Vaccine (2008) 26, 1011—1024
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Vaccines and immunotherapy against Pseudomonas aeruginosa Gerd D¨ oring a,∗, Gerald B. Pier b,∗∗ a
Institute of Medical Microbiology and Hygiene, University of T¨ ubingen, Wilhelmstraße 31, D-72074 T¨ ubingen, Germany Channing Laboratory, Brigham and Women’s Hospital, Harvard Medical School, 181 Longwood Avenue, Boston, MA 02115, USA
Received 28 September 2007; received in revised form 28 November 2007; accepted 5 December 2007 Available online 26 December 2007
KEYWORDS Pseudomonas aeruginosa; Vaccines; Monoclonal antibodies
Summary A number of different vaccines and several monoclonal antibodies have been developed in the last decades for active and passive vaccination against the Gram-negative opportunistic pathogen Pseudomonas aeruginosa. These approaches include vaccine antigens such as lipopolysaccharide, surface polysaccharides, polysaccharide—protein conjugates, ﬂagella, outer membrane proteins, pili, whole formalin-killed P. aeruginosa cells, live-attenuated P. aeruginosa and Salmonella enterica strains expressing P. aeruginosa antigens and DNA sequences. While many of these experimental vaccines and monoclonal antibodies have been tested in preclinical trials, only a few have reached clinical phases and none of these vaccines has obtained market authorization. The purpose of this review is to provide a brief summary of the present state of the development of vaccines and immunotherapies against P. aeruginosa infections. According to the different types of infection caused by P. aeruginosa — localized on mucosal surfaces such as the airways or systemic infection in the blood stream — several potential routes suggesting optimal means to administer the experimental vaccines are presented. Finally, the inherent problem of testing P. aeruginosa candidate vaccines in patient populations is discussed. © 2007 Elsevier Ltd. All rights reserved.
Contents Introduction ............................................................................................................ Lipopolysaccharide, polysaccharide and polysaccharide-conjugate vaccines ............................................ Preclinical studies..................................................................................................
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Abbreviations: KLH, keyhole limpet hemocyanin; LPS, lipopolysaccharide; mAbs, monoclonal antibodies; MEP, mucoid exopolysaccharide; Opr, outer membrane proteins; PS, polysaccharides. ∗ Corresponding author. Tel.: +49 7071 298 2069; fax: +49 7071 293011. ∗∗ Corresponding author. Tel.: +1 617 525 2269; fax: +1 617 525 2510. E-mail addresses: [email protected]
(G. D¨ oring), [email protected]
(G.B. Pier). 0264-410X/$ — see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2007.12.007
G. D¨ oring, G.B. Pier
Phase I studies in humans .......................................................................................... Phases II and III studies............................................................................................. Flagella vaccines........................................................................................................ Preclinical studies.................................................................................................. Phase I studies ..................................................................................................... Phases II and III studies............................................................................................. Outer membrane protein vaccines ...................................................................................... Preclinical studies.................................................................................................. Phases I/II studies .................................................................................................. Killed whole cell and live-attenuated vaccines .......................................................................... Preclinical studies.................................................................................................. Phase I studies ..................................................................................................... Pili and type III secretion system vaccines............................................................................... Preclinical studies.................................................................................................. DNA and viral vector vaccines........................................................................................... Preclinical studies.................................................................................................. Passive immunization against P. aeruginosa infections................................................................... Preclinical studies.................................................................................................. Phases I/II studies .................................................................................................. Phases II/III studies................................................................................................. Conclusions ............................................................................................................. References ............................................................................................................
Introduction Pseudomonas aeruginosa, a Gram-negative bacterial pathogen which is found mostly in water reservoirs in the environment, causes severe nosocomial and communityacquired infections at various body sites including the urinary tract, surgical or burn wounds, the cornea and the lower respiratory tract [1,2]. As an opportunist, P. aeruginosa takes advantage of various underlying host conditions to establish acute or chronic infections. Patient groups at risk for acquisition of P. aeruginosa infections include those with the hereditary disease cystic ﬁbrosis (CF), paraplegic and burn patients, patients hospitalized in intensive care units and those undergoing mechanical ventilation, and patients who are immunosuppressed due to underlying disease or therapy, such as patients with cancer  or AIDS. Although antibiotic therapy has considerably improved the management of infectious diseases in general, many P. aeruginosa infections are not fully treated or eradicated by the application of anti-pseudomonal drugs and can thus become chronic infections. For instance, burn patients that survive the initial burn trauma can become colonized with antibiotic-resistant, hospital-derived P. aeruginosa strains that are not easily eradicated with antibiotic therapy [3,4]. In CF patients, when the strains are eventually selected out by antibiotic therapy to become multiply-resistant, an increase in the rate of decline in lung function is seen when compared to patients infected with antibioticsusceptible strains [5—7]. The multiple-antibiotic-resistant P. aeruginosa Liverpool epidemic strain, which also displays increased virulence , has infected the non-CF parents of a CF patient, causing signiﬁcant morbidity . Other infections, such as P. aeruginosa keratitis, can develop too fast for effective chemotherapy. Thus, as an alternative in this context, vaccine development against P. aeruginosa may indeed be useful.
