Safety and immunogenicity of a Pseudomonas aeruginosa outer membrane protein I vaccine in human volunteers

Elsevier PII: SO264-4lOX(96)00054-0

ELSEVIER

L’accine, Vol. 14, No. 12, pp. 1111-1117, 1996 Copyright 0 1996 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0264-41 OX/96 $15+0.00

Safety and immunogenicity of a Pseudomonas aeruginosa outer membrane protein I vaccine in human volunteers Bernd-Ulrich von Specht*§, Hanns Christian Licking*, Barbara Anja Schmitt *, Klaus Dieter Hungerert and Horst Domdey’f

Blum?,

The outer membrane protein I (OprI) of Pseudomonas aeruginosa was expressed in Escherichia coli and purtjied by Ni’+ chelate-afJinit_v chromatography. After safety and pyrogenicity evaluation in animals, four groups of seven adult human volunteers were vaccinated three times at four week intervals with either 500 ,ug, 200 ,ug, 50 pg or 20 ,ug of Oprl adsorbed onto Al( OH),. All vaccinations were well tolerated and no systemic side eflects were detected. A sigmJicant rise of antibody titers against OprI could be measured in the serum of all volunteers who had received the 500 ,ug, 200 ,ug or 50 ,ug doses. Elevated untibod_y titers against OprI could still be measured 30 Mfeeksafter the final vaccination. Binding of the complement component Clq to the elicited antibodies could be demonstrated, showing the ability of the latter to promote antibody-mediated complement-dependent opsonization. Copyright 0 1996 Elsevier Science Ltd. Keywords: P. uwuginosu;

outer

membrane

protein;

vaccine;

Clq

INTRODUCTION P. aeruginosa is a common cause of nosocomial infection in hospitals’. The pathogen particularly affects immunocompromised patients, such as cancer or transplant patients undergoing cytostatic or immunosuppressive therapy’,3. However, the risk for patients suffering from trauma such as severe burns, or after major surgery, is also greatly increasedh6. Antibiotic treatment is frequently unsuccessful, because of the resistance of P. aeruginmu to many antibiotics7s. For this reason research has been concentrated on immunotherapy’,“. The two major antigenic surface-associated components of P. aeruginosa are the lipopolysaccharides (LPS) and the outer membrane proteins (OPRs). Vaccine preparations based on P. aeruginosa LPS itself’ ’ I3 have not met with approval for clinical routine use, because of the toxicity caused by the lipid A portion of the LPS. Subunit vaccines based on oligosaccharides purified from LPS conjugated to P. aeruginosa exotoxin or mucoid exopolysaccharide (alginate) of P.

*Chirurgische Universitatsklinik, Chirurgische Forschung, Hugstetterstrasse 55, D-79106 Freiburg im Breisgau, Germany. -fGenzentrum der LMU Mtinchen, Wurmtalstrasse 221, D-81 375 Mtinchen,Germany. $Forschungslaboratorien der Behringwerke AG, Postfach 1140, D-35001 Marburg, Germany. $To whom correspondence should be addressed. (Received 11 November 1995; revised 5 February 1996; accepted 27 February 1996)

ueruginosn’4m’8 were shown to be less toxic, and have been used successfully to elicit antibodies in a number of volunteers and patient groups. We have directed our attention towards cloning and the expression of P. aeruginosa OPR genes, and have been interested in the potential of OPRs as immunoprophylactic tools, because OPRs are antigenically cross-reactive among all 17 known sero roups of the International Antigenic Typing Scheme’ B.20. We have cloned the genes coding for OprF” and OprI’“, and have successfully used recombinant OPRs expressed in Escherichia coli for vaccinating mice”-z5. A vaccine based on outer membrane proteins of P. aeruginosa could have several advantages. In addition to the observation that OPRs induce a cross-reactive immunity, a successful defence against the pathogen may require the induction of an immune response at specific sites, such as the mucosa of the intestine or lung16. Furthermore, directing the immune response towards a certain antibody class such as secretory IgA (s-IgA), or inducing a T cell-dependent immune response, have been regarded as ways of achieving protective immunity in cases of chronic lung colonization in patients with cystic fibrosis*‘. We have recently been able to show in experimental animal models that antibodies of the class s-IgA and a T-helper class l(T 1) specific immune response can be elicited against OprI 3 8.29.Cloned genes coding for OPRs would also be relevant for a recent approach in the development of vaccines which employs immunization with “naked DNA”30.3’. This method of immunization

