients and is the major contributor to morbidity in individuals with cystic fibrosis (CF). The antibiotic resistance shown by this pathogen and morbidity in patients with chronic infection has encouraged investigations into the development of a vaccine. This study reports the purification of a 60 kDa protein, isolated from a mucoid strain of P. aeruginosa, identified by amino acid sequence analysis as the catalase protein (KatA). A rat model of acute P. aeruginosa respiratory infection was used to investigate the immunogenicity of KatA and determine the potential of mucosal immunization with KatA to protect against infection. Immunization regimens compared a single intra-Peyer's patch (IPP) immunization with an IPP primary inoculation followed by an intratracheal boost to the lungs. Mucosal immunization with KatA resulted in significant pulmonary clearance of both homologous (p<0.001) and heterologous (p<0.05) strains of P. aeruginosa. Both immunization regimens enhanced bacterial clearance, increased the rate of recruitment of phagocytes to the bronchoalveoli and induced KatA-specific antibody. However, the regimen that included a boost induced a more effective immune response that also resulted in better clearance of P. aeruginosa from the lungs. Mucosal immunization induced KatA- specific antibodies in the serum and the bronchoalveolar lavage, and KatA-specific lymphocyte proliferation in vitro in cells isolated from the mesenteric lymph nodes of immunized rats. The data presented suggests that KatA has the potential to afford a protective immune response against pulmonary infection by P. aeruginosa

Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen associated with otitis media and the exacerbation of chronic bronchitis. This study reports the vaccine potential of three peptides representing conserved regions of the NTHi P5 outer membrane protein which have been fused to a promiscuous measles virus F protein T-cell eptitope (MVF). The peptides correspond to a region in surface loop one (MVF/L1A), the central region of loop four (MVF/L4), and a C-terminal region homologous to peptide 10 of OprF from Pseudomonas aeruginosa (MVF/H3). Immunization of rats with MVF/H3 was the most efficacious in significantly reducing the number of viable NTHi in both the broncho-alveolar lavage fluid (74%) and lung homogenates (70%), compared to control rats. Importantly, despite significantly increased rates of clearance, immunization with MVF/H3 elicited poor antibody responses, suggesting that cell-mediated rather than humoral responses play an important role in the enhanced clearance of NTHi in this model.

Pseudomonas aeruginosa is an environmentally ubiquitous, extracellular opportunistic gram-negative bacteria that causes significant morbidity and mortality to a disproportionately high degree for infections with this bacteria compared with other gram-negative bacteria. Patients at particular risk of infection are those with compromised respiratory function, in intensive-care support and taking immunocompromising pharmaceutical agents. Once acquired, infection is difficult to eradicate with chemotherapy and attempts to vaccinate against infection have been of little success. Over the past five years, we have pursued the concept of mucosal immunisation against respiratory infection with P. aeruginosa. Initial studies in an acute animal model clearly demonstrated that mucosal immunisation with a killed whole bacterial cell preparation could induce protective immune responses in the lung. Subsequent studies have shown that the protective immune mechanisms were dependent on antigen specific CD4+ T cells, the activation of alveolar macrophages, the recruitment and activation of polymorphs, predominantly neutrophils, the controlled secretion of TNF-alpha, IL-1 and IFN gamma and the presence of antibody. We have hypothesised that the protective response is under the control of T cells. A pre-clinical human trial of an oral whole killed cell preparation has been completed with no adverse side effects. A limited open trial in patients with bronchiectasis has also been completed. Preliminary analysis of the results has demonstrated that after oral vaccination, specific lymphocyte responses were observed to P. aeruginosa.

The aim of this study was to determine the efficacies of prior mucosal (oral, intra-Peyer's patch, or intratracheal) and systemic (subcutaneous) immunizations with killed Pseudomonas aeruginosa in clearance of an acute P. aeruginosa respiratory infection in rats. Rats were immunized with paraformaldehyde-killed P. aeruginosa at various doses, and 2 weeks later, the rats were challenged with a log10 dose of 8.7 live bacteria. This dose was fatal for unimmunized rats, with death occurring approximately 12 h after challenge. The numbers of surviving bacteria in the airways and lung tissue were determined by analyses of bronchoalveolar lavage fluid (BAL) and lung homogenate samples, respectively. Enhanced bacterial clearance was associated with survival of intra-Peyer's patch-immunized rats. Determination of bacterial clearance in BAL 4 h after challenge demonstrated that the use of all immunization routes led to significant clearance compared with the bacterial levels in unimmunized controls (the order of effectiveness was intra-Peyer's patch > oral-intratracheal > intratracheal > subcutaneous > oral). Bacterial clearance in the lung homogenate was also significantly greater for all immunization routes than in the unimmunized controls (the order of effectiveness was intra-Peyer's patch > subcutaneous > oral-intratracheal > oral = intratracheal). Prior oral immunization with killed P. aeruginosa also induced enhanced bacterial clearance of heterologous strains of P. aeruginosa, Haemophilus influenzae, and to a lesser extent, Klebsiella pneumoniae. Because of the ease of antigen delivery, oral immunization with killed P. aeruginosa may be an important route of immunization for induction of enhanced bacterial clearance of subsequent acute respiratory infection with P. aeruginosa and other gram-negative bacteria.

This paper investigates the effector mechanisms of immune clearance in the lungs of rats immunized against mucoid Pseudomonas aeruginosa. After the gut-associated lymphoid tissue was primed and after a subsequent pulmonary challenge with live bacteria, significantly accelerated bacterial clearances from the lung and raised levels of anti-P. aeruginosa antibodies in sera (immunoglobulin G [IgG], IgA, and IgM) and bronchoalveolar lavages (IgG and IgA) were observed for all immune animals. These changes were associated with enhanced recruitment, chemotaxis, chemokinesis, phagocytic indices, and chemiluminescence of pulmonary polymorphonuclear neutrophils (PMN). In the alveolar spaces of immune animals, an increase in the level of PMN recruitment was not associated with higher levels of leukotriene B4 (LTB4). In contrast, in nonimmune animals that were intratracheally infected with P. aeruginosa, the levels of recruitment and activity of alveolar PMN were lower than those in immune rats but PMN infiltration correlated with a significant increase in the synthesis of LTB4 in the alveolar space. In pulmonary tissue, LTB4 synthesis for both groups was elevated. These findings suggest that accelerated clearance of mucoid P. aeruginosa from the lungs of intestinally immunized rats is due at least in part to factors that induce the enhancement of PMN recruitment and activity in the alveolar space. The mediators that regulate this enhanced response remain unknown but do not seem to include LTB4. The high levels of LTB4 measured in the bronchoalveolar lavages and pulmonary tissues from nonimmune animals infected with live bacteria implicate LTB4 as an important amplifier of the inflammatory response during acute pulmonary infections with mucoid P. aeruginosa in unimmunized hosts.