Safety and Immunogenicity of an Oral Inactivated Whole-Cell Pseudomonas aeruginosa Vaccine Administered to Healthy Human Subjects
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感染与免疫杂志 2006年第2期
School of Medicine, Griffith University, Queensland, Australia
Provalis PLC, Deeside, Flintshire, United Kingdom
University of Newcastle, Newcastle, New South Wales, Australia
Protein Reference and Immunopathology Unit, St. George's Hospital Medical School, London, United Kingdom
Centres for Studies in Drug Disposition, Department of Medicine, Royal Brisbane Hospital, Queensland, Australia
ABSTRACT
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.
INTRODUCTION
Pseudomonas aeruginosa is a gram-negative bacterium with a ubiquitous distribution within the biosphere. In the compromised host, it is capable of establishing opportunistic infections, and this is particularly common in subjects with lung dysfunction. P. aeruginosa is particularly well adapted to the conditions found in the lungs of cystic fibrosis (CF) patients, where a defective chloride channel transport protein results in an increased viscosity of secretions, making it difficult to clear airway mucous. Colonization takes place at an early age, often in the absence of any overt clinical presentation or culture-positive sputum and throat swabs (3). Because the bacteria are not effectively eradicated from the CF lungs, chronic colonization occurs. P. aeruginosa adapts through gene switching to undergo a number of phenotypic changes. These include the loss of lipopolysaccharide (LPS) O antigen, which renders the strain nontypeable or polyagglutinating, and the production of excessive amounts of an alginate polysaccharide capsule (14), which allows the microbe to exist in microcolonies (biofilms) within the lungs. In addition, these nontypeable, mucoid colonies exhibit a reduced susceptibility to antibiotics and frustrated phagocytosis, where excessive amounts of alginate prevent phagocytosis by polymorphonuclear neutrophils and macrophages. The resulting excessive production of proteases, superoxide radicals, and inflammatory mediators contributes to the subsequent destruction of normal lung tissue. The major antigen of immune complexes in the sputum of CF patients has been shown to be LPS (17). Outer membrane protein F (OprF) and outer membrane protein H2 (OprH2) in particular have been shown to induce strong antibody activity, while OprI, OprF, and OprH2 are highly conserved in P. aeruginosa (31, 33).
Current therapies with antibiotics are targeted at controlling bacterial load of P. aeruginosa and other bacteria. These frequently fail to adequately clear established infections, while low antibiotic concentrations in the airways are ineffective and may lead to the development of resistant bacterial strains. A vaccine which could prevent or delay initial colonization with P. aeruginosa in the lungs may have a positive impact on CF patients and contribute to improvement in quality of life and survival in these patients. In addition, it is also feasible that immunization may reduce bacterial loads in patients who have become chronically colonized with P. aeruginosa. Vaccine candidates for P. aeruginosa have been under study for some 30 years or more, but progress has been slow (9). The potential to vaccinate against P. aeruginosa infection has been recently reviewed, and a number of exciting opportunities have been identified including mucosal immunization (27). Most studies have focused on burn patients and CF patients, and many havenot progressed beyond initial proof-of-concept stages. ACochranereview in 1999 (16) concluded that there was a paucity of randomized clinical trials assessing the effectiveness of vaccination against P. aeruginosa in CF patients. The only trial to meet their inclusion criteria was one evaluating a blended LPS administered to children, which showed no clinical benefit at the 10-year follow-up. There was also a suggestion that the vaccine may have been detrimental, with the immunized group appearing to have more severe pulmonary exacerbations than the control group (18). Although this does not preclude a vaccine approach to P. aeruginosa infection in the management of CF, it may have added to the reluctance and slow progress in developing a vaccine.
This study reports on a phase 1 safety and immunogenicity study using an oral inactivated whole-cell P. aeruginosa vaccine administered to healthy volunteers. The vaccine has previously been shown to protect against acute P. aeruginosa challenges to the lungs of rodents (2, 7) and may be suitable for the development of an oral formulation for CF infants to prevent or delay colonization by P. aeruginosa (7). Successful mucosal and systemic immune stimulation mediated via gastrointestinally associated lymphoid tissue would allow for the development of oral vaccines to be used as a prophylactic and therapeutic tool against chronic P. aeruginosa infections.
MATERIALS AND METHODS
Clinical trial rationale. The aim of the study was to assess the safety of Pseudostat as an oral vaccine in humans and to obtain preliminary information on antibody responses to P. aeruginosa following oral vaccine administration. For the safety study, treatment was selected as the lowest scaled-up by-weight dose derived from mice studies which had induced a protective response against a P. aeruginosa lung challenge and statistically significant increases in serum P. aeruginosa-specific antibody titers. Based on animal data, a "priming" and "stimulatory" dose regime was chosen as it could produce a greater response than a single exposure.
Study design. This was a single-center, open-label, phase 1 study to assess the safety and immune response to Pseudostat, an oral preparation of whole-cell, formaldehyde-inactivated P. aeruginosa, in healthy human volunteers. Thirty healthy subjects between the ages of 18 and 50 years who met the inclusion criteria were recruited for the study. Subjects were excluded if they had any clinically relevant medical condition including a past history of Pseudomonas infection requiring medical treatment, a history of alcohol or drug abuse, any allergic sensitivity or intolerance to vaccines, and impaired oral absorption. They were also excluded if they were immunocompromised, had participated in any study during the last 2 months, were pregnant, or were of childbearing age and had not been taking a combined oral contraceptive pill for at least 3 months.
Vaccination and safety. The Pseudostat vaccine consisted of enteric-coated hard gelatin capsules, each containing 150 mg of lyophilized, formaldehyde-inactivated P. aeruginosa strain 385, serotype 2, phage type 21/44/109/110X/1214, which was equivalent to 2 x1011 bacteria per capsule. The vaccine was manufactured for the trial by Boehringer-Ingelheim. Strain 385 is a clinical isolate of a mucoid phenotype. In extensive preclinical animal experiments, immunization with this strain gave optimal and reproducible responses, and immune protection was observed against challenge with other serotypes (7). All subjects were dosed over a 3-day period to avoid any possibility of seasonal effects. All subjects were asked to fast 6 h prior to the clinic visit for dosing and 1 h after. Eligibility of subjects was established at screening (day –14), and each subject received a single 150-mg dose of Pseudostat on day 0 and day 28 with follow-up visits 2 weeks after each dose (day 14 and day 42, respectively) and a final visit on day 56. At each of the five visits and prior to dosing, 30 ml of blood and 10 ml of saliva were collected into plain tubes. Serum prepared from blood samples and saliva were stored at –80°C until analyzed. At each visit, vital signs, concomitant medications, and any adverse events were noted; on day 0 and day 56, a clinical examination was undertaken by a qualified physician, and blood samples were collected for routine hematology and biochemistry analysis at an accredited pathology laboratory. An adverse event was recorded for any change from the subject's baseline (pretreatment) condition, including any clinical or laboratory test value abnormality of clinical significance which occurred after the start of the study, whether it was considered related to the study medication or not.