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Despite high numbers of patients who may develop P. aeruginosa infections and the threat of antibiotic treatment failure due to bacterial resistance, there is surprisingly no P. aeruginosa vaccine currently available on the market, although many attempts have been made in the past. Most of published studies are preclinical, some describe results from phases I and II studies and only two vaccines have made it to phase III studies in CF patients [10,11]. This raises the question as to whether P. aeruginosa is an antigenically variable microorganism that can escape immune recognition and/or induce immunological non-responsiveness as is seen with other bacteria such as Borrelia, Bordetella or Neisseria. However, if this is the case, the basis for the host’s inability to mount an effective immune response to these pathogens has not been well documented. For P. aeruginosa there is better evidence that the organism has the ability to undergo phenotypic variation due to changing environmental conditions such as in the airways of CF patients. It is well known that most often, an initially non-mucoid P. aeruginosa phenotype associated with low or no production of alginate changes to a variant overproducing alginate not synthesizing lipopolysaccharide (LPS) O side chains and forming macrocolonies in the lungs that some refer to as bioﬁlms . Although among P. aeruginosa strains there is chemical and thus antigenically variable vaccine candidate antigens such as LPS O-antigens and ﬂagella, the overall variability among strains seems to be fairly limited in comparison to inﬂuenza or other microorganisms mentioned above. Additionally, P. aeruginosa expresses some highly conserved antigens such as outer membrane proteins (Opr). In the light of highly efﬁcient technologies to express proteins from cloned genes using various heterologous systems and to purify protein and carbohydrate antigens in high yields under good manufacturing practices standards, the lack of a protective P. aeruginosa vaccine is not adequately explained by insufﬁcient technologies to produce such vaccines. The basis
Vaccines and immunotherapy against Pseudomonas aeruginosa Table 1
Pseudomonas aeruginosa antigens used for vaccine development
Alone Embedded in liposomes Alginate LPS-based O-PS Exotoxin A, tetanus Exotoxin A DNA F, I proteins, DNA Adenovirus, Salmonella Subtypes a, b PcrV Type IV/exotoxin A Caulobacter
[19,33—38] [20,21]   [11,25—31,39—41] [116,117] [69—73,76—83,85—90] [114,115,118,119] [74,75,116] [10,52—56] [108,109] [102—105,107]   [92,95,96] [75,93,94,97]
Polysaccharides PS-protein conjugates Extracellular proteins Outer membrane proteins
Flagella Type III secretion Pili Killed whole cell Attenuated P. aeruginosa S. enterica SL3261
aroA O11, OprF/I
for lack of progress in P. aeruginosa vaccine development may be more related to the difﬁculties in testing the efﬁcacy of potential vaccines in randomized, double-blinded, placebo-controlled phase III trials in patient groups at risk for acquiring P. aeruginosa infections which can enroll a sufﬁcient number of patients to produce statistically relevant conclusions. In this review, the present state of the development of vaccines and immunotherapies against P. aeruginosa infections is described, including such vaccine candidates as LPS, polysaccharides (PS), PS-protein conjugates, ﬂagella, Oprs, pili, whole formalin-killed P. aeruginosa cells, live-attenuated P. aeruginosa and Salmonella enterica and virus strains expressing P. aeruginosa antigens and DNA sequences (Table 1). Several new results of clinical trials have been published recently [10,11] or reported on web sites and newer approaches to vaccination involving the live-attenuated P. aeruginosa and S. enterica strains expressing P. aeruginosa LPS O-antigens have also appeared recently, adding new insights and approaches to those discussed in prior reviews of vaccination against P. aeruginosa. According to the different types of infection caused by P. aeruginosa — localized on mucosal surfaces such as the airways or systemic infection in the blood stream — several potential routes of administration of the experimental vaccines that could enhance their effectiveness are also discussed. Finally, the inherent problem of testing P. aeruginosa candidate vaccines in patient populations is analyzed. The interested reader is also referred to other reviews on this topic [13—16].
Lipopolysaccharide, polysaccharide and polysaccharide-conjugate vaccines LPS, the major component of the outer leaﬂet of Gramnegative bacteria , is generally pyrogenic and toxic when administered in a puriﬁed state and therefore pure LPS, or vaccines containing LPS, are generally considered too
toxic to be used as vaccines in humans. Indeed, when a P. aeruginosa LPS-based vaccine (Pseudogen® )  was tested in patients with leukemia and CF, febrile responses were observed in 20—40% of the patients . Thus, it is unlikely that similar strategies will be repeated in the future. The toxicity of LPS is speciﬁcally linked to the lipid moiety and is absent when this structure is removed from the core region and the O-PS region . One strategy to circumvent the problem of toxicity is to introduce LPS into liposomes to mask the toxic lipid A moieties [20,21]. Indeed, the endotoxic activities of these LPS-containing liposomes were less than 0.1% of the endotoxicity of the original free LPS as measured by reactivity in the Limulus amoebocyte lysate assay . Another approach is to use only the non-toxic PS part of the LPS in vaccine preparations. The O-PS portions of P. aeruginosa LPS molecules differ structurally, and at least 20 different structures can be distinguished , although only about 10 of these are expressed in the majority of clinical P. aeruginosa isolates. Consequently O-PS-based vaccines have to be multivalent due to the speciﬁcity of LPS to P. aeruginosa serotypes. Alternately, alginate (sometimes referred to as mucoid exopolysaccharide, MEP) is structurally less variable than LPS and has been considered as a vaccine candidate . Alginate is a random ␤1—4 linked polymer of D-mannuronic acid (M) and L-guluronic acid (G) residues . Alginate preparations differ principally in molecular size, the ratio of M:G residues from 10:1 to 6:4, and the level of O-acetylation at the M residues on C-2 and C3. All of these factors may affect the immune response, notably in the ability to generate broadly reactive, opsonic antibody. However, puriﬁed P. aeruginosa PS and alginate vaccine candidates are often poorly immunogenic, either eliciting poorly protective antibody or inducing some antagonistic immune responses when multiple antigens are combined into a multivalent vaccine [22,24]. Poor immunogenicity of P. aeruginosa O-PS molecules and alginate have led to pursuing strategies of conjugation of the sugar moieties to proteins to induce enhanced immune responses.
