Clinico-immunological trials of Pseudomonas aeruginosa vaccine E.S. S t a n i s l a v s k y *~, S.S. B a l a y a n t, A l e x a n d r a I. S e r g i e n k o t, T a t j a n a A. M a k a r e n k o * , Liliya S. E d v a b n a y a * , M a r i n a A. K r o h i n a ~ and V.M. Rusanov ~ P s e u d o n o m a s aeruginosa vaccine (PV) containing predominantly cell-wall protein protective antigens was tested for safety and immunogenicity by immunization of 119 volunteers. The criteria for safety and immunogenicity were the absence of serious post-vaccinal reactions or complications either during immunization or 12 months later. There were mild (19 donors or 15.9%) and moderate (three donors or 2.5%) febrile reactions after immunization and in two volunteers the body temperature increased up to 38°C, however it decreased to normal values within 24 h. We observed in 43 (36.1%) of volunteers mild and in five (4.2%) moderate local reactions which disappeared within 24 h. Using the ELISA and passive mouse protection test it was shown that P V induces the formation of specific antibodies. A high level of specific antibodies persisted for the 5-month period of observation. The antibody titres increased in 94-97% of volunteers and moreover in 45.6% the antibody titres (the number of ELISA units) increased 2.5-3-fold and more. Anti-P. aeruginosa plasma was used for the treatment of 46 patients with severe forms of P. aeruginosa infection (40 adults and six infants aged up to 2 years) and 87% of the patients recovered. Keywords:Pseudomonas aeruginosa; vaccine; clinical trials
INTRODUCTION
MATERIALS AND METHODS
Pseudomonas aeruginosa infection is a serious problem for surgical and other patients 1-5. Hospital strains of P. aeruginosa are highly resistant to the majority of antibiotics 2'4'5. Consequently, immunotherapy of P. aeruginosa infection may be assumed to be of practical importance. Although P. aeruginosa vaccines are being prepared in several laboratories 6-8, commercial preparations are not yet available. We developed an experimental technology of P. aeruginosa vaccine (PV) on the basis of cell-wall protein protective antigens. PV was shown to be of low toxicity in laboratory animals and induced protective immunity in mice against experimental P. aeruginosa infection 9. This study illustrates the results of clinico-immunological trials of PV, prepared in semi-industrial conditions. L.M. Tarasevitch National Control Institute, Moscow, USSR, permitted limited studies of PV on volunteers. Preliminary clinical results are also shown which were obtained by anti-P, aeruginosa plasma treatment.
P. aeruginosa vaccine (PV)
*1.1. Mechnikov Research Institute for Vaccine and Sera, Academy of Medical Sciences, Moscow, USSR. tS.M. Kirov Army Medical Academy, Leningrad, USSR. tHaematology Research Centre, Moscow, USSR. °~To whom correspondence should be addressed at: Mechnikov Lane 5a, 103064 Moscow, USSR. (Received 3 September 1990; revised 14 January 1991; accepted 14 January 1991) 0264-410X/91/070491-04 © 1991Butterworth-HeinemannLtd
PV was prepared from four P. aeruginosa strains: no. 868 (02 serogroup 1° or Fisher 11 immunotype 3), no. 170015 (011 serogroup or immunotype 2), no. 170041 (06 serogroup or immunotype 1) and no. 170046 (07 serogroup or immunotype 6). Strains nos 170015, 170041 and 170046 were donated by Dr B. Lanyi from the National Institute of Hygiene, Budapest, Hungary. Strain no. 868 was obtained from Dr Irina Grishina from Vishnevsky Surgery Institute, Academy of Medical Sciences of the USSR, Moscow, USSR. PV contains 44.6 + 7.06% (w/v) protein (Mr = 20 000-100 000), 0.06 + 0.02% (w/v) lipopolysaccharide (according to ELISA), 2.36 + 0.3% (w/v) general carbohydrates and 27.2 + 0.2% (w/v) nucleic acids 9. PV is a liquid preparation, containing no preservatives. Each ampoule contains 1 ml PV which is equal to a single human dose (HD): 1.2+0.1 mg Lowery protein per ml. Three lots of PV were prepared under semi-industrial conditions 9 in I.I. Mechnikov Research Institute for Vaccine and Sera, and checked for sterility and toxicity on laboratory animals and good specific protective activity for mice in the Department of Biological Control of BIOMED Industrial Enterprise, USSR Ministry of Health, Petrovo-Dalnee, Moscow Region, and in L.M. Tarasevitch National Control Institute, Moscow, USSR.
Vaccine, Vol. 9, July 1991 491
Trials of Pseudonomas aeruginosa vaccine: E.S. Stanislavsky et al.