Determination of antibodies to whole-cell extract of P. aeruginosa and outer membrane proteins (OprF and OprI). Enzyme-linked immunosorbant serum assays (ELISAs) were developed and validated for measuring antibody responses to whole-cell P. aeruginosa extract and outer membrane proteins OprF and OprI. Microtiter plates were coated with 100 μl of a 10-μg/ml solution of either soluble whole-cell P. aeruginosa extract or recombinant His-tagged OprF or OprI in 50 mM sodium bicarbonate buffer at pH 9.6. Plates were then blocked with 0.1% (vol/vol) Tween 20 in phosphate-buffered saline (PBS) at pH 7.0. Serum samples were diluted at 1:50 for immunoglobulin A (IgA), 1:200 for IgM, and 1:2,000 for IgG. Triplicate samples were dispensed into microwells for incubation overnight at 2 to 8°C. After incubation, plates were washed twice in PBS containing 0.05% (vol/vol) Tween 80 and incubated for a further 1 h at 37°C with 100 μl of the appropriate peroxidase conjugated anti-human immunoglobulins diluted in PBS containing 0.05% (vol/vol) Tween 20 (1:10,000 for IgG and 1:2,000 for IgM and IgA). After being washed with PBS in 0.05% (vol/vol) Tween 80, the plates were developed by addition of 100 μl of tetramethyl benzidine chromogen for 10 min at room temperature (controlled at 22°C). Reactions were stopped by the addition of 50 μl of 25% (vol/vol) phosphoric acid, and absorbency values were measured at 450 nm with a microtiter plate spectrophotometer. Endpoint titers were calculated as the mean absorbencies against the baseline with coefficients of variations typically of <5% for all triplicate determinations and trend analysis at both dilutions.
Determination of antibodies to lipopolysaccharide. LPS was prepared from the P. aeruginosa vaccine strain by the method of Westphal and Jann (32). Microtiter plates were coated with 100 μl of 25-μg/ml LPS in 10 mM PBS at pH 7.4 containing 20 mM MgCl2 and incubated overnight at 37°C. The plates were then blocked with 200 μl of 10 mM PBS (pH 7.4) containing 5% (wt/vol) bovine serum albumin and 0.05% (vol/vol) Tween 20 for 15 min at 22°C. Plates were washed twice in PBS containing 0.05% (vol/vol) Tween 80, and 100 μl of pooled serum diluted in PBS (1:200 for IgA and IgM and 1:2,000 for IgG) was added to each well and incubated overnight at 4°C. After being washed in PBS containing 0.05% (vol/vol) Tween 80, the plates were incubated with the appropriate peroxidase-conjugated anti-human immunoglobulin (1:2,500 dilution with PBS containing 0.05% [vol/vol] Tween 20) for 1 h at 37°C and developed as above.
Measurement of total serum immunoglobulins. The total concentrations of nonspecific IgG, IgA, and IgM were determined for the serum and saliva samples of each individual by the use of a Beckman Array 360 nephelometer and standard reagents (Beckman Coulter, Inc., Fullerton, Calif.).
Detection of saliva antibody responses. Measurement of antibody responses was determined for saliva samples by essentially the same ELISAs as used above for the whole-cell P. aeruginosa extract, OprF, and OprI but with lower sample dilutions. Total salivary albumin was also measured by nephelometry.
Opsonization and phagocytosis assays. The sera of all subjects were pooled for each of the time points during the study and used to determine opsonophagocytosis of live P. aeruginosa 385 with a human monocytic cell line (THP-1). The THP-1 cell line (5 x 104) was seeded into tissue culture flasks and grown in RPMI-1640 medium containing 10% (vol/vol) fetal calf serum and 1(wt/vol) penicillin-streptomycin for 3 days. The cells were then activated to produce a more macrophage-like phenotype by the addition of 50-ng/ml myristyl phorbol ester for 24 h. After activation, nonadherent cells were removed by being washed, and the adherent cells were harvested with a cell scraper and suspended in Hanks balanced salt solution (HBSS). Bacteria were opsonized by combining 50 μl of bacteria (equivalent to 109/ml) to 20 μl of pooled serum for each of the time points and incubating for 30 min at 25°C. Cells were washed twice in PBS and made to 1 ml, of which 50 μl was added to 500 μl of activated THP-1 cells and again made to 1 ml with HBSS. The cells were incubated for 30 min at 37°C, washed in HBSS containing 250-μg/ml gentamicin sulfate, and further incubated for 10 min to kill adherent nonphagocytosed bacteria. Cells were then washed in PBS, and a 50-μl sample was removed and lysed in 450 μl of water. An additional 10-fold dilution of the sample was made, and 50 μl of this dilution was plated onto nutrient agar to determine the number of CFU. The remaining macrophages with phagocytosed P. aeruginosa were incubated for a further 60 min (total time, 90 min) and plated as above to determine the extent of killing of P. aeruginosa by the macrophages. Non-serum samples were used as negative controls, and a control sample of pooled serum from cystic fibrosis subjects known to be colonized with P. aeruginosa was also included to represent a high-titer sample to P. aeruginosa antigens. The assay was repeated for each time point on five occasions, and a mean value was determined for the results.
Statistical analysis. Serum and saliva-specific antibody levels as determined by ELISA absorbance at 450 nm were compared at each time point against day 0 by using a paired two-tailed t test. Statistical significance was defined as occurring when P values were <0.05. Salivary antibody data were also expressed as a ratio of salivary albumin levels to adjust for possible salivary flow rate differences between individual subjects. For the purpose of the immunogenicity study, responders were defined as those showing at least two or more postdose antibody titer increases with an increase of at least >15% above predose baseline levels.
Opsonization was extrapolated from colony counts immediately after phagocytosis. Bactericidal effect was measured 1 h postphagocytosis and was expressed as a percentage of difference in the final colony counts.
For the purpose of analyzing the serum IgM-specific antibody response to the whole-cell extract, one subject was removed as an outlier. For this subject, values determined on day 42 and 56 were three to six times the group mean. Removal of this subject from the IgM analysis did not alter the statistical significance observed for the group mean.
No significant differences were observed for any of the parameters determined between day –14 and day 0; therefore, day 0 was taken as the baseline measure throughout.