Preclinical studies Several vaccines based on P. aeruginosa PS showed promising results in animal studies. To increase its immunogenicity, alginate from P. aeruginosa was conjugated to exotoxin A . The non-toxic and non-pyrogenic conjugate induced high antibody levels against alginate and exotoxin A in rabbits. The antibodies promoted opsonophagocytosis and killing of mucoid strains of P. aeruginosa by human neutrophils . However, further investigations of this vaccine were not pursued. A similar approach was used when P. aeruginosa alginate was conjugated to keyhole limpet hemocyanin (KLH), resulting in improved immunogenicity in C3H-HeN mice and in a rabbit . No data on animal protection have been published yet, however, preclinical trials with the KLH-conjugate are ongoing. Finally, a P. aeruginosa alginate—tetanus toxoid conjugate vaccine has been produced which protected mice against a lethal dose of an alginate overproducing P. aeruginosa strain . Using puriﬁed P. aeruginosa O-PS molecules, an octavalent conjugate vaccine was synthesized. The O-PS moieties derived from LPS of IATS (or Habs) serotypes 1—6, 11 and 12 were covalently coupled to exotoxin A . This vaccine, later called Aerugen® , conferred signiﬁcant protection after intramuscular administration in animals against subsequent challenge with the P. aeruginosa serotype strains contained in the vaccine. Since a proposed route of P. aeruginosa infection in CF patients is via the oropharynx, two subunit O-PS-exotoxin A vaccines were given intranasally to mice to test whether the vaccine would also protect the animals against P. aeruginosa challenge when given by this route . While this route of immunization provided effective protection to the mice, it is still unclear whether intranasal administration of Aerugen® is equivalent or superior in comparison to an intramuscular administration of the vaccine in terms of generating protective immunity. The liposomal complete-core LPS vaccine when injected into rabbits was devoid of pyrogenicity or overt toxicity and induced cross-reactive antibodies to a large panel of LPSrough and LPS-smooth molecules from numerous clinically relevant Gram-negative bacteria, including E. coli, P. aeruginosa, Klebsiella pneumoniae, B. fragilis, and Bacteroides vulgatus . Active immunization with this vaccine protected mice against a lethal challenge with E. coli O18 LPS . Whether the vaccine can protect against lethal bacterial challenges including P. aeruginosa has not been reported.
Phase I studies in humans Although puriﬁed alginate was injected into a large number of healthy volunteers in the 1990s, only low opsonic antibody titers were produced . Thus, the non-conjugated alginate vaccine is no longer being investigated. Furthermore, none of the alginate-conjugate vaccines or the liposomal complete-core LPS vaccine have been reported to have entered phase I trials in healthy human individuals. However, the safety and immunogenicity of Aerugen® was demonstrated in plasma donors, bone marrow transplant and CF patients [25,28,30,31]. Opsonic and toxin A neutralizing IgG antibody titers were initially induced to all vaccine anti-
G. D¨ oring, G.B. Pier gens. Still, conjugation of the PS to exotoxin A did not prevent antibody titers from decreasing substantially 12 months after immunization, demanding yearly boosters of the vaccine in subsequent clinical trials.
Phases II and III studies When the LPS-based Pseudogen® vaccine  was tested in CF patients already infected with P. aeruginosa , this organism was not eliminated from the airways and the patients even did worse clinically compared with nonvaccinated controls, perhaps due to exacerbation of damage from increased inﬂammation engendered by vaccination. As already discussed, this negative outcome is most probably due to the known adverse reactions to LPS-based vaccines. In addition, the vaccine may have increased immune complex formation in the infected CF patients, thereby triggering further inﬂammatory reactions via recruited neutrophils . Therefore, later P. aeruginosa vaccine studies were performed in CF patients free of the pathogen. Pseudogen® was also tested in other patient groups including patients with cancer , burns  and those in intensive care settings . However, the major drawback to the clinical application of Pseudogen® was the high adverse reaction rate. Further development of Pseudogen® has therefore been stopped. Improvements in a different vaccine preparation, termed PEV-01, wherein LPS was still the major antigenic component [36,37], led to a study in 28 CF patients who were not colonized with P. aeruginosa . In this prospective study, the PEV-01 vaccine stimulated-speciﬁc antibody production. However, neither the acquisition of P. aeruginosa nor the course of the disease was changed when compared with a non-vaccinated control group. Possibly, the vaccine did not protect against a sufﬁcient range of LPS serotypes of P. aeruginosa. Unexpectedly, the overall clinical status of the vaccine group was worse than that of the control group for several years, after which both groups became clinically indistinguishable. The PEV-01 vaccine was also tested in burned patients, however, the results of the studies were not conclusive. Further development of the PEV-01 vaccine has also been stopped. Additionally, phases II and III clinical studies with the Aerugen® vaccine have been carried out in CF patients not infected with P. aeruginosa. In a small open study involving 26 patients, the vaccine was safe and induced antibodies to the O-PS which promoted the opsonophagocytic killing of P. aeruginosa by human neutrophils [30,31]. Six years after immunization, 75% of control but signiﬁcantly less immunized subjects (35%) were found to be infected with P. aeruginosa. Infection of immunized patients was apparently correlated with a decline in speciﬁc antibody titers between years 2 and 3 of follow-up. Ten years after the ﬁrst human immunizations with Aerugen® , a signiﬁcant difference in P. aeruginosa infection between control and immunized subjects was still present. Only 32% of immunized patients suffered from chronic infection at the end of the 10-year observational period compared to 72% in the matched non-immunized control group (8/25 versus 18/25) . Additionally, lung function and weight was higher in immunized CF patients than in controls . The protective
Vaccines and immunotherapy against Pseudomonas aeruginosa capacity of speciﬁc serum IgG antibodies was linked to its high afﬁnity and to speciﬁcity for O-PS serotypes rather than for LPS core epitopes . Furthermore, Aerugen® stimulated some form of cell-mediated immunity . Since the results from this open, non-randomized study were not adequate to obtain regulatory approval for clinical use, a placebo-controlled, randomized and double-blinded multicenter trial was started with Aerugen® in European CF patients. The study was stopped in 2006 by the manufacturer, Crucell, Berne, Switzerland, since no signiﬁcant differences between the two groups in the clinical parameters chosen for evaluation were observed. Results from this study have not been published yet, and it is therefore difﬁcult to understand the reasons for the negative outcome. One obvious reason, however, is that the current strategy of antibiotic treatment against P. aeruginosa in European CF patients may have kept the non-immunized control group from acquiring a signiﬁcant level of infection during the study period. This problem is discussed below (Flagella vaccines: Phases II and III studies) in more detail. Another reason that may underlie the failure of vaccines targeting P. aeruginosa LPS O-antigens in CF patients is that P. aeruginosa isolates from CF patients are often LPS-rough and not producing O-side chains . While the clinical trials with Aerugen® were targeted to CF patients without evidence of P. aeruginosa colonization, it is possible that the switch to the LPS-rough phenotype occurs rapidly in the CF lung, rendering the vaccine-induced antibodies ineffective. Furthermore, LPS-rough strains may also be responsible for initiating colonization of the CF airways. Hopefully, the publication of the phase III Aerugen® trial will shed some light on these issues. Whether future trials with Aerugen® in CF patients or in other risk groups for P. aeruginosa infections will be carried out is unclear at present.