Volunteers Volunteers were selected from men aged 19 to 40 years according to the regulations for donor selection in the USSR ~2. All 119 volunteers were immunized with PV by subcutaneous injection in a subscapular area. The injections were repeated thrice at intervals of 7 days: 0.5, 1, 2 HD, or ~0.6, 1.2, 2.4 mg of protein per injection, respectively. Before and after the immunization, volunteers were subjected to plasmapheresis 12 with a withdrawal of 250-500ml plasma, at 14 day intervals according to the Instructions for Plasmapheresisl 2. Each donor underwent six to nine plasmaphereses. The immune plasma was kept at -40°C until used.
Patients and use of immune plasma Anti-P. aeruginosa plasma was used for the treatment of 46 patients with severe forms of P. aeruoinosa infection: 40 adults (average age 36.1_+5.1 years, x_+tS~) and six infants aged up to 2 years (for characteristics of patients see Results). The diagnosis of P. aeruginosa infection was based on bacteriological examination. The therapeutic effect of immune plasma was assessed by clinical symptoms and microbe isolation from clinical specimens. Anti-P. aeruginosa plasma was injected intravenously in a dose of 125-400ml to adults and in a dose of 50-125 ml to infants (3-5 ml kg -~ body weight). The treatment course consisted of three to six transfusions at intervals of 2-5 days. The immune plasma was used in cases where there was no response to conventional therapy. Conventional therapy comprised transfusions of normal donor's plasma, which was used in almost the same dosages as the immune plasma, synthetic plasma substitutes effective against infection, electrolyte solutions, broad spectrum antibiotics (gentamicin, cephalosporins, tetracyclines, semi-synthetic penicillins, etc.), vitamins, etc. 13. The period of conventional therapy served as an internal control and the period of conventional therapy together with the immune plasma was taken as a test period.
Evaluation of PV safety and reactogenicity in volunteers In the course of observation of immunized volunteers the following safety criteria were applied: general condition and febrile and local reactions (hyperaemia, regional lymphadenitis, tenderness on the site of injection). Febrile reactions were ranked as mild (37.1-37.4°C), moderate (37.5-37.9°C) and severe (over 38°C). Local reactions were ranked as mild (slight hyperaemia on the injection site with a diameter ~ 5 cm, intensive pain and thickening of derma on the site of injection, enlargement of regional lymphatic nodes and pain at the site of palpation). In addition, clinico-laboratory studies on red blood cell (RBC) and white blood cell (WBC) counts from peripheral blood, transaminase activity, thymol, total protein, direct and indirect bilirubin tests were performed1 a. 14 prior to the immunization, on days 4-7 after immunization and before each plasmapheresis. Before each injection of PV, volunteers underwent careful
492
Vaccirie, Vol. 9, July 1991
physical examination, their arterial blood pressure and body temperature were measured. ELISA The test was carried out in polystyrene plates (Nunc Immuno Plate I, Denmark). The results were read using a Titertek Multiskan (Labsystems, Helsinki, Finland). PV (0.1 #g protein, w/v) was used as an antigen for the coating of microplates. The antigen was dissolved in 0.05 M carbonate-bicarbonate buffer, pH 9.5, and 100 #1 of the antigen solution was transferred by pipette into each well. Microplates were coated for 16-18 h at room temperature. After antigen sorption the wells were washed five times with phosphate buffered saline (PBS), containing 0.05% Tween 20 (PBS-T), pH 7.3. Plasma (serum) of volunteers was diluted with PBS-T from 1:400 to 1:3200. Each plasma (serum) dilution (100#1) was added to the microplate wells and incubated for 3.5 h at room temperature. The wells were then washed as above. Rabbit anti-human IgG (100 #1) labelled with horseradish peroxidase (Labsystems, Helsinki, Finland) diluted with PBS-T to 1:1000 was added to each well, incubated overnight at room temperature, and then washed as above. Orthophenylenediamine (100 #1) (Sigma, St Louis, USA) dissolved in 0.1 M citrate buffer, pH 6.0, was added to the wells and the reaction was stopped after 50 min incubation at room temperature by 50 #1 1 M sulphuric acid. Absorbances were measured at 492 nm. Antibody titre calculations in ELISA units prior to and after the third immunization were described elsewhere 13.