RESULTS
Safety. Thirty subjects were recruited as eligible for the study, of which 28 subjects received the first dose and 24 subjects completed the study to day 56. Subjects were removed from the study if they received routine or prohibited medications, did not receive both doses, or did not attend key visits. Twenty-one subjects were male (70%) and nine subjects were female (30%). The mean age ± standard deviation was 23.1 ± 4.2 years, with a range of 18 years to 35 years of age, and the mean weight ± standard deviation was 69.9 ± 12.0 kg. Pseudostat was well tolerated by all subjects, although 14 subjects reported 20 adverse events during the study, none of which were considered to be possibly or probably related to the use of Pseudostat. Adverse events were grouped by body systems; the preferred terms followed the World Health Organization classification system. These identified 2 individuals with musculoskeletal system disorders, 2 individuals with central nervous system disorders (headache and convulsion), 3 individuals with gastrointestinal disorders (diarrhea, dyspepsia, and vomiting), and 13 individuals with respiratory system disorders (pharyngitis, rhinitis, and upper respiratory tract infection). Only one adverse event was considered clinically significant, although not related to the study drug. Two reported adverse events (gastritis and upper respiratory tract infection) occurred prior to treatment; among the posttreatment adverse events, twice the number of upper respiratory tract infections followed the second dose versus the first dose. There was no close temporal relationship to either day of dosing or any consistency between adverse event reports with regard to time of occurrence (ranging from 9 to 21 days after day 0 and 4 to 22 days after the day 28 dose). This was also true of other adverse event reports except for the reported headache, which occurred 1 day after the initial dose. One serious adverse event involved a subject who had an epileptic seizure (described as being of moderate severity) and was hospitalized overnight for treatment. This subject was subsequently found to have had a seizure some months previously, which had been not reported to the investigators, and a family history of epilepsy. No abnormal hematological or biochemical test results of clinical significance were found at screening or at the day 56 follow-up visit for any subjects. There were no clinically significant changes in vital signs observed throughout the course of the study. All patients felt well at the completion of the study.
Total serum immunoglobulins. Total serum immunoglobulins showed no statistically significant changes or trends throughout the course of the study. Values for IgA and IgM are presented in Table 1.
Specific antibody responses to whole-cell P. aeruginosa. Serum whole-cell P. aeruginosa extract-specific IgA increased by day 14 after the first dose, and remained above the day 0 level for the remainder of the observation period (Table 1). The group mean showed a statistically significant increase in P. aeruginosa-specific IgA titers compared to day 0 for day 14 (P= 0.009), day 28 (P = 0.017), and day 42 (P = 0.010) but failed to reach significance on day 56 (P = 0.053). Whole-cell P. aeruginosa extract-specific IgM antibody titers also reached a statistically significant group mean increase from baseline atday 14 (P = 0.019) (Table 1). No statistically significant changes were observed for whole-cell P. aeruginosa extract-specific serum group mean IgG levels (data not shown).
Of the subjects tested, 39 to 65% were positive responders for all antibody classes and proteins tested. The least number of responders was to OprI at 39% and 48% for IgA and IgM, respectively. All other categories showed >50% responders; however, for the purpose of this analysis, responders were defined as those showing at least two or more postdose antibody titer increases with a least one increase of >15% above the predose baseline levels. Hence, of the 24 volunteers who completed the study, 29% were considered IgA responders, 25% were considered IgM responders, and 33% IgG were considered responders against whole-cell P. aeruginosa extracts (Table 2).
There were no significant changes in the levels of P. aeruginosa-specific IgA salivary antibody levels following immunization. Salivary IgG antibodies against whole-cell P. aeruginosa extract demonstrated an increase from baseline levels at day 14, which waned by day 42 before a more significant second increase at day 56 (P = 0.027) (data not shown). No statistically significant changes were observed for either salivary IgA or IgG antibodies against any of the P. aeruginosa antigens tested when the antibody data were expressed as a ratio of the salivary albumin levels. Salivary IgM-specific antibodies were not measured.
Outer membrane protein (OprF and OprI)-specific IgA responses. No significant group mean IgA, IgM, or IgG serum responses were observed for either OprF or OprI. Of the 24 volunteers, 46% were OprF-specific IgA responders, and 33% were OprI-specific IgG responders. To a lesser degree and in decreasing order, the responders were as follows: OprF-specific IgM responders (29%), OprI-specific IgA responders (21%), OprI-specific IgM responders (17%), and OprF-specific IgG responders (4%) (Table 2).
LPS. LPS-specific serum IgA titers increased significantly (P< 0.05) from baseline levels on day 14, day 28, day 42, and day 56 with maximum increases occurring 28 days post-first dose (Fig. 1). LPS-specific serum IgM titers showed no significant change from baseline on day 14; however, there were statistically significant (P < 0.05) decreases in responses for days 28, 42, and 56. LPS-specific serum IgG titers showed a pattern similar to that of LPS-specific serum IgA, compared to day 0, achieving statistically significant increases (P < 0.05) on all days postdose except day 14 (Fig.1C).
Opsonophagocytosis assays. The number of colonies grown after opsonization and phagocytosis by activated THP-1 monocytes in the presence of pooled serum from Pseudostat-vaccinated individuals is represented in Fig. 2. The results show an increase (34 to 62%) in the number of colonies compared to day 0, with the highest macrophage capture of live P. aeruginosa cells in the presence of CF sera, which was used as a control. After a further 60-min incubation period, the percentage of opsonized colonies killed ranged from 45 to 82%, compared to 40% on day 0 (Fig. 3). Killing of bacteria was particularly enhanced on day 14 (82%), at which maximal anti-P. aeruginosa IgA responses in serum samples were also observed. After day 14, the levels dropped progressively but were still maintained above baseline values.
DISCUSSION
Studies in a rodent model of acute infection have demonstrated that mucosal immunization with a whole-cell killed P. aeruginosa vaccine results in enhanced clearance of the bacteria from the lung, as well as reduced mortality (2, 7). In a preliminary study of nine bronchiectasis patients, oral immunization with an enteric coated whole-cell killed P. aeruginosa vaccine resulted in the detection of circulating antigen-reactive peripheral blood leukocytes, as well as a significant reduction in the levels of P. aeruginosa in the sputum (8). Further evidence to support the development of a whole-cell killed vaccine and an oral immunization strategy for P. aeruginosa comes from previous studies of nontypeable Haemophilus influenzae (NTHi), where patients with recurrent acute exacerbations of chronic bronchitis were orally immunized with a whole-cell killed NTHi vaccine (4, 5, 6, 20, 28). A recent Cochrane review of six NTHi trials of 440 subjects reported that oral immunization significantly reduced the number and severity of acute exacerbations (13). In those studies, bacterial load was reduced, as determined by the incidence of throat colonization and/or quantitative or semiquantitative bacteriology of sputum samples. In addition, NTHi-specific cellular responses were detected in peripheral blood lymphocytes, following oral immunization with whole-cell killed NTHi vaccine.