Flagella vaccines The polar ﬂagella of P. aeruginosa is a polymer of a protein ﬁlament which can be divided into one group that is a 53 kDa protein referred to as the b-type and a heterogeneous 45—52 kDa structure referred to as the a-type. The a-type ﬂagellin protein contains a serologically conserved region referred to as a0 and serologically variable regions comprising subtypes a1 , a2 , a3 and a4 [43—46]. Unfortunately, the Ansorg typing system can no longer be used, since type strains and absorbed rabbit sera used to deﬁne the initial serologic groups are no longer available. Thus, antisera raised to ﬂagella of known types, such as from strains PAK (a-type) and PAO1 (b-type) must be used but what speciﬁc sub-type antigenic determinants are expressed on these ﬂagellin monomers is not known. However, a set of monoclonal antibodies against ﬂagellin antigens expressed by various clinical isolates of P. aeruginosa has been generated, which may allow reliable ﬂagella typing once epitope speciﬁcities have been deﬁned (Brian Crowe, Baxter Inc., Orth an der Donau, Austria, personal communication). The ﬂagella synthesis genes of P. aeruginosa are clustered in three non-contiguous regions of the chromosome and the expression of the ﬂagellum is coordinately regulated by four classes of regulators, including the alternative
1015 sigma factor 54, encoded by the rpoN gene . Besides providing mobility and contributing to invasiveness of P. aeruginosa, ﬂagella proteins have also been found to be involved in adhesion to host cells and molecules in vitro. It may bind to mucins , the glycolipid asialoGM1, as well as to toll-like receptor 5, inducing inﬂammation [46,49,50]. Due to hypoxic environmental conditions  alginate overproducing mucoid variants of P. aeruginosa develop during chronic infection in CF patients. These variants are characterized by a loss of ﬂagella production. Responsible for the loss of ﬂagella synthesis is the alternative sigma factor AlgT, which induces the mucoid phenotype and also represses the ﬂagella regulator ﬂeQ . Therefore, for CF patients, ﬂagella vaccines have to be administered prior to emergence of the non-ﬂagellated variants of P. aeruginosa.
Preclinical studies Animal studies in a burn-wound infection model and a neonatal mouse model of acute P. aeruginosa pneumonia have shown that immunization against the ﬂagellum protects against the lethal effects of P. aeruginosa infection [52—54]. Protection was attributed in the burned-mouse model to immobilization of the pathogen in the local burn infection site and prevention or delay of systemic spread. This notion was based on the fact that similar numbers of bacteria were found in the infected skins of both immunized and control groups, but signiﬁcantly fewer bacteria were observed in the livers of immunized mice compared to controls. However, protection by anti-ﬂagella immunization may also have involved improved opsonophagocytosis.
Phase I studies Monovalent P. aeruginosa ﬂagella vaccines, prepared from puriﬁed ﬂagella protein, have been tested in healthy human adults. High and long-lasting circulating antibody titers against the ﬂagella antigen have been noted following intramuscular immunization and adverse effects were mild . Intramuscular administration of a monovalent ﬂagella vaccine elicited not only elevated serum IgG and IgA antibody titers against ﬂagella, but also ﬂagella-speciﬁc IgG, IgA and sIgA antibodies in the secretory immune system. Evidence was provided that P. aeruginosa-speciﬁc antibodies, detected in the bronchoalveolar lavage samples after immunization, were not derived by transudation from serum but locally produced in the respiratory tract . Thus, from an immunological standpoint, intramuscular administration is comparable to intranasal or aerosol administration. However, the effective level of antibody titer in the respiratory tract to provide protection against P. aeruginosa infection is unknown at present (see also Killed whole cell and live-attenuated vaccines: Preclinical studies). According to the hypothesis, developed by Robbins et al. , speciﬁc serum IgG is all that is needed to protect against microbial lung infections. Nevertheless, a local vaccine administration is less invasive than the intramuscular route and thus may be preferred if an acceptable delivery system can be developed.