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vaccine in humans: B.-U. von Specht et al.

seems to have, amongst others, the important advantage of being able to elicit both humoral and cellular immune responses. We have recently published a fast and simple procedure for the production and purification of recombinant OprI from E. COZIES. The phase one clinical trial study presented here provides evidence that OprI isolated by this method can be used to vaccinate healthy human subjects against P. aeruginosa without any apparent side effects.

MATERIALS

AND

METHODS

OprI preparation Recombinant OprI was prepared from E. cob and purified by metal chelate-affinity chromatography, as recently described in detai129. After elution from Ni2‘ NTA (nitrilo-tri-acetic acid: Diagen, Diisseldorf, Germany) with 250 mM imidazole buffer, the protein was extensively dialysed against distilled water, using a 3500 molecular weight cut-off dialysis tube (Reichelt, Heidelberg, Germany), and lyophilized. Fractions were pooled, dissolved in pyrogen-free sterile physiological saline solution at a concentration of 2000 ,ug ml-’ and passed through a 0.22 pm filter (Millipore, Molsheim, France).

Vaccine preparation Recombinant OprI was adsorbed to AI(O and Thimerosal (Caesar and Lorenz, Hilden, Germany) added as a preservative. A Thimerosal stock solution was prepared, using a sterile, pyro en-free physiological saline solution. For the 1 mg ml- Bvaccine preparation, a dispersion of 3% (w/v) of Al(OH), (AlhydrogelR, Superfos, Vedbaek, Denmark) was mixed with the OprI solution and the Thimerosal stock solution to yield final concentrations of OprI: 1 mg ml- ‘, Al(OH),: 3 mg ml- ’ and Thimerosal: 0.05 mg ml- ‘. Al(OH), and the OprI solution were mixed and stirred for 30 min, and the Thimerosal solution was then added. This was followed by additional stirring for 10 min. For the 0.1 mg ml-’ OprI vaccine preparation, pyrogen-free physiological saline solution was added to yield final concentrations of 0.1 mg ml-’ OprI, 0.3 mg ml-‘AI( and 0.05 mg ml-’ Thimerosal. Aliquots of 1 ml were aseptically introduced into sterile pyrogen-free glass vials, and the vials stoppered and sealed.

Quality assessment 10% of the samples of both vaccine preparations were sent for routine testing for sterility. Five samples each of the 1000 pg ml-’ preparation were analyzed at the Chemische Landesuntersuchungsanstalt Freiburg, Germany, by atom adsorption spectrometry for the content of nickel, mercury and aluminum. The amount of contaminating DNA from one of the samples of the 1000 ,ug preparation was analyzed after extraction with chloropane and PCR amplification. The pyrogenicity of the OprI preparation was evaluated in three samples, each taken before the addition of Al(OH), and thimerosal. One ml of OprI solution, diluted to 1 mg ml- ‘, was injected into the ear