This study represents a clinical pilot study designed to demonstrate the immunogenicity and safety of an oral vaccine against P. aeruginosa in healthy volunteers. The dose chosen was the lowest dose expected to induce an immune response in humans, based on a scaled-up dose from successful dose-ranging studies in rodents. Future placebo-controlled studies will determine efficacy and optimal dose and treatment regimes with human subjects.
Safety of the vaccine in healthy individuals was confirmed in the absence of any probable or possible adverse events, and no hematological or biochemical profile derangements were recorded for any subjects throughout the study. While 20 adverse events were recorded, these were not considered of clinical significance (the exception being one case of epilepsy missed on initial screening), and none were attributable to the study vaccine being administered. Upper respiratory tract infection represented 60% of the adverse events reported with twice the number of upper respiratory tract infections following the second dose compared to the first dose. However, there was no temporal relationship to either the day of dosing or in the time of occurrence.
The vaccine was demonstrated to be immunogenic. Of particular note is the significant increase in IgA-specific antibody responses against both whole-cell extract and LPS in the post-immunization observation period. Confidence in the consistency and specificity of these changes was further supported by comparison with group mean total serum IgA, which did not follow a similar trend. A sustained-specific IgG LPS antibody response was also observed postimmunization. The opsonization results clearly showed a substantial increase in ability of pooled sera from the study volunteers to promote phagocytosis of P. aeruginosa by a human macrophage cell line. The results confirm the specificity of the response observed and that antibodies induced were opsonizing for P. aeruginosa. Opsonizing antibody responses have been shown to be important for protection against P. aeruginosa infection in CF patients (24, 30). Hostoffer et al. (15) reported on the importance of IgA receptors (Fc alpha R) on mucosal phagocytes and suggested an important role in defense of mucosal surfaces. They demonstrated high levels of expression of Fc alpha R on neutrophils obtained from bronchoalveolar lavage fluid of cystic fibrosis patients and showed that neutrophil superoxide production was enhanced by IgA. Significant IgA responses following pneumococcal polysaccharide vaccination showed that the IgA induced a comparable receptor-mediated phagocytosis response, as did IgG, and that the IgA holds an important leukocyte receptor which provides immunity against Streptococcus pneumoniae (25). In this study, both opsonization and killing peaked at day 14 post-first immunization; although there was a decline in functional activity after this observation point, functional activity remained greater than the preimmunization levels. The two booster immunizations did not enhance either opsinization or killing. Further studies are required to investigate the kinetics of the functional antibody data, particularly in response to booster immunization.
The overall salivary immune responses did not reach statistical significance and were variable at each time point of the study. These observations probably reflect the inherent difficulties of measuring antibody responses in mucosal secretions, particularly following oral immunization. Despite the absence of a detectable and consistent salivary antibody response to the vaccine, this does not preclude the possibility of a mucosal immune response in the lungs or on other mucosal surfaces. If memory T cells and B cells are migrating to other mucosal surfaces from the gut, a measurable response may not be observed until booster encounter with antigen at these surfaces. IgA can reach external secretions by passive paracellular diffusion and may have a role in local defense on mucosal surfaces in addition to locally produced IgA antibodies. Furthermore, leakage of serum IgA onto mucosal surfaces is suspected to increase in inflammatory situations such as those that exist in the CF lung (22). In the context of the results of this study, Berstad et al. (1) noted an absence of salivary antibody response to a whole-cell pertussis vaccine administered nasally to human volunteers, despite strong serum antibody responses for IgA and IgG. Recently, a hybrid OprF-OprI P. aeruginosa vaccine was tested with human volunteers by intranasal application, which resulted in serum IgA and IgG responses in 75% of the volunteers (19). Although saliva and nasal washings were taken for analysis in that study, the results were not reported, implying that no responses may have been detected. Studies of oral whole-cell vaccination with an animal model have shown that protection against P. aeruginosa challenge can be observed in the absence of any significant serum or bronchoalveolar lavage specimen antibody response (7). These findings are also consistent with results observed for whole-cell killed NTHi oral immunization of subjects with chronic bronchitis, which did not stimulate a specific antibody response in saliva, although the vaccine was highly efficacious (6). Dose-ranging studies of mice (M. Dunkley, unpublished data) have shown that enhanced clearance at the lowest dose was achieved in the absence of any detectable serum antibody response. Increasing the dose by 1 log, which is equivalent to the single doses given in this study (2.6 mg/kg versus 2.1 mg/kg), resulted in better clearance in the mouse and the detection of statistically significant increases in serum IgA and IgM, as was indeed observed in this trial. These results may suggest an important role for serum IgA in the protection against P. aeruginosa lung infections.
Studies of this vaccine with an animal model have indicated that both antibody and T cells are important in protecting rodents from an acute lethal challenge of P. aeruginosa to the lungs (2, 7, 10, 11). However, some evidence points to a detrimental effect, at least in chronically colonized CF patients where increasing serum IgG antibody levels to P. aeruginosa LPS and exotoxin A and the resulting immune complexes formed have been associated with a poor prognosis (21). Kronborg et al. (17) showed that in sputum, immune complexes were almost entirely composed of LPS-specific IgG. Studies have shown that the sera of colonized patients have high titers of poorly opsonizing antibodies (26) and that the opsonizing ability of sera decreases upon conversion to the mucoid form and the onset of chronic colonization (29, 30). Of importance, the results from this study clearly demonstrate that the antibody response induced was opsonogenic. The specificity of opsonizing antibodies and their protective role requires further study (12, 23, 24).
In a recent review (27), we concluded that mucosal immunization was a potential viable option for protection against P. aeruginosa infections in at-risk patients. This study has demonstrated that oral immunization against P. aeruginosa is safe and induces a significant serum antibody response, notably of the IgA isotype. In addition, an increase in functional opsonization and killing by macrophages was demonstrated in vitro with postvaccination serum. Proof of concept with human subjects, based on the extensive preclinical studies with animal models, has been established. Further studies will involve dose-ranging studies and characterization of the immune response induced with respect to serotype specificity, cell-mediated immune mechanisms, the identification of the functional opsonins and their antigenic specificity, and the kinetics of the response.
ACKNOWLEDGMENTS
This study was supported by Provalis PLC, Deeside, Flintshire, United Kingdom.
Thanks are extended to Shrikant Jondhale for help in developing the phagocytosis assay; Sharon Griffiths, Helen Rawden, and Jane Evans for assistance in developing the ELISAs; Mark Smith and Suzanne Thomson for cloning and purifying the OprF and OprI; and Pamela Riches for advice given and discussions.
Present address: Teva Pharmaceuticals, Ltd., Aylesbury, Bucks, United Kingdom.
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20. Lehmann, D., K. J. Coakley, C. A. Coakley, V. Spooner, J. M. Montgomery, A. Michael, I. D. Riley, T. Smith, R. L. Clancy, and A. W. Cripps. 1991. Reduction in the incidence of acute bronchitis by an oral Haemophilus influenzae vaccine in patients with chronic bronchitis in the highlands of Papua New Guinea. Am. Rev. Resp. Dis. 144:324-330.