Passive mice protection test Plasma (serum) of volunteers was tested before and after the third immunization as follows: 0.1 ml plasma was injected intra-abdominally into Swiss mice (weight 18-20 g). Two hours later the mice were infected intraabdominally by highly virulent test strain no. 8 of P. aeruginosa (02 serogroup or immunotype 3), which was antigenically identical to the vaccine strain no. 868 of P. aeruginosa. Protective potency of volunteer plasma (serum) was assessed using the index of efficacy (IE), i.e. the ratio between LDso for the mice that receive plasma (test animals) and LDso for untreated mice (control animals). The IE values of non-immune and immune plasma (serum) of each volunteer were compared.
Methods of bacterial investigation Specimens obtained from patients, i.e. pus and blood, were subjected to bacteriological examination. Pure P. aeruginosa culture was isolated from clinical material by previously described methods ~s.
Methods of clinico-laboratory investigation of blood The investigation of peripheral blood was performed as described in Ref. 15.
Statistical methods Mean 02) and standard error of deviation (sO were calculated and Student's t-test was used. The difference was considered to be significant at p
INTRODUCTION
MATERIALS AND METHODS
Pseudomonas aeruginosa infection is a serious problem for surgical and other patients 1-5. Hospital strains of P. aeruginosa are highly resistant to the majority of antibiotics 2'4'5. Consequently, immunotherapy of P. aeruginosa infection may be assumed to be of practical importance. Although P. aeruginosa vaccines are being prepared in several laboratories 6-8, commercial preparations are not yet available. We developed an experimental technology of P. aeruginosa vaccine (PV) on the basis of cell-wall protein protective antigens. PV was shown to be of low toxicity in laboratory animals and induced protective immunity in mice against experimental P. aeruginosa infection 9. This study illustrates the results of clinico-immunological trials of PV, prepared in semi-industrial conditions. L.M. Tarasevitch National Control Institute, Moscow, USSR, permitted limited studies of PV on volunteers. Preliminary clinical results are also shown which were obtained by anti-P, aeruginosa plasma treatment.
P. aeruginosa vaccine (PV)
*1.1. Mechnikov Research Institute for Vaccine and Sera, Academy of Medical Sciences, Moscow, USSR. tS.M. Kirov Army Medical Academy, Leningrad, USSR. tHaematology Research Centre, Moscow, USSR. °~To whom correspondence should be addressed at: Mechnikov Lane 5a, 103064 Moscow, USSR. (Received 3 September 1990; revised 14 January 1991; accepted 14 January 1991) 0264-410X/91/070491-04 © 1991Butterworth-HeinemannLtd
PV was prepared from four P. aeruginosa strains: no. 868 (02 serogroup 1° or Fisher 11 immunotype 3), no. 170015 (011 serogroup or immunotype 2), no. 170041 (06 serogroup or immunotype 1) and no. 170046 (07 serogroup or immunotype 6). Strains nos 170015, 170041 and 170046 were donated by Dr B. Lanyi from the National Institute of Hygiene, Budapest, Hungary. Strain no. 868 was obtained from Dr Irina Grishina from Vishnevsky Surgery Institute, Academy of Medical Sciences of the USSR, Moscow, USSR. PV contains 44.6 + 7.06% (w/v) protein (Mr = 20 000-100 000), 0.06 + 0.02% (w/v) lipopolysaccharide (according to ELISA), 2.36 + 0.3% (w/v) general carbohydrates and 27.2 + 0.2% (w/v) nucleic acids 9. PV is a liquid preparation, containing no preservatives. Each ampoule contains 1 ml PV which is equal to a single human dose (HD): 1.2+0.1 mg Lowery protein per ml. Three lots of PV were prepared under semi-industrial conditions 9 in I.I. Mechnikov Research Institute for Vaccine and Sera, and checked for sterility and toxicity on laboratory animals and good specific protective activity for mice in the Department of Biological Control of BIOMED Industrial Enterprise, USSR Ministry of Health, Petrovo-Dalnee, Moscow Region, and in L.M. Tarasevitch National Control Institute, Moscow, USSR.
Vaccine, Vol. 9, July 1991 491
Trials of Pseudonomas aeruginosa vaccine: E.S. Stanislavsky et al.
Volunteers Volunteers were selected from men aged 19 to 40 years according to the regulations for donor selection in the USSR ~2. All 119 volunteers were immunized with PV by subcutaneous injection in a subscapular area. The injections were repeated thrice at intervals of 7 days: 0.5, 1, 2 HD, or ~0.6, 1.2, 2.4 mg of protein per injection, respectively. Before and after the immunization, volunteers were subjected to plasmapheresis 12 with a withdrawal of 250-500ml plasma, at 14 day intervals according to the Instructions for Plasmapheresisl 2. Each donor underwent six to nine plasmaphereses. The immune plasma was kept at -40°C until used.