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veins of three white New Zealand rabbits. The body temperature of the animals was continuously monitored for 3 h by a rectal temperature recorder. Local tolerance of the vaccine was assessed in Wistar rats. Eight rats (250 g, four male, four female) were anesthetized with ether and each animal given an injection of 0.25 ml of vaccine (1 mg ml-‘) into the left and 0.5 ml into the right rectus femoris. Four control animals received injections of equal volumes of sterile saline solution. The injection sites were shaved and disinfected beforehand. After 13 days the animals were killed by CO2 inhalation, and after macroscopic inspection both quadriceps were fixed in formalin and sent for histological examination. Vaccination study Twenty-eight healthy volunteers (male; 18 years of age) gave informed consent in accordance with institutional review board approved protocols. As laid down by the German regulations for the conduct of vaccination studies, the protocols concerning the preparation, laboratory and animal safety testing of the vaccine were deposited at the Paul Ehrlich Institute, Langen, Germany. Volunteers were randomly allotted to 4 groups, and each received three injections of vaccine into the deltoid muscle of the left arm of either 20 pg (0.2 ml of 100 g ml-‘), 50 pg (0.5 ml of 100 pug ml-‘), 200 pug (0.2 ml of 1000 pg ml-‘) or 500 pg (0.5 ml of 1000 pug ml- ‘) OprI at 4 week intervals. All volunteers underwent physical examination and had histories taken to rule out any conditions which would have necessitated exclusion. Before, and two and 14 days after each vaccination, blood samples were taken and sent to the clinical laboratory for a complete blood count and evaluation of the liver specific enzymes, creatinine and urea. Reactions to the vaccine were assessed for 5 consecutive days and documented by the volunteers. The local and systemic responses were graded on a subjective scale of 0 to 3, with the respective scores representing absent, mild, moderate and severe reactions. Vaccinees were instructed to take their temperature before and 12, 24, 48 and 72 h after vaccination. In addition, each volunteer underwent a physical examination two days after vaccination. For the determination of OprI specific antibodies, venous blood samples were taken on days 0 (prior to immunization), 14, 42 and 70, and 6, 15 and 30 weeks after the final immunization. Serum was stored in aliquots at -20°C. Analysis of immune response The antibody response against OprI was determined by an enzyme-linked immunosorbent assay (ELISA) as previously described in detai123. Briefly, 96-well round bottom plates (Falcon, Microtest III, Becton Dickinson, Oxnard, USA) were coated overnight at 4°C with 50 ~1 of OprI solution (1~ g ml - ’ in phosphate buffered saline containing 0.05% sodium azide). The plates were washed and the remaining binding sites blocked with 0.05% Tween 20 (Serva, Heidelberg, Germany) and 0.25% gelantin (Merck, Darmstadt, Germany) in borate buffered saline (pH 8.5) for 1 h at 37°C. Aliquots (50~1) of serial 1:2 dilutions of serum were added in duplicates. Serial dilutions of a serum with a high antibody titer

P. aeruginosa

against OprI (obtained from a patient suffering from cystic fibrosis and with a long history of colonization by P. aeruginosa) were added as a standard. Binding was visuaiized with peroxidase-conjugated isotype-specific rabbit anti-human secondary antibodies (DAKO, Hamburg,Germany) diluted 1:8000 in dilution buffer [0.05X Tween 20, 2% bovine serum albumin (USB, Cleveland, USA) in PBS] and ortho-phenylendiamine (OPD) (Sigma, Deisenhofen, Germany) as a substrate. The specific antibody content against OprI is expressed in ELISA units (EU). The absorbance of the standard serum measured at 492 nm at a I:512 dilution (which was in the linear range in all tests) was defined as 100 EU. The absorbance of the volunteers’ sera measured at a I:128 dilution was compared with this value and the EU calculated. Seroconversion was defined as an increase of calculated EU, exceeding 2-fold the EU calculated for preimmunization serum. Clq binding was assayed as previously described32. Briefly, microtiter plates were coated with recombinant OprI as described above. After blocking nonspecific binding sites, 50~1 of heat-inactivated diluted serum and 50 ~1 of complement source were added. Human AB serum from an unvaccinated blood donor tested for a low titer of OprI antibodies was used as the complement source, the optimal concentrations of complement source and serum dilution having been evaluated beforehand by serial dilutions. After incubation for 1 h at 37”C, the plates were washed and 50~1 of diluted peroxidase-linked anti Clq antibodies added. Binding was visualized as described above with OPD as a substrate. Ig class and IgG subclass distribution was analyzed with monospecific peroxydase-conjugated sheep antibodies against human IgA (u-chain), IgM b-chain), diluted 1:6000, IgG (a-chain) diluted I:8000 or antibodies against IgG,, IgG,, IgG,, or IgG,, diluted 1:200 (The Binding Site, Birmingham, UK) Western blot analysis of recombinant and native P. aeruginosa OyrI with the OprI specific monoclonal antibody 2A1 22.3 and with sera of vaccinated volunteers was carried out as described24.