21. Moss, R. B., Y.-P. Hsu, N. J. Lewiston, J. G. Curd, H. Milgrom, S. Hart, B. Dyer, and J. W. Larrick. 1986. Association of systemic immune complexes, complement activation and antibodies to Pseudomonas aeruginosa lipopolysaccharide and exotoxin A with mortality in cystic fibrosis. Am. Rev. Respir. Dis. 133:648-652.
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Provalis PLC, Deeside, Flintshire, United Kingdom
University of Newcastle, Newcastle, New South Wales, Australia
Protein Reference and Immunopathology Unit, St. George's Hospital Medical School, London, United Kingdom
Centres for Studies in Drug Disposition, Department of Medicine, Royal Brisbane Hospital, Queensland, Australia
ABSTRACT
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.
INTRODUCTION
Pseudomonas aeruginosa is a gram-negative bacterium with a ubiquitous distribution within the biosphere. In the compromised host, it is capable of establishing opportunistic infections, and this is particularly common in subjects with lung dysfunction. P. aeruginosa is particularly well adapted to the conditions found in the lungs of cystic fibrosis (CF) patients, where a defective chloride channel transport protein results in an increased viscosity of secretions, making it difficult to clear airway mucous. Colonization takes place at an early age, often in the absence of any overt clinical presentation or culture-positive sputum and throat swabs (3). Because the bacteria are not effectively eradicated from the CF lungs, chronic colonization occurs. P. aeruginosa adapts through gene switching to undergo a number of phenotypic changes. These include the loss of lipopolysaccharide (LPS) O antigen, which renders the strain nontypeable or polyagglutinating, and the production of excessive amounts of an alginate polysaccharide capsule (14), which allows the microbe to exist in microcolonies (biofilms) within the lungs. In addition, these nontypeable, mucoid colonies exhibit a reduced susceptibility to antibiotics and frustrated phagocytosis, where excessive amounts of alginate prevent phagocytosis by polymorphonuclear neutrophils and macrophages. The resulting excessive production of proteases, superoxide radicals, and inflammatory mediators contributes to the subsequent destruction of normal lung tissue. The major antigen of immune complexes in the sputum of CF patients has been shown to be LPS (17). Outer membrane protein F (OprF) and outer membrane protein H2 (OprH2) in particular have been shown to induce strong antibody activity, while OprI, OprF, and OprH2 are highly conserved in P. aeruginosa (31, 33).
Current therapies with antibiotics are targeted at controlling bacterial load of P. aeruginosa and other bacteria. These frequently fail to adequately clear established infections, while low antibiotic concentrations in the airways are ineffective and may lead to the development of resistant bacterial strains. A vaccine which could prevent or delay initial colonization with P. aeruginosa in the lungs may have a positive impact on CF patients and contribute to improvement in quality of life and survival in these patients. In addition, it is also feasible that immunization may reduce bacterial loads in patients who have become chronically colonized with P. aeruginosa. Vaccine candidates for P. aeruginosa have been under study for some 30 years or more, but progress has been slow (9). The potential to vaccinate against P. aeruginosa infection has been recently reviewed, and a number of exciting opportunities have been identified including mucosal immunization (27). Most studies have focused on burn patients and CF patients, and many havenot progressed beyond initial proof-of-concept stages. ACochranereview in 1999 (16) concluded that there was a paucity of randomized clinical trials assessing the effectiveness of vaccination against P. aeruginosa in CF patients. The only trial to meet their inclusion criteria was one evaluating a blended LPS administered to children, which showed no clinical benefit at the 10-year follow-up. There was also a suggestion that the vaccine may have been detrimental, with the immunized group appearing to have more severe pulmonary exacerbations than the control group (18). Although this does not preclude a vaccine approach to P. aeruginosa infection in the management of CF, it may have added to the reluctance and slow progress in developing a vaccine.
This study reports on a phase 1 safety and immunogenicity study using an oral inactivated whole-cell P. aeruginosa vaccine administered to healthy volunteers. The vaccine has previously been shown to protect against acute P. aeruginosa challenges to the lungs of rodents (2, 7) and may be suitable for the development of an oral formulation for CF infants to prevent or delay colonization by P. aeruginosa (7). Successful mucosal and systemic immune stimulation mediated via gastrointestinally associated lymphoid tissue would allow for the development of oral vaccines to be used as a prophylactic and therapeutic tool against chronic P. aeruginosa infections.
MATERIALS AND METHODS
Clinical trial rationale. The aim of the study was to assess the safety of Pseudostat as an oral vaccine in humans and to obtain preliminary information on antibody responses to P. aeruginosa following oral vaccine administration. For the safety study, treatment was selected as the lowest scaled-up by-weight dose derived from mice studies which had induced a protective response against a P. aeruginosa lung challenge and statistically significant increases in serum P. aeruginosa-specific antibody titers. Based on animal data, a "priming" and "stimulatory" dose regime was chosen as it could produce a greater response than a single exposure.
Study design. This was a single-center, open-label, phase 1 study to assess the safety and immune response to Pseudostat, an oral preparation of whole-cell, formaldehyde-inactivated P. aeruginosa, in healthy human volunteers. Thirty healthy subjects between the ages of 18 and 50 years who met the inclusion criteria were recruited for the study. Subjects were excluded if they had any clinically relevant medical condition including a past history of Pseudomonas infection requiring medical treatment, a history of alcohol or drug abuse, any allergic sensitivity or intolerance to vaccines, and impaired oral absorption. They were also excluded if they were immunocompromised, had participated in any study during the last 2 months, were pregnant, or were of childbearing age and had not been taking a combined oral contraceptive pill for at least 3 months.
Vaccination and safety. The Pseudostat vaccine consisted of enteric-coated hard gelatin capsules, each containing 150 mg of lyophilized, formaldehyde-inactivated P. aeruginosa strain 385, serotype 2, phage type 21/44/109/110X/1214, which was equivalent to 2 x1011 bacteria per capsule. The vaccine was manufactured for the trial by Boehringer-Ingelheim. Strain 385 is a clinical isolate of a mucoid phenotype. In extensive preclinical animal experiments, immunization with this strain gave optimal and reproducible responses, and immune protection was observed against challenge with other serotypes (7). All subjects were dosed over a 3-day period to avoid any possibility of seasonal effects. All subjects were asked to fast 6 h prior to the clinic visit for dosing and 1 h after. Eligibility of subjects was established at screening (day –14), and each subject received a single 150-mg dose of Pseudostat on day 0 and day 28 with follow-up visits 2 weeks after each dose (day 14 and day 42, respectively) and a final visit on day 56. At each of the five visits and prior to dosing, 30 ml of blood and 10 ml of saliva were collected into plain tubes. Serum prepared from blood samples and saliva were stored at –80°C until analyzed. At each visit, vital signs, concomitant medications, and any adverse events were noted; on day 0 and day 56, a clinical examination was undertaken by a qualified physician, and blood samples were collected for routine hematology and biochemistry analysis at an accredited pathology laboratory. An adverse event was recorded for any change from the subject's baseline (pretreatment) condition, including any clinical or laboratory test value abnormality of clinical significance which occurred after the start of the study, whether it was considered related to the study medication or not.