Phases II and III studies In a phase III, placebo-controlled, randomized and doubleblinded multicenter trial including 483 CF patients, not infected with P. aeruginosa, a bivalent ﬂagella vaccine has been tested during an observational period of 2 years . The patients received four intramuscular injections of the vaccine or placebo over a 14-months period and were evaluated quarterly for P. aeruginosa positive throat cultures and development of serum antibody titers to alkaline proteinase, elastase and exotoxin A, indicative of a P. aeruginosa infection. The vaccine was well tolerated and the patients developed high serum IgG titers to ﬂagella subtypes included in the vaccine. Whereas in the intention-to-treat group (all patients enrolled), the statistical difference between vaccinated patients and placebo patients did not reach signiﬁcance in regard to having a ﬁrst positive culture for P. aeruginosa, a signiﬁcant reduction in the numbers of immunized patients with a ﬁrst positive culture for P. aeruginosa was obtained for this primary endpoint in the per-protocol group, which encompassed the patients who received all four of the intended vaccinations. In this group, 37 of 189 patients in the vaccine group had documented infection compared to 59 of 192 patients in the placebo group. The degree of protection against P. aeruginosa infection, calculated from the relative risk , was 34%. Of note, among the P. aeruginosa strains recovered and available for analysis of the ﬂagella subtype, the large majority were ﬂagella positive. Furthermore, there were signiﬁcantly fewer P. aeruginosa strains exhibiting ﬂagella subtypes included in the vaccine isolated from vaccinated patients, compared to those strains isolated from patients in the placebo group. The second primary endpoint, tested in this study, i.e., prevention of chronic P. aeruginosa infection, as deﬁned by three-positive bacterial cultures and/or positive serum antibodies against the pathogen, was not achieved due to a much lower than expected rate of development of chronic infection in the placebo group. The expected rate was based on rates of infection observed in European CF patients in the 1990s. This reduction was attributed to the institution of much more frequent use of antibiotic treatments following an initial episode of P. aeruginosa colonization in many of the CF patients enrolled in the trial. This antibiotic treatment strategy has successfully eradicated P. aeruginosa and prolonged onset of chronic infection in many European CF centers [58—65]. Nevertheless, calculated from the relative risk  in the per-protocol group, the degree of protection against chronic P. aeruginosa infection was 51%. Although the ﬂagella vaccine trial  established early immunization as a reasonable strategy for CF patients with a vaccine antigen that can be readily produced, the bivalent ﬂagella vaccine may not be optimal. The analysis of the ﬂagella types by immunoﬂuorescence using monoclonal antibodies in P. aeruginosa isolates from vaccinated patients suggested that some strains of P. aeruginosa express ﬂagella antigens not included in the vaccine. Therefore, the inclusion of other P. aeruginosa ﬂagella types in a future multivalent vaccine preparation may improve protection against P. aeruginosa in CF patients. Whether the abnormal high viscosity of the mucus layer on the respiratory epithelium  which has been shown to inhibit neutrophil migration in vitro  impairs the protection rate of the ﬂagella vaccine, or P.
G. D¨ oring, G.B. Pier aeruginosa vaccines in general, is not known. At present, the production of the bivalent ﬂagella vaccine, manufactured previously by IMMUNO, Vienna, Austria, has been terminated and the development of a multivalent vaccine is not envisioned by the pharmaceutical industry.
Outer membrane protein vaccines Compared to the high heterogeneity of O-PS and moderate serologic variation among ﬂagella molecules, the P. aeruginosa major outer membrane proteins F (OprF) and I (OprI) are highly conserved . Therefore immunization may induce protective antibodies reactive to all of the known P. aeruginosa serotypes. Classical protein preparation and recombinant DNA technology has been used to construct Opr vaccines and test them to prevent P. aeruginosa infections . A recombinant hybrid protein consisting of the entire OprI molecule fused to the carboxy terminal sequence (aa 190—342) of OprF has been constructed and puriﬁed for use as a vaccine with Al(OH)3 as adjuvant . In addition, a nasally applied gel was prepared by mixing the vaccine with sodium dodecylsulfate and aerosil, a fumed silica material used in making pharmaceutical tablets among other uses. The fusion protein is expressed at high levels in a soluble form in an E. coli-based bacterial expression system using a plasmid containing a kanamycin resistance cassette. A nickel chromatography puriﬁcation step of a his-tagged protein was described for the vaccine preparation and according to the published studies the his-tag has been left in place in the recombinant vaccine. This recombinant approach avoids contamination of the vaccine with P. aeruginosa LPS which may be present in a conventional Opr vaccine as can be assumed from the preparation process . Other vaccine preparations have tested naked Opr DNA [72,73] (see DNA and viral vector vaccines: Preclinical studies), and linkage of an 11-amino acid residue of OprF to the hemagglutinin of the inﬂuenza virus to produce a chimeric virus . Furthermore, an attenuated aroA mutant of S. enterica serovar Typhimurium strain SL3261 was used to express P. aeruginosa OprF/I for evaluation as a vaccine  (see Killed whole cell and live-attenuated vaccines: Preclinical studies).
Preclinical studies In preclinical studies, several tested Opr vaccine preparations generated protection against different P. aeruginosa challenges in various animal models. An OprF vaccine protected rats against the development of severe lesions caused by pulmonary P. aeruginosa infection  and the recombinant OprF/I hybrid P. aeruginosa vaccine protected immunocompromised mice against intraperitoneal challenge with P. aeruginosa [70,77,78]. Also the chimeric P. aeruginosa protein vaccine, composed of the receptor binding and membrane translocation domains of exotoxin A with the OprI and F, revealed protection in a burned mouse model  and the chimeric inﬂuenza virus-immunized mice developed signiﬁcantly fewer severe lung lesions than did control mice immunized with the wild-type inﬂuenza virus . Finally, native and recombinant preparations of other non-integral Oprs of P. aeruginosa have been tested in a
Vaccines and immunotherapy against Pseudomonas aeruginosa rat model of acute pulmonary infection , although challenge experiments only used the one strain from which the non-recombinant proteins were isolated.