OPR vaccine in humans: B.-U. von Specht et al.

106 000 80 000 49 500

The Wilcoxon signed-rank test was used for intragroup comparison of immune responses. The MannWhitney U test was used for intergroup comparison of immune responses. A P value co.05 was considered to be significant.

RESULTS Purity, safety and pyrogenicity vaccine

testing of the OprI

Recombinant OprI-(His), fusion protein was expressed in E. coli and purified by Ni +-chelate affinity chromatography. From one liter of IPTG-induced E. coli culture, 30 mg of OprI with a purity of 99”/0was isolated, as estimated after SDS-PAGE separation by silver staining (Figure 1, left side). Figure I shows that under non-reducing conditions (lanes 1 and 3) OprI (calculated M,=8 kDa) migrates as a trimer, whereas under reducing conditions the main fraction of the protein migrates in the monomeric form (lanes 2 and 4).

49 500

32 500

32 500

27 500

27 500 (> 18 500

18 500 1=G

1

2

3

M

4

M

Figure 1 SDS polyacrylamide gel electrophoresis of recombinant Oprl-(His), expressed in 15.co/iand purified by Ni*’ chelate-affinity chromatography.Left side: silver staining. Right side: Western blot analysis using the Oprl-specific monoclonal antibody 2 A122,24. Lanes 1 and 3: separation under non-reducing conditions; lanes 2 and 4: separation under reducing conditions. M: Low molecular weight marker kid (BioRad, Munich, Germany)

The nickel content was measured with 150 ng per 1000 pug of OprI. The content of mercury (0.0242 mg ml-‘) and aluminum (1.03 mg ml-‘) was calculated to correspond to the amounts added to the vaccine. All samples assessed for sterility were found to be negative with respect to microbial growth. After i.v. injection of 1000 ,ug (1 ml) of OprI solution into three rabbits, no signs of a pyrogenic reaction (rise in body temperature) was observed. Local tolerance of the vaccine was evaluated in rats after intramuscular injection of 0.25 ml and 0.5 ml of vaccine (1OOOpgml- ’ OprI). Histological examination of the injection sites showed signs of inflammation with focal infiltration of lymphocytes due to the alum adjuvant. Response to vaccination

Statistics

106 000 80 000

in human volunteers

Two of the 28 vaccinated volunteers complained of a mild pain at the injection site which disappeared after three days. No local side effects such as an inflammatory response could be recognized. Systemic reactions such as a rise in body temperature, headache or general illness were not observed to follow any vaccination. During the safety clinical laboratory evaluations, none of the observed alterations in any of the volunteers could be attributed to the vaccination. In accordance with the protocol, two volunteers had to be excluded from the study. One suffered from a viral infection which started in the second week after the first vaccination, and the other developed a swelling of the inguinal lymph nodes two weeks after the second vaccination. This was attributed to an infection superimposed upon an allergic eczema of his left foot, and was treated successfully with antibiotics. No sensitization to any of the vaccine components could be detected with epicutaneous testing. Antibody induction in vaccinated volunteers

Before vaccination, serum antibody titers against OprI below 5 EU were measured in 20/26 volunteers.

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P. aeruginosa Table 1

OPR vaccine in humans: B.-U. von Specht et al.

