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GMP-grade pneumococcal whole-cell vaccine injected subcutaneously protects mice from nasopharyngeal colonization and fatal aspiration-sepsis

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GMP-grade pneumococcal whole-cell vaccine injected subcutaneously protects mice from nasopharyngeal colonization and fatal aspiration-sepsis
  GMP-grade pneumococcal whole-cell vaccine injectedsubcutaneously protects mice from nasopharyngealcolonization and fatal aspiration-sepsis Ying-Jie Lu 1 , Luciana Leite 2 , Viviane Maimoni Gonçalves 2 , Waldely de Oliveira Dias 2 , CeliaLiberman 2 , Fernando Fratelli 2 , Mark Alderson 3 , Andrea Tate 3 , Jean-FrançoisMaisonneuve 3 , George Robertson 3 , Rita Graca 1 , Sabina Sayeed 1 , Claudette M.Thompson 4 , Porter Anderson 1 , and Richard Malley 1 1  Division of Infectious Diseases, Children’s Hospital, Harvard Medical School, Boston, MA 2  Instituto Butantan, Sao Paulo, Brazil 3  PATH, Seattle, WA 4  Department of Epidemiology, Harvard School of Public Health, Boston, MA Abstract Mucosal immunization with a killed whole-cell pneumococcal vaccine, given with enterotoxin-related adjuvants, has been shown to confer multi-serotype protection against colonization of thenasopharynx and middle ear in mice. However, because novel mucosal immunization strategiesmay be difficult to implement, here we evaluated subcutaneous injection. Strain RM200 wasengineered to be capsule-negative, autolysin-negative, and to express a non-toxic mutantpneumolysoid. Liter-scale and 60-L Good Manufacturing Practice (GMP) cultures were grown inbovine-free soy-based medium, killed with chloroform or beta-propiolactone, and injected intoC57Bl/6 mice without or with aluminum adjuvant. The adjuvant Al(OH) 3  strongly increasedresponses, particularly if pre-treated with phosphate. Protection was found in several tested modelinfections: nasal colonization with a serotype 6B strain and fatal aspiration-sepsis with strains of serotype 3 and 5. Protection against colonization was mechanistically dependent on the presenceof CD4+ T cells at the time of challenge; in contrast, in the type 3 aspiration-sepsis model, CD4+T cells were not required for protection at the time of challenge, suggesting that antibody alonewas sufficient to protect against death in this model. Rabbits receiving sequential intramuscularinjections in a pilot toxicity study displayed local reactogenicity at injection sites but no clinicalsigns. The rabbit antiserum thus produced was active in an in vitro  phagocytic killing assay andpassively protected mice in the type 3 aspiration-sepsis model. Approval is being sought forhuman trials of this vaccine. Keywords Streptococcus pneumoniae ; vaccine; colonization; sepsis Address correspondence to: Richard Malley, MD, Division of Infectious Diseases, Children’s Hospital, 300 Longwood Avenue,Boston MA 02115, Phone: 617-919-2902, Fax: 617-730-0255, Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to ourcustomers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may bediscovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author Manuscript Vaccine . Author manuscript; available in PMC 2011 November 3. Published in final edited form as: Vaccine  . 2010 November 3; 28(47): 74687475. doi:10.1016/j.vaccine.2010.09.031. N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t    INTRODUCTION Streptococcus pneumoniae  (pneumococcus) persists as a major pathogen, particularlyamong children in low-income countries [1]. Capsular polysaccharide conjugate vaccinesprovide type-specific immunity, but have the disadvantages of limited serotype coverage,increasing disease from non-vaccine serotypes, and relatively high cost [2,3]. Therefore, potentially more economical serotype-independent vaccines based upon species-commonprotein antigens are being investigated [4]. We have been investigating the use of killed cellsof non-capsulated pneumococci, maximizing the exposure of a variety of species-commonsub-capsular antigens, thus potentially providing synergistic immunity to multiplepneumococcal targets. This preparation, designated “whole cell antigen” (WCA) or, whengiven with suitable adjuvant, whole cell vaccine (WCV), was initially intended for mucosaladministration to reduce colonization. Intranasal (i.n.) vaccination using cholera toxin asadjuvant prevents fatal serotype 3 pneumonia in rats and reduces nasopharygeal (NP) andmiddle ear colonization in mice by strains of serotype 6B or 23F [5,6]. Serum antibodies areraised, but the accelerated pneumococcal clearance in mice can be induced in the absence of antibodies by a CD4+ T cell-dependent, IL-17A-mediated mechanism [7,8].Considering the potency and low cost, PATH supported the further development of WCVand generation under “Good Manufacturing Practice” (GMP) at Instituto Butantan (SaoPaulo, Brazil). Anticipating human testing, WCA was made from cells expressing a non-toxic variant of pneumolysin (carrying three mutations -- W433F, D385N, C428G -- whichgreatly reduce hemolytic capacity) and cultured in medium lacking bovine components[9,10]. Potency was increased by killing with agents such as chloroform or beta-propiolactone so that released soluble components are retained. In preclinical studies, wehave investigated several mucosal adjuvants other than cholera toxin, and different routes(such as buccal, sublingual or transcutaneous) [9]. However, at present, mucosaladministration may be difficult to implement in developing countries and currentinternational recommendations favor injection, so here we have tested WCA in mice by thesubcutaneous (s.c.) route, evaluating aluminum salts as an adjuvant. In addition tonasopharyngeal colonization with serotype 6B, protection was tested in models of fatalaspiration-sepsis with isolates of serotype 3 and 5. A pilot toxicology study including animmunogenicity endpoint was conducted with multiple intramuscular injections in rabbits,and the antiserum produced was shown to induce phagocytic killing in vitro  and protectionfollowing passive transfer to mice in the pneumonia model. MATERIALS AND METHODS Materials Aluminum hydroxide (alum) was from Brenntag North America (2% Alhydrogel). Beta-propiolactone (BPL) was from Fisher, and saline was from B. Braun Medical Inc.(Bethlehem, PA). All other reagents were obtained from Sigma. Antigen preparations For non-GMP grade material, pneumococcal strain RM200 (a capsule-negative, autolysin-negative, pneumolysoid-expressing strain derived from Rx1 as described in [9]) was grownat 37°C with 5% CO 2  to A 600  1.0 in animal protein-free medium [10] at which viable countwas approximately 6×10 8  CFU/ml. Further steps were at 4 °C. The cells were collected bycentrifugation, and washed twice with Lactated Ringer’s solution (LR) (102 mM NaCl, 28mM NaC 3 H 5 O 3 , 1.5 mM CaCl 2  and 4 mM KCl). For killing with chloroform, washed cellsin LR with 0.2% glucose at A 600 =32 were mixed with chloroform 1/40 v/v for 2 hours (thispreparation is named WCC). Killed cells were then lyophilized, which eliminates residual Lu et al.Page 2 Vaccine . Author manuscript; available in PMC 2011 November 3. N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t    organic solvent. For killing with BPL, washed cells in LR with 10% sucrose were mixedwith BPL, 1/4000 v/v for 24 hours at 4°C followed by a 2-hour incubation at 37°C toinactivate BPL; cells were subsequently frozen or lyophilized (this preparation is calledWCB). Protein concentration was determined using the Total Protein Kit with bovine serumalbumin as standard (Sigma).One day prior to immunization, vaccines were prepared as follows. Frozen aliquots werethawed or lyophilized vials were reconstituted with sterile water, diluted to the appropriateconcentration, and mixed with Al(OH) 3  at the indicated concentration in a 15 ml conicaltube, which was then tumbled overnight at 4°C to allow for adsorption. Immunization and challenge of mice C57BL/6J mice (Jackson Laboratories, Bar Harbor, Maine) were used in all the experiments.The age at time of first immunization was between 4–6 weeks. Gently restrained,nonanesthetized mice received 2 or 3 subcutaneous injections of 200 μ l of adjuvant with orwithout antigen in the lower part of the back at 2-week intervals. Blood was drawn 1 or 2weeks after the last immunization, and assayed for antibody and for IL-17A production invitro  after stimulation with WCA. NP colonization model— To determine susceptibility to NP colonization, i.n. challengewith live encapsulated pneumococci was performed as described [5]: 3 weeks after the lastimmunization, mice were challenged with 10 7  colony-forming units (CFU) of serotype 6Bstrain 0603 in 10 μ l of PBS applied as described. To determine NP colonization, an upperrespiratory culture was done 10 days later by instilling sterile saline retrograde through thetransected trachea, collecting the first 6 drops (about 0.1 ml) from the nostrils, and platingneat or diluted samples on blood agar plates containing 2.5 μ g gentamicin/ml. The figuresshow the CFU per nasal wash sample of individual mice; the geometric means (GM) aredisplayed as a horizontal bar. For ease of statistical analysis, a sterile sample was assignedhalf the lower limit of detection (1.6 CFU/nasal wash), or 0.8 CFU/nasal wash. Aspiration-sepsis challenge model— Two weeks after the last immunization, micewere gently anesthetized with isoflorane, held supine, and given a 100 μ l intranasalinoculation containing an inoculum of type 3 strain WU-2 or type 5 strain DBL5 (a kind giftof Dr. David Briles) using a model we have described before [11] but with the modificationthat mice were not intranasally exposed to pneumococcus 2 days prior to aspirationchallenge. This model induces sepsis and death within 3–4 days in nonimmunized mice.Mice are monitored twice daily and sacrificed by C02 inhalation and terminalexsanguination when demonstrating signs of illness following which a blood culture isobtained; in all cases, these blood cultures reliably demonstrate the presence of pneumococcal bacteremia