Phases I/II studies Only two Opr vaccines have been administered to humans: the recombinant OprF/I vaccine, manufactured previously by Behringwerke, Germany, and the Opr vaccine CFC101, manufactured conventionally by extraction from P. aeruginosa cells by CheilJedang Corp., Ichon, Korea. The administration of the recombinant OprF/I vaccine, to healthy volunteers and patients with severe burns was safe and the vaccine induced speciﬁc antibodies [81—83]. Apparently, potential problems associated with burned patients indicating that cellular immunity might be impaired and thus compromise the vaccine’s immunogenicity , did not preclude the development of humoral immunity to the vaccine. In a study of eight patients suffering from second or third degree burn who were immunized three times with 100 g of the OprF—OprI vaccine, seven of the patients produced serum antibodies speciﬁc to the vaccine antigens . However, it is not known whether antibodies to OprI/F mediate neutrophil phagocytosis and killing of P. aeruginosa strains. With regard to speciﬁc antibody production, nasal immunization using the recombinant OprF/I vaccine was inferior to systemic immunization in healthy volunteers  (see also Flagella vaccines: Phase I studies and Killed whole cell and live-attenuated vaccines: Preclinical studies). In a group of 12 CF patients, initially immunized intranasally with the gel formulation then divided into two groups of six patients each and boosted either systemically or intranasally, serum, sputum and BAL antibodies were induced . The systemically boosted group had better serum IgG antibodies but the nasally immunized and boosted group had better bronchial IgG and IgA antibodies 1-year post-immunization. The P. aeruginosa CFC-101 vaccine, shown to be safe and immunogenic in healthy volunteers  was also tested in a double-blind, randomized and placebo-controlled clinical trial in burn patients . A total of 95 patients were enrolled in the study and 16 patients in the placebo group and 72 in the immunization groups completed the study. No severe adverse reactions were observed in the vaccines and Opr-speciﬁc antibody titers in the immunization groups were signiﬁcantly higher than those in the placebo group [89,90]. Since antibiotics were administered to burn patients which rendered conventional blood culture testing mostly negative, a nested polymerase chain reaction (PCR) assay of blood specimens were performed to determine the protective efﬁcacy against P. aeruginosa. The overall detection rate of P. aeruginosa in blood was signiﬁcantly lower among immunized patients than placebo patients suggesting that the P. aeruginosa Opr vaccine may be effective in conferring protection against P. aeruginosa bacteremia in burn patients .
1017 ). The aroA gene encodes an enzyme essential for the synthesis of aromatic amino acids (5-enolpyruvylshikimate 3-phosphate synthase of the shikimate pathway). Mutations in the aroA gene have been utilized with several other pathogens, including Salmonella species  for the production of live, attenuated vaccine strains. An attenuated aroA mutant of S. enterica serovar Typhimurium (strain SL3261) was used to express OprF/I from P. aeruginosa . This strain was also used to express the serogroup O11 O-antigen of P. aeruginosa . However, in contrast to P. aeruginosa, single aroA deletion mutants in strain SL3261 retain sufﬁcient virulence to make them unacceptable as human vaccines.
Preclinical studies The intrinsically low virulence of P. aeruginosa allowed single aroA deletion mutants to be sufﬁciently attenuated to permit study in animal models. Indeed, PAO1 aroA is highly attenuated in mice and doses up to 5 × 109 CFU, given intranasally or intraperitoneally, had no apparent adverse effects . Intranasal application of PAO1 aroA to mice elicited high titers of antibodies to whole bacterial cells and to heat-stable bacterial antigens of all seven variant P. aeruginosa serogroup O2/O5 strains. The vaccine provided protection against challenge of mice with O-antigen homologous, but not against LPS-serogroup heterologous strains . The vaccine was also protective in a murine corneal infection model  but in this setting protected against several LPS serotypes, which was attributed to crossreactive antibodies to outer membrane proteins elicited by the vaccine. The attenuated aroA mutant of the Salmonella strain SL3261, expressing OprF/I from P. aeruginosa, induced speciﬁc antibodies in mice when administered with a systemic booster following an oral immunization schedule . The SL3261 strain, expressing the serogroup O11 O-antigen of P. aeruginosa, when administered orally or intraperitoneally, increased the survival of mice, challenged with a P. aeruginosa O11 strain, but not with a heterologous P. aeruginosa strain . In a further study , intranasal immunization with this construct provided full protection against challenge in three infection models: pneumonia, burns and corneal infections. This route of immunization induced a more effective immune response, particularly in local tissues, compared to the oral or intraperitoneal routes, which may account for its superior efﬁcacy.
Phase I studies An oral, killed whole-cell P. aeruginosa vaccine, when tested in 30 healthy volunteers was demonstrated to be safe and immunogenic . No further studies of this vaccine have been reported.
Killed whole cell and live-attenuated vaccines
Pili and type III secretion system vaccines
Besides a killed whole-cell P. aeruginosa vaccine , a liveattenuated P. aeruginosa vaccine was constructed in strain PAO1 carrying a deletion in the aroA gene (PAO1aroA)
Type IV pili are long, ﬂexible ﬁlaments that extend from the surface of Gram-negative bacteria and are formed by the polymerization of pilin monomers . Type IV pili medi-
1018 ate bacterial adhesion to asialoGM1 on cultured cells, but whether this interaction actually happens in vivo is not established. Pili also cause twitching motility, and play a role in bioﬁlm formation on abiotic surfaces. A factor complicating vaccination with pili preparations is that different bacterial strains produce distinct and sometimes highly divergent pilin variants. P. aeruginosa type IV pili can be divided into ﬁve distinct phylogenetic groups . A predominance of group 1 pili has been demonstrated in isolates from CF patients . Crystal structures of pilin from the P. aeruginosa strains PAK and K122-4 have been published [100,101]. Puriﬁed pili have been used to vaccinate mice  and a P. aeruginosa pilin/exotoxin A vaccine, using gene sequences of PilAII , has been prepared and characterized . Furthermore, several PilAII peptides have been coupled to tetanus toxoid and tested for immunogenicity in mice . The C-terminal receptor-binding region of the P. aeruginosa pilin protein (residues 128—144) from strain PAK, expressing PilAII , has been used to construct a P. aeruginosa vaccine . Sequences from the type IV pilin of P. aeruginosa were inserted into a vector encoding a non-toxic version of exotoxin A resulting in a chimeric protein, termed PE64553pil. Another strategy involved pilus tip epitopes, thought to be the major targets of protective immunity. These epitopes were fused to the C-terminus or inserted into the full-length surface protein RsaA of Caulobacter crescentus . Finally, since the pilin of P. aeruginosa 1244 is glycosylated with an oligosaccharide that is structurally identical to the O-antigen repeating unit of this organism, it has been proposed to utilize the 1244 pilin glycosylation system in vaccine engineering . To deliver exoenzymes into eukaryotic cells, P. aeruginosa uses a type III secretion system analogous to that of Yersinia pestis and Salmonella spp. P. aeruginosa toxins, including ExoS ExoT, ExoU and ExoY, are injected directly into eukaryotic cells via a needle-like structure that pierces the plasma membrane of the target cells. Complex structures form on the bacterial surface to assemble the delivery system, and one component of this structure is the PcrV protein. Vaccination against PcrV increased the survival of challenged mice and decreased lung inﬂammation and injury . Antibodies to PcrV also inhibited the translocation of type III toxins . Also, burned P. aeruginosa-infected mice immunized against PcrV showed signiﬁcantly enhanced survival compared to controls for two of three strains tested, whereas for the third stain immunity to exotoxin A was also needed to achieve full protection. Survival was nonO serotype speciﬁc and correlated with a reduced systemic microbial load .