Mean antibody

level (ELISA units)+S.D.

(range)

Group

Dose 019)

Day 0

Day 14

Day 42

Day 70

Week 16

Week 25

Week 40

1

20

2

50 200

4

500

21.6k29.3 (0.4-85.1) 9.3k7.5 (2.2-20.6) 15.1k12.4 (1.2-40.8) 22.4*23.9 (2.7-70.6)

21.8k28.6 (0.4-88) 12.3k11.8 (2.5-34.8) 26k20.2 (3.9-61) 27.4i21.8 (4.7-60.4)

26.9*29.3 (0.9-90.8) 23.8i22.4

3

8.7k12.9 (0.4-39.1) 3.2k2.6 (1 .O-8.5) 2.6k2.7 (0.5-8.8) 4.2k3.8 (0.6-12.1)

26.8229.1 (0.4-86.8) 19.Oi18.9 (4.5-54.6) 38.4k37.4 (2.6-90.7) 27.5k21.2 (6.5-60)

24.3k28.3 (0.3-85.7) 13.4i11.8 (3.2-32.5) 33.2*32 (2.4-81.4) 21.6kl8.6 (2.1-51.8)

20.8i25.1 (0.3-75.4) 9.6k7.6 (1.3-21.1) 20.7i24.4 (0.7-72.2) 18.3kl7.8 (0.4-47)

I

60

s

40

3 w

20

(4.;-100.1) 38i25.5 (9.8-77.2)

In order to determine how long the antibody response against P. aeruginosa persists in the serum after the final immunization, serum antibody titers against OprI were measured in the volunteers 16,25 and 40 weeks after the first immunization. The titers measured in samples taken 40 weeks after the first immunization (32 weeks after the final revaccination) were found to be still significantly (PcO.05) above the preimmunization values.

80

g

!9_4$F)

0 1

3

5

7

0 10 13

16 18 20

23 26 28

VOLUNTEER Figure 2 Oprl specific antibody response of volunteers before 0 and Q after three vaccinations at four week intervals with recombinant Oprl. Antibody titers are expressed in ELBA units in comparison with a serum (100 EU) obtained from a case of cystic fibrosis with a long history of colonization by P aeruginosa. Volunteers No 11 and 24 were excluded from the trial in accordance with the protocol1 due to intercurrent disease.Volunteers l-7: 20~9 Oprl; 8-14: 50pg Oprl; 15-21: 200 pg Oprl; 22-28: 500 pg Oprl

Western blot analysis Figure 3 shows the Western blot analyses of a preimmunization serum sample (lane l), and a serum sample (lane 2) obtained from volunteer No. 3 on day 70 after the third vaccination with 20 ,ug of OprI against SDS PAGE separated bands of P. aeruginosa serogroup 1 (Figure 3”a), and against recombinant OprI 0. Only with the immune serum sample could the 8 kDa OprI band be visualized. Identical results were obtained with serum samples taken from volunteers immunized with higher doses of the vaccine.

4126 showed titers below 10 EU and 2/26 titers above

10 EU. All volunteers received three doses of either 20 yg, 50 yg, 200 ,ug or 500 yg of recombinant OprI at 28 day intervals. The antibody level against OprI in the serum before and two weeks after each immunization-and 16, 25 and 40 weeks after the first immunization-was measured by ELISA. and the ELISA Units (EU) calculated. Table I summarizes the ELISA values for each dosage group; namely, the mean value. the standard deviation and the range of the antibody levels measured. Within each group the first immunization with OprI led to a significant increase in the mean antibody levels (PcO.05). After each revaccination, an increase in the mean antibody levels could be measured; this increase was, however, only significant for the second revaccination in groups 1, 2 and 4 (PcO.05). These findings mean that, for all the different dosage groups investigated, a significant increase in the mean antibody level (i.e. above the preimmunization level) was measured after three immunizations. Analysis of the individual antibody response against OprI showed considerable variations within the different dosage groups, as seen in Figure 2. Whereas 417 volunteers vaccinated with the 20 ,ug dose did not respond to immunization with OprI (less than a twofold increase in EU), seroconversion was observed in all volunteers vaccinated three times with the 50 pg. 200 ,ug or 500 ,ug doses. However, in some of the vaccinees of these groups, only low responses against OprI could be elicited.