Determination of antibodies to whole-cell extract of P. aeruginosa and outer membrane proteins (OprF and OprI). Enzyme-linked immunosorbant serum assays (ELISAs) were developed and validated for measuring antibody responses to whole-cell P. aeruginosa extract and outer membrane proteins OprF and OprI. Microtiter plates were coated with 100 μl of a 10-μg/ml solution of either soluble whole-cell P. aeruginosa extract or recombinant His-tagged OprF or OprI in 50 mM sodium bicarbonate buffer at pH 9.6. Plates were then blocked with 0.1% (vol/vol) Tween 20 in phosphate-buffered saline (PBS) at pH 7.0. Serum samples were diluted at 1:50 for immunoglobulin A (IgA), 1:200 for IgM, and 1:2,000 for IgG. Triplicate samples were dispensed into microwells for incubation overnight at 2 to 8°C. After incubation, plates were washed twice in PBS containing 0.05% (vol/vol) Tween 80 and incubated for a further 1 h at 37°C with 100 μl of the appropriate peroxidase conjugated anti-human immunoglobulins diluted in PBS containing 0.05% (vol/vol) Tween 20 (1:10,000 for IgG and 1:2,000 for IgM and IgA). After being washed with PBS in 0.05% (vol/vol) Tween 80, the plates were developed by addition of 100 μl of tetramethyl benzidine chromogen for 10 min at room temperature (controlled at 22°C). Reactions were stopped by the addition of 50 μl of 25% (vol/vol) phosphoric acid, and absorbency values were measured at 450 nm with a microtiter plate spectrophotometer. Endpoint titers were calculated as the mean absorbencies against the baseline with coefficients of variations typically of <5% for all triplicate determinations and trend analysis at both dilutions.
Determination of antibodies to lipopolysaccharide. LPS was prepared from the P. aeruginosa vaccine strain by the method of Westphal and Jann (32). Microtiter plates were coated with 100 μl of 25-μg/ml LPS in 10 mM PBS at pH 7.4 containing 20 mM MgCl2 and incubated overnight at 37°C. The plates were then blocked with 200 μl of 10 mM PBS (pH 7.4) containing 5% (wt/vol) bovine serum albumin and 0.05% (vol/vol) Tween 20 for 15 min at 22°C. Plates were washed twice in PBS containing 0.05% (vol/vol) Tween 80, and 100 μl of pooled serum diluted in PBS (1:200 for IgA and IgM and 1:2,000 for IgG) was added to each well and incubated overnight at 4°C. After being washed in PBS containing 0.05% (vol/vol) Tween 80, the plates were incubated with the appropriate peroxidase-conjugated anti-human immunoglobulin (1:2,500 dilution with PBS containing 0.05% [vol/vol] Tween 20) for 1 h at 37°C and developed as above.
Measurement of total serum immunoglobulins. The total concentrations of nonspecific IgG, IgA, and IgM were determined for the serum and saliva samples of each individual by the use of a Beckman Array 360 nephelometer and standard reagents (Beckman Coulter, Inc., Fullerton, Calif.).
Detection of saliva antibody responses. Measurement of antibody responses was determined for saliva samples by essentially the same ELISAs as used above for the whole-cell P. aeruginosa extract, OprF, and OprI but with lower sample dilutions. Total salivary albumin was also measured by nephelometry.
Opsonization and phagocytosis assays. The sera of all subjects were pooled for each of the time points during the study and used to determine opsonophagocytosis of live P. aeruginosa 385 with a human monocytic cell line (THP-1). The THP-1 cell line (5 x 104) was seeded into tissue culture flasks and grown in RPMI-1640 medium containing 10% (vol/vol) fetal calf serum and 1(wt/vol) penicillin-streptomycin for 3 days. The cells were then activated to produce a more macrophage-like phenotype by the addition of 50-ng/ml myristyl phorbol ester for 24 h. After activation, nonadherent cells were removed by being washed, and the adherent cells were harvested with a cell scraper and suspended in Hanks balanced salt solution (HBSS). Bacteria were opsonized by combining 50 μl of bacteria (equivalent to 109/ml) to 20 μl of pooled serum for each of the time points and incubating for 30 min at 25°C. Cells were washed twice in PBS and made to 1 ml, of which 50 μl was added to 500 μl of activated THP-1 cells and again made to 1 ml with HBSS. The cells were incubated for 30 min at 37°C, washed in HBSS containing 250-μg/ml gentamicin sulfate, and further incubated for 10 min to kill adherent nonphagocytosed bacteria. Cells were then washed in PBS, and a 50-μl sample was removed and lysed in 450 μl of water. An additional 10-fold dilution of the sample was made, and 50 μl of this dilution was plated onto nutrient agar to determine the number of CFU. The remaining macrophages with phagocytosed P. aeruginosa were incubated for a further 60 min (total time, 90 min) and plated as above to determine the extent of killing of P. aeruginosa by the macrophages. Non-serum samples were used as negative controls, and a control sample of pooled serum from cystic fibrosis subjects known to be colonized with P. aeruginosa was also included to represent a high-titer sample to P. aeruginosa antigens. The assay was repeated for each time point on five occasions, and a mean value was determined for the results.
Statistical analysis. Serum and saliva-specific antibody levels as determined by ELISA absorbance at 450 nm were compared at each time point against day 0 by using a paired two-tailed t test. Statistical significance was defined as occurring when P values were <0.05. Salivary antibody data were also expressed as a ratio of salivary albumin levels to adjust for possible salivary flow rate differences between individual subjects. For the purpose of the immunogenicity study, responders were defined as those showing at least two or more postdose antibody titer increases with an increase of at least >15% above predose baseline levels.
Opsonization was extrapolated from colony counts immediately after phagocytosis. Bactericidal effect was measured 1 h postphagocytosis and was expressed as a percentage of difference in the final colony counts.
For the purpose of analyzing the serum IgM-specific antibody response to the whole-cell extract, one subject was removed as an outlier. For this subject, values determined on day 42 and 56 were three to six times the group mean. Removal of this subject from the IgM analysis did not alter the statistical significance observed for the group mean.
No significant differences were observed for any of the parameters determined between day –14 and day 0; therefore, day 0 was taken as the baseline measure throughout.