Patients and use of immune plasma Anti-P. aeruginosa plasma was used for the treatment of 46 patients with severe forms of P. aeruoinosa infection: 40 adults (average age 36.1_+5.1 years, x_+tS~) and six infants aged up to 2 years (for characteristics of patients see Results). The diagnosis of P. aeruginosa infection was based on bacteriological examination. The therapeutic effect of immune plasma was assessed by clinical symptoms and microbe isolation from clinical specimens. Anti-P. aeruginosa plasma was injected intravenously in a dose of 125-400ml to adults and in a dose of 50-125 ml to infants (3-5 ml kg -~ body weight). The treatment course consisted of three to six transfusions at intervals of 2-5 days. The immune plasma was used in cases where there was no response to conventional therapy. Conventional therapy comprised transfusions of normal donor's plasma, which was used in almost the same dosages as the immune plasma, synthetic plasma substitutes effective against infection, electrolyte solutions, broad spectrum antibiotics (gentamicin, cephalosporins, tetracyclines, semi-synthetic penicillins, etc.), vitamins, etc. 13. The period of conventional therapy served as an internal control and the period of conventional therapy together with the immune plasma was taken as a test period.
Evaluation of PV safety and reactogenicity in volunteers In the course of observation of immunized volunteers the following safety criteria were applied: general condition and febrile and local reactions (hyperaemia, regional lymphadenitis, tenderness on the site of injection). Febrile reactions were ranked as mild (37.1-37.4°C), moderate (37.5-37.9°C) and severe (over 38°C). Local reactions were ranked as mild (slight hyperaemia on the injection site with a diameter ~ 5 cm, intensive pain and thickening of derma on the site of injection, enlargement of regional lymphatic nodes and pain at the site of palpation). In addition, clinico-laboratory studies on red blood cell (RBC) and white blood cell (WBC) counts from peripheral blood, transaminase activity, thymol, total protein, direct and indirect bilirubin tests were performed1 a. 14 prior to the immunization, on days 4-7 after immunization and before each plasmapheresis. Before each injection of PV, volunteers underwent careful
492
Vaccirie, Vol. 9, July 1991
physical examination, their arterial blood pressure and body temperature were measured. ELISA The test was carried out in polystyrene plates (Nunc Immuno Plate I, Denmark). The results were read using a Titertek Multiskan (Labsystems, Helsinki, Finland). PV (0.1 #g protein, w/v) was used as an antigen for the coating of microplates. The antigen was dissolved in 0.05 M carbonate-bicarbonate buffer, pH 9.5, and 100 #1 of the antigen solution was transferred by pipette into each well. Microplates were coated for 16-18 h at room temperature. After antigen sorption the wells were washed five times with phosphate buffered saline (PBS), containing 0.05% Tween 20 (PBS-T), pH 7.3. Plasma (serum) of volunteers was diluted with PBS-T from 1:400 to 1:3200. Each plasma (serum) dilution (100#1) was added to the microplate wells and incubated for 3.5 h at room temperature. The wells were then washed as above. Rabbit anti-human IgG (100 #1) labelled with horseradish peroxidase (Labsystems, Helsinki, Finland) diluted with PBS-T to 1:1000 was added to each well, incubated overnight at room temperature, and then washed as above. Orthophenylenediamine (100 #1) (Sigma, St Louis, USA) dissolved in 0.1 M citrate buffer, pH 6.0, was added to the wells and the reaction was stopped after 50 min incubation at room temperature by 50 #1 1 M sulphuric acid. Absorbances were measured at 492 nm. Antibody titre calculations in ELISA units prior to and after the third immunization were described elsewhere 13.
Passive mice protection test Plasma (serum) of volunteers was tested before and after the third immunization as follows: 0.1 ml plasma was injected intra-abdominally into Swiss mice (weight 18-20 g). Two hours later the mice were infected intraabdominally by highly virulent test strain no. 8 of P. aeruginosa (02 serogroup or immunotype 3), which was antigenically identical to the vaccine strain no. 868 of P. aeruginosa. Protective potency of volunteer plasma (serum) was assessed using the index of efficacy (IE), i.e. the ratio between LDso for the mice that receive plasma (test animals) and LDso for untreated mice (control animals). The IE values of non-immune and immune plasma (serum) of each volunteer were compared.
Methods of bacterial investigation Specimens obtained from patients, i.e. pus and blood, were subjected to bacteriological examination. Pure P. aeruginosa culture was isolated from clinical material by previously described methods ~s.
Methods of clinico-laboratory investigation of blood The investigation of peripheral blood was performed as described in Ref. 15.
Statistical methods Mean 02) and standard error of deviation (sO were calculated and Student's t-test was used. The difference was considered to be significant at p