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OprI specific antibody class and subclass distribution Serum samples from volunteers after receiving each of the four different dose levels, in which a high rise of OprI specific antibody titers following vaccination was measured, were further analyzed in relation to the antibody class and subclass distribution. An increase in IgG and IgA specific antibody titers was measured, but we could not detect any preferential increase in one particular IgG subclass. The individual subclass responses also varied considerably between volunteers (data not shown). Clq binding assay In order to determine whether the antibody response induced by OprI vaccination could promote antibody-mediated complement-dependent opsonization, the Clq-binding capacity of two antisera from each of the different OprI dosage groups with the highest increase of OprI specific antibody titers following vaccination was measured, and analyzed by a Clq binding ELISA”‘. Figure 4 shows the Clq-binding capacity of serum from volunteers 3 and 5 (20 pugOprI), 10 and 13 (50 yg OprI), 17 and 18 (200 ,ug OprI) and 27 and 28 (500 pg OprI) before and after the third vaccination. Whereas no significant Clq binding could be detected before vaccination, the binding of Clq by the induced OprI specific antibodies could be measured after vaccination in the immune serum of all volunteers tested.

P. aeruginosa OPR vaccine in humans: B.-U. von Specht et al.

(a)

3

5

10

13 17 VOLUNTEER

18

27

28

Figure 4 Clq binding analysis of preimmune serum Band Oaf serum obtained after three vaccinationsTwo specimens of serum from each dosage group in which the highest increase of Oprl specific antibody titers had been measured were further analyzed for ClQ binding. Because sufficient serum was not available from volunteer No 19, that of volunteer 18 was analyzed.Plates were coated with Oprl, incubated with 50 ~1 of the serum (diluted 1:4) and 50 ~1 of complement source. Binding of Clq was measured with peroxidase-linked anti Clq antibodies

1

4 Ho. 3

w

Figure 3 Western blot analysis of antibodies raised after vaccination with 20 pg Oprl (volunteer No. 3) against P. aeruginosa serogroup 1 (figure 3a) or against recombinant Oprl (Figure 3b). Lane 1: preimmune serum. Lane 4: serum obtained after the third vaccination. M: Low molecular weight marker kid (BioRad, Munich, Germany)

DISCUSSION Conditions such as cystic fibrosis, neutropenia, major burns and paraplegia, or even the the continued use of extended-wear contact lenses, may result in an increased morbidity rate from P. aeruginosa infection1,4.5333-38.In spite of therapeutic advances, the mortality rates from P. aeruginosa bacteremia and pneumonia have remained unchanged over the past two decades’. Because the majority of affected patients show a local or systemic