RESULTS
Safety. Thirty subjects were recruited as eligible for the study, of which 28 subjects received the first dose and 24 subjects completed the study to day 56. Subjects were removed from the study if they received routine or prohibited medications, did not receive both doses, or did not attend key visits. Twenty-one subjects were male (70%) and nine subjects were female (30%). The mean age ± standard deviation was 23.1 ± 4.2 years, with a range of 18 years to 35 years of age, and the mean weight ± standard deviation was 69.9 ± 12.0 kg. Pseudostat was well tolerated by all subjects, although 14 subjects reported 20 adverse events during the study, none of which were considered to be possibly or probably related to the use of Pseudostat. Adverse events were grouped by body systems; the preferred terms followed the World Health Organization classification system. These identified 2 individuals with musculoskeletal system disorders, 2 individuals with central nervous system disorders (headache and convulsion), 3 individuals with gastrointestinal disorders (diarrhea, dyspepsia, and vomiting), and 13 individuals with respiratory system disorders (pharyngitis, rhinitis, and upper respiratory tract infection). Only one adverse event was considered clinically significant, although not related to the study drug. Two reported adverse events (gastritis and upper respiratory tract infection) occurred prior to treatment; among the posttreatment adverse events, twice the number of upper respiratory tract infections followed the second dose versus the first dose. There was no close temporal relationship to either day of dosing or any consistency between adverse event reports with regard to time of occurrence (ranging from 9 to 21 days after day 0 and 4 to 22 days after the day 28 dose). This was also true of other adverse event reports except for the reported headache, which occurred 1 day after the initial dose. One serious adverse event involved a subject who had an epileptic seizure (described as being of moderate severity) and was hospitalized overnight for treatment. This subject was subsequently found to have had a seizure some months previously, which had been not reported to the investigators, and a family history of epilepsy. No abnormal hematological or biochemical test results of clinical significance were found at screening or at the day 56 follow-up visit for any subjects. There were no clinically significant changes in vital signs observed throughout the course of the study. All patients felt well at the completion of the study.
Total serum immunoglobulins. Total serum immunoglobulins showed no statistically significant changes or trends throughout the course of the study. Values for IgA and IgM are presented in Table 1.
Specific antibody responses to whole-cell P. aeruginosa. Serum whole-cell P. aeruginosa extract-specific IgA increased by day 14 after the first dose, and remained above the day 0 level for the remainder of the observation period (Table 1). The group mean showed a statistically significant increase in P. aeruginosa-specific IgA titers compared to day 0 for day 14 (P= 0.009), day 28 (P = 0.017), and day 42 (P = 0.010) but failed to reach significance on day 56 (P = 0.053). Whole-cell P. aeruginosa extract-specific IgM antibody titers also reached a statistically significant group mean increase from baseline atday 14 (P = 0.019) (Table 1). No statistically significant changes were observed for whole-cell P. aeruginosa extract-specific serum group mean IgG levels (data not shown).
Of the subjects tested, 39 to 65% were positive responders for all antibody classes and proteins tested. The least number of responders was to OprI at 39% and 48% for IgA and IgM, respectively. All other categories showed >50% responders; however, for the purpose of this analysis, responders were defined as those showing at least two or more postdose antibody titer increases with a least one increase of >15% above the predose baseline levels. Hence, of the 24 volunteers who completed the study, 29% were considered IgA responders, 25% were considered IgM responders, and 33% IgG were considered responders against whole-cell P. aeruginosa extracts (Table 2).
There were no significant changes in the levels of P. aeruginosa-specific IgA salivary antibody levels following immunization. Salivary IgG antibodies against whole-cell P. aeruginosa extract demonstrated an increase from baseline levels at day 14, which waned by day 42 before a more significant second increase at day 56 (P = 0.027) (data not shown). No statistically significant changes were observed for either salivary IgA or IgG antibodies against any of the P. aeruginosa antigens tested when the antibody data were expressed as a ratio of the salivary albumin levels. Salivary IgM-specific antibodies were not measured.
Outer membrane protein (OprF and OprI)-specific IgA responses. No significant group mean IgA, IgM, or IgG serum responses were observed for either OprF or OprI. Of the 24 volunteers, 46% were OprF-specific IgA responders, and 33% were OprI-specific IgG responders. To a lesser degree and in decreasing order, the responders were as follows: OprF-specific IgM responders (29%), OprI-specific IgA responders (21%), OprI-specific IgM responders (17%), and OprF-specific IgG responders (4%) (Table 2).
LPS. LPS-specific serum IgA titers increased significantly (P< 0.05) from baseline levels on day 14, day 28, day 42, and day 56 with maximum increases occurring 28 days post-first dose (Fig. 1). LPS-specific serum IgM titers showed no significant change from baseline on day 14; however, there were statistically significant (P < 0.05) decreases in responses for days 28, 42, and 56. LPS-specific serum IgG titers showed a pattern similar to that of LPS-specific serum IgA, compared to day 0, achieving statistically significant increases (P < 0.05) on all days postdose except day 14 (Fig.1C).
Opsonophagocytosis assays. The number of colonies grown after opsonization and phagocytosis by activated THP-1 monocytes in the presence of pooled serum from Pseudostat-vaccinated individuals is represented in Fig. 2. The results show an increase (34 to 62%) in the number of colonies compared to day 0, with the highest macrophage capture of live P. aeruginosa cells in the presence of CF sera, which was used as a control. After a further 60-min incubation period, the percentage of opsonized colonies killed ranged from 45 to 82%, compared to 40% on day 0 (Fig. 3). Killing of bacteria was particularly enhanced on day 14 (82%), at which maximal anti-P. aeruginosa IgA responses in serum samples were also observed. After day 14, the levels dropped progressively but were still maintained above baseline values.
DISCUSSION
Studies in a rodent model of acute infection have demonstrated that mucosal immunization with a whole-cell killed P. aeruginosa vaccine results in enhanced clearance of the bacteria from the lung, as well as reduced mortality (2, 7). In a preliminary study of nine bronchiectasis patients, oral immunization with an enteric coated whole-cell killed P. aeruginosa vaccine resulted in the detection of circulating antigen-reactive peripheral blood leukocytes, as well as a significant reduction in the levels of P. aeruginosa in the sputum (8). Further evidence to support the development of a whole-cell killed vaccine and an oral immunization strategy for P. aeruginosa comes from previous studies of nontypeable Haemophilus influenzae (NTHi), where patients with recurrent acute exacerbations of chronic bronchitis were orally immunized with a whole-cell killed NTHi vaccine (4, 5, 6, 20, 28). A recent Cochrane review of six NTHi trials of 440 subjects reported that oral immunization significantly reduced the number and severity of acute exacerbations (13). In those studies, bacterial load was reduced, as determined by the incidence of throat colonization and/or quantitative or semiquantitative bacteriology of sputum samples. In addition, NTHi-specific cellular responses were detected in peripheral blood lymphocytes, following oral immunization with whole-cell killed NTHi vaccine.