Preclinical studies Intratracheal immunization of mice with P. aeruginosa pili resulted in a signiﬁcant improvement of survival and signiﬁcantly decreased bacterial numbers in the lungs of the animals after intratracheal challenge with P. aeruginosa strain PAO1 . Also, the pilin peptide—tetanus toxoid conjugate vaccine in which the peptide was conjugated to the carrier at both ends provided protection in mice challenged with lethal doses of P. aeruginosa . The chimeric PE64Delta553pil protein bound speciﬁcally to asialoGM1,
G. D¨ oring, G.B. Pier and, when injected into rabbits, produced antibodies that reduced bacterial adherence and neutralized the cell killing activity of exotoxin . None of the various type IV pili vaccines or the PcrV vaccine have yet been tested in clinical studies.
DNA and viral vector vaccines DNA vaccines have emerged as an attractive approach for antigen-speciﬁc immunotherapy. DNA vaccines offer many advantages over other conventional vaccines such as peptide or attenuated live pathogens. For example, DNA vaccines are relatively stable and can be easily prepared and harvested in large quantities. Additionally, naked plasmid DNA is relatively safe and can be repeatedly administered without adverse effects. Moreover, DNA is able to be maintained in cells for long-term expression of the encoded antigen; therefore, maintenance of immunologic memory is possible [73,110]. Intradermal administration of DNA vaccines via a gene gun can be used to efﬁciently deliver genes of interest to professional antigen-presenting cells in vivo . The skin contains numerous bone marrow-derived antigenpresenting cells (the Langerhans cells) that are able to move through the lymphatic system from the site of injection to draining lymph nodes, where they prime antigen-speciﬁc T cells [112,113]. Gene gun immunization therefore provides the opportunity to test vaccine strategies that require direct delivery of DNA to antigen-presenting cells. A P. aeruginosa DNA vaccine composed of a plasmid containing the gene for OprF , a bivalent vaccine, containing a plasmid encoding a fused OprF and OprI protein , and a multivalent DNA vaccine, containing the OprF/OprI construct, PcrV, or PilA  have been prepared. Another strategy involved the expression of OprF in an adenovirus vector: a 14-amino acid epitope of P. aeruginosa OprF was introduced in a replication-deﬁcient adenovirus vector . Other approaches to construct P. aeruginosa vaccines utilized a plasmid, containing a mutated toxA gene .
Preclinical studies When the OprF DNA vaccine was administered intradermally, it induced protection against P. aeruginosa challenge in a mouse model of chronic pulmonary infection [114,118]. Eight days post-challenge, a signiﬁcant reduction in the presence of macroscopic lesions, as well as in the number of bacteria present in the lungs, was observed. The bivalent OprF/OprI DNA vaccine induced antibody-mediated and cellmediated protection against P. aeruginosa challenge in mice [74,119]. When the multivalent DNA vaccine was administered to mice via intramuscular electroporation, the vaccine induced protection against lethal pneumonia. When mice were immunized with a OprF/adenovirus construct, speciﬁc serum antibodies against OprF were observed, and the animals were protected against a lethal pulmonary challenge with agar-encapsulated P. aeruginosa. Also the plasmid, containing a mutated toxA gene, conferred protection against challenge of mice with exotoxin A . None of the various DNA vaccines was tested in clinical studies until now.
Vaccines and immunotherapy against Pseudomonas aeruginosa
Passive immunization against P. aeruginosa infections Many patients at risk for severe P. aeruginosa infections would not be candidates for active vaccination either because a medical condition that predisposes them to infection has a sudden onset or an underlying immune compromise would interfere with immunization. This idea is also consistent with the situation that the immune response could be ineffective, as seen in the antibody response to alginate made by most, but not all, CF patients wherein most of the antibodies are ineffective in opsonic killing and protection [120,121]. Such situations have prompted attempts to develop passive immunotherapies that could provide patients with immediate resistance to infection. Early attempts to use immunoglobulin derived from multiple donors suggested some efﬁcacy in animal studies  but long term use likely would present problems associated with the variable antibody titers to P. aeruginosa antigens found among human plasma donors, so this was not a good source for potential passive therapeutic antibody. In a phase III clinical trial, a hyperimmune globulin derived from the plasma of healthy individuals immunized with the Aerugen® vaccine failed to offer signiﬁcant protection in patients in intensive care units in federal hospitals in the United States . Another phase III trial involving a hyperimmune immunoglobulin preparation derived from the plasma of donors immunized with alginate to induce opsonic antibodies was stopped when no signiﬁcant differences were found between the hyperimmune immunoglobulin treatment group and the control group. An innovative approach has been to use transgenic mice whose murine immunoglobulin genes have been removed and replaced with a limited array of human immunoglobulin loci. These mice can be used to generate human mAb, and Schreiber and colleagues [124,125] have shown this potential by producing such antibodies against P. aeruginosa O-PS antigens. More recently, a human mAb to P. aeruginosa LPS O6a,d antigen has been expressed in transgenic tobacco plants and can mediate effective in vitro opsonic killing of the target strain . Fully human mAb against P. aeruginosa O-PS have also been developed using other systems [127,128]. This latter approach is based on active immunization with P. aeruginosa O-PS, conjugated to toxin A, and antigen-speciﬁc panning of B lymphocytes. However, one major drawback to targeting the O-PS with passive antibodies is that a panel of 7—10 different antibodies would need to be given to cover the major O-serotypes associated with P. aeruginosa infection. An alternative approach would be to target conserved epitopes on a less variable antigen such as alginate. Fully human mAbs to alginate have been produced from the plasma cells of a volunteer immunized with puriﬁed alginate . These mAbs were shown to be opsonic against both mucoid and non-mucoid strains, the latter producing low levels of alginate . One major concern is how therapeutic human mAbs to P. aeruginosa are delivered to infected airways other than by an aerosol. Such a requirement could result in technical barriers to delivering passive protection to some patients.