impairment of the body’s own defence systems, immunotherapy seems to be a promising tool. Most of the vaccines that have so far been evaluated in humans were based on anti enic determinants expressed on the LPS 0 side chain”- $’ . The finding that a limited number (10) of serogroups of P. aeruginosa account for 90% of clinical isolates has led to the development of vaccines composed either of conjugates of 0 side chains purified from those serogroups and exotoxin Al4 or of high molecular weight polysaccharide versions of the 0 side chains16. However, the identification of subtype epitopes within serogroups has raised the questions (1) whether the current oligovalent vaccine formulation contains sufficient components to provoke human antibodies reactive to the majority of clinical strains of P. aeruginosa, and (2) to what extent subtype-specific antibodies are needed for protection39. In addition to this, clinical isolates of P. urruginosu have been identified which do not fit serologically into the 17 known ATCC strains (A. Bauernfeind, Max von Pettenkofer Institute, University of Munich, personal communication). It has, however, also been suggested that the failure of 0 side-chain based vaccines to prevent lung colonization in cystic fibrosis may be due to insufficient stimulation of the lung macrophages b cytokines secreted by the vaccine-specific T,l cells 27. Already in the early eighties, the use of outer membrane proteins as a vaccine against P. aeruginoscz was suggested by Mutharia and co-workers”. The work of several groups including our own has provided evidence that OPRs can be used for the induction of a cross-protective immunity in animal models. OPRs are highly conserved among all serogroups of P. aeruginosa. After sequencing the OprI gene from all 17 ATCC strains, only one single base exchange in one serogroup was observed which did not lead to an amino-acid exchange (H. Domdey: unpublished results). So far, however, no data have become available on the safety and immunogenicity of OPRs in humans, because considerable contamination by LPS was found in the classical purified OPR preparations. Using recombinant molecular biology techniques and metal chelate-affinity chromatography, we have been able to produce

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OPR vaccine in humans: B.-U. von Specht et al.

sufficient amounts of highly purified OprI for a vaccination phase one clinical trial in healthy volunteers. This technique was first described for the purification of clinical grade proteins by Takacs et a14’. As depicted in the results section, vaccination with OprI purified according to this protocol is well tolerated, with no side effects observed even after vaccination with the 500 ,ug dose. This is in contrast to the erythema and tenderness at the injection site, or fever and headache, seen after immunization with 0 side chain based vaccinesl6.is.41~42. Since no data on the immunogenicity of purified P. aeruginosa outer membrane proteins in humans had previously been evaluated, four different dose ranges (500 ,ug, 200 pug, 50 pg and 20 ,ug) were tested. With each of the different doses used, a significant increase of OprI-specific antibodies could be measured after only one vaccination. However, because of considerable individual variations in the antibody response and the rather small numbers of volunteers in each group, no significant difference between the doses applied could be demonstrated, although after the 20 pg dose 4/7 nonresponders were observed. Similar variations in the individual responses by human beings have been described after immunization with a mucoid exopolysaccharide (alginate) vaccine by Hatano and colleagues39, and after vaccination with the P. aeruginosn 0-polysaccharide-toxin A conjugate vaccine by Cryz

2

3

4

5

6

7 8

9 10 11

12

13

et aL4-.

The relevance of Clq binding to antibodies for prohas been demonstrated tection a ainst P. aeruginosa before32.4$. During previous investigations carried out on mice, we were able to show that the protectivity of monoclonal antibodies against P. aeruginosa outer membrane roteins can be predicted by the assay used in this study-3P. In conclusion; we have been able to demonstrate for the first time that a recombinant outer membrane protein of P. aeruginosu can be used safely on human beings for vaccination, eliciting an antibody response which is able to promote phagocytosis. We have recently cloned hybrid antigens of protective epitopes of P. aeruginosa outer membrane protein F and outer membrane protein I which showed a highly increased protectivity in mice in comparison with the single outer membrane protein?. Our demonstration that an outer membrane protein vaccine is extremely well tolerated in humans encourages us to continue with the development of an efficient expression and purification system for this hybrid vaccine, and to test its safety and immunogenicity in man.

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16

17

18

19

20

21

ACKNOWLEDGEMENTS This work was supported by Grant No. OlKI8910/4 from the Bundesministerium fur Forschung und Technologie to H. Domdey and B.-U. von Specht, and E/B41 G/L0407/L592 1 from the by Grant No. Bundesministerium fur Verteidigung to B.-U. von Specht. The authors wish to thank Dr B. Hein for carrying out the histological examinations. REFERENCES 1

Gallagher, P.G. and Watanakunakorn, Ch. Pseudomonas bacteremia in a community teaching hospital, 1980-1984. Rev. Infect. Dis. 1989, 11, 848-852

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