This study represents a clinical pilot study designed to demonstrate the immunogenicity and safety of an oral vaccine against P. aeruginosa in healthy volunteers. The dose chosen was the lowest dose expected to induce an immune response in humans, based on a scaled-up dose from successful dose-ranging studies in rodents. Future placebo-controlled studies will determine efficacy and optimal dose and treatment regimes with human subjects.
Safety of the vaccine in healthy individuals was confirmed in the absence of any probable or possible adverse events, and no hematological or biochemical profile derangements were recorded for any subjects throughout the study. While 20 adverse events were recorded, these were not considered of clinical significance (the exception being one case of epilepsy missed on initial screening), and none were attributable to the study vaccine being administered. Upper respiratory tract infection represented 60% of the adverse events reported with twice the number of upper respiratory tract infections following the second dose compared to the first dose. However, there was no temporal relationship to either the day of dosing or in the time of occurrence.
The vaccine was demonstrated to be immunogenic. Of particular note is the significant increase in IgA-specific antibody responses against both whole-cell extract and LPS in the post-immunization observation period. Confidence in the consistency and specificity of these changes was further supported by comparison with group mean total serum IgA, which did not follow a similar trend. A sustained-specific IgG LPS antibody response was also observed postimmunization. The opsonization results clearly showed a substantial increase in ability of pooled sera from the study volunteers to promote phagocytosis of P. aeruginosa by a human macrophage cell line. The results confirm the specificity of the response observed and that antibodies induced were opsonizing for P. aeruginosa. Opsonizing antibody responses have been shown to be important for protection against P. aeruginosa infection in CF patients (24, 30). Hostoffer et al. (15) reported on the importance of IgA receptors (Fc alpha R) on mucosal phagocytes and suggested an important role in defense of mucosal surfaces. They demonstrated high levels of expression of Fc alpha R on neutrophils obtained from bronchoalveolar lavage fluid of cystic fibrosis patients and showed that neutrophil superoxide production was enhanced by IgA. Significant IgA responses following pneumococcal polysaccharide vaccination showed that the IgA induced a comparable receptor-mediated phagocytosis response, as did IgG, and that the IgA holds an important leukocyte receptor which provides immunity against Streptococcus pneumoniae (25). In this study, both opsonization and killing peaked at day 14 post-first immunization; although there was a decline in functional activity after this observation point, functional activity remained greater than the preimmunization levels. The two booster immunizations did not enhance either opsinization or killing. Further studies are required to investigate the kinetics of the functional antibody data, particularly in response to booster immunization.
The overall salivary immune responses did not reach statistical significance and were variable at each time point of the study. These observations probably reflect the inherent difficulties of measuring antibody responses in mucosal secretions, particularly following oral immunization. Despite the absence of a detectable and consistent salivary antibody response to the vaccine, this does not preclude the possibility of a mucosal immune response in the lungs or on other mucosal surfaces. If memory T cells and B cells are migrating to other mucosal surfaces from the gut, a measurable response may not be observed until booster encounter with antigen at these surfaces. IgA can reach external secretions by passive paracellular diffusion and may have a role in local defense on mucosal surfaces in addition to locally produced IgA antibodies. Furthermore, leakage of serum IgA onto mucosal surfaces is suspected to increase in inflammatory situations such as those that exist in the CF lung (22). In the context of the results of this study, Berstad et al. (1) noted an absence of salivary antibody response to a whole-cell pertussis vaccine administered nasally to human volunteers, despite strong serum antibody responses for IgA and IgG. Recently, a hybrid OprF-OprI P. aeruginosa vaccine was tested with human volunteers by intranasal application, which resulted in serum IgA and IgG responses in 75% of the volunteers (19). Although saliva and nasal washings were taken for analysis in that study, the results were not reported, implying that no responses may have been detected. Studies of oral whole-cell vaccination with an animal model have shown that protection against P. aeruginosa challenge can be observed in the absence of any significant serum or bronchoalveolar lavage specimen antibody response (7). These findings are also consistent with results observed for whole-cell killed NTHi oral immunization of subjects with chronic bronchitis, which did not stimulate a specific antibody response in saliva, although the vaccine was highly efficacious (6). Dose-ranging studies of mice (M. Dunkley, unpublished data) have shown that enhanced clearance at the lowest dose was achieved in the absence of any detectable serum antibody response. Increasing the dose by 1 log, which is equivalent to the single doses given in this study (2.6 mg/kg versus 2.1 mg/kg), resulted in better clearance in the mouse and the detection of statistically significant increases in serum IgA and IgM, as was indeed observed in this trial. These results may suggest an important role for serum IgA in the protection against P. aeruginosa lung infections.
Studies of this vaccine with an animal model have indicated that both antibody and T cells are important in protecting rodents from an acute lethal challenge of P. aeruginosa to the lungs (2, 7, 10, 11). However, some evidence points to a detrimental effect, at least in chronically colonized CF patients where increasing serum IgG antibody levels to P. aeruginosa LPS and exotoxin A and the resulting immune complexes formed have been associated with a poor prognosis (21). Kronborg et al. (17) showed that in sputum, immune complexes were almost entirely composed of LPS-specific IgG. Studies have shown that the sera of colonized patients have high titers of poorly opsonizing antibodies (26) and that the opsonizing ability of sera decreases upon conversion to the mucoid form and the onset of chronic colonization (29, 30). Of importance, the results from this study clearly demonstrate that the antibody response induced was opsonogenic. The specificity of opsonizing antibodies and their protective role requires further study (12, 23, 24).
In a recent review (27), we concluded that mucosal immunization was a potential viable option for protection against P. aeruginosa infections in at-risk patients. This study has demonstrated that oral immunization against P. aeruginosa is safe and induces a significant serum antibody response, notably of the IgA isotype. In addition, an increase in functional opsonization and killing by macrophages was demonstrated in vitro with postvaccination serum. Proof of concept with human subjects, based on the extensive preclinical studies with animal models, has been established. Further studies will involve dose-ranging studies and characterization of the immune response induced with respect to serotype specificity, cell-mediated immune mechanisms, the identification of the functional opsonins and their antigenic specificity, and the kinetics of the response.
ACKNOWLEDGMENTS
This study was supported by Provalis PLC, Deeside, Flintshire, United Kingdom.
Thanks are extended to Shrikant Jondhale for help in developing the phagocytosis assay; Sharon Griffiths, Helen Rawden, and Jane Evans for assistance in developing the ELISAs; Mark Smith and Suzanne Thomson for cloning and purifying the OprF and OprI; and Pamela Riches for advice given and discussions.
Present address: Teva Pharmaceuticals, Ltd., Aylesbury, Bucks, United Kingdom.
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