Preclinical studies Several antibodies, speciﬁc to various antigens of P. aeruginosa have been tested, including antibodies against ﬂagella, LPS O-PS, PcrV type IV pili and alginate. Passive anti-ﬂagellar immunotherapy including human mAbs was effective in protecting mice or rats challenged in different animal models from P. aeruginosa infection, although high doses had to be given [54,131—137]. The human mAbs to both O-PS [124,125] and alginate  have also shown efﬁcacy in animal infection models. Importantly, a subset of the mAbs to alginate showed protective efﬁcacy against some nonmucoid and mucoid strains in murine models of pneumonia . Non-mucoid strains produce only low amounts of alginate under aerobic conditions but alginate production is rapidly increased under anaerobic conditions [129,130]. Passive immunization studies were also successfully carried out with P. aeruginosa pilus-speciﬁc and cross-reactive mAbs in mice . Furthermore, rabbit antisera raised against PcrV were protective in several P. aeruginosa challenge animal models [138,139]. Similarly, a murine mAb to PcrV, mAb166, mapped to the carboxyl-terminus of PcrV, prevented sepsis and death from P. aeruginosa infection in mice . Finally, the degree of lung tissue injury and inﬂammation improved in rats, challenged with P. aeruginosa strain PA103 in the respiratory tract, that had received intratracheal instillations of a murine monoclonal anti-PcrV IgG mAb166 or mAb166 Fab-fragments in comparison with the control group .
Phases I/II studies Hunt and Perdue  administered a tetravalent hyperimmune globulin to 10 burn patients with P. aeruginosa sepsis, and reported clinical improvement, associated with a 3- to 125-fold increase in antibody titers. No control group was used.
Phases II/III studies Jones et al.  used both active and passive immunization in a controlled clinical trial in burned patients at Safdarjang Hospital, New Delhi, India. Immunoglobulin derived from healthy human volunteers who received the 16-valent PEV01 vaccine was administered to children with burns, and among the passively immunized groups there was 0%, (0/18) mortality compared with 21% (9/42) in controls. Adults that received the immunoglobulin had a 10% (3/30) mortality rate whereas 36% (22/61) of controls died. However, further human trials with this vaccine have not been published in the past 25 years. A large multicenter study failed to show a decrease in the incidence or severity of P. aeruginosa infections in patients admitted to intensive care units  who were given 100 mg/kg of an intravenous hyperimmune globulin, derived from donors immunized with a 24-valent Klebsiella capsular polysaccharide plus the octavalent P. aeruginosa O-polysaccharide—toxin A conjugate vaccine (Aerugen® ; ). In addition, patients receiving hyperimmune globulin had more adverse reactions. Thus at the moment no passive therapeutic reagent targeting P. aeruginosa infec-
1020 Table 2
G. D¨ oring, G.B. Pier Experimental Pseudomonas aeruginosa Vaccines
LPS Killed whole cell Pili OprF/I PS-exotoxin A conjugate Flagella subtypes a, b P. aeruginosa aroA S. enterica O11/OprF/I DNA OprF/I, Exotoxin A, PcrV, PilA
Phases II/III Phase I Preclinical Phases I/II Phase III Phases I—III Preclinical Preclinical Preclinical Preclinical
1971—1984 2006 1995—2006 1997—2004 1989—2006 1986—2007 2002—2006 2004 1998—2006 1998—2006
[19,33—35,38]  [102—107] [81—83,85—90] [11,28—31,39—41] [10,55,56] [95,96] [75,94,97] [74,114—119] [117,119]
tion has been successful enough in clinical trials to warrant licensure.
Conclusions Taken together, it seems not to be a problem to deﬁne speciﬁc P. aeruginosa antigens for vaccine development, since many different antigens have been successfully tested in preclinical trials including animal model of experimental P. aeruginosa infections (Table 1). Rather, problems occur in testing a given vaccine or a monoclonal antibody for its efﬁcacy in a patient group at risk for P. aeruginosa infection. This has been demonstrated by the two vaccine trials in CF patients in which improved antibiotic therapy strategies against P. aeruginosa impaired a proper evaluation of the vaccine’s efﬁcacies. Similar problems have occurred in vaccine trials involving patients with burns or intubated patients in intensive care units. In addition to better antibiotic therapy, P. aeruginosa infections may become more rare due to improved hygiene, which will make future vaccine studies more difﬁcult. The low numbers of patients with underlying diseases who are at risk for infection with the opportunistic pathogen P. aeruginosa may not be high enough for pharmaceutical companies involved in vaccine development to initiate clinical trials. Consequently, there is a paucity of randomized controlled trials assessing the effectiveness of vaccination against P. aeruginosa infections in CF and other patient risk groups. While many experimental vaccines and monoclonal antibodies have been tested in preclinical trials, only a few have reached clinical phases and none of these vaccines has obtained market authorization (Table 2). Whether the lack of efﬁcacious P. aeruginosa vaccines will be overcome in the future, remains an open question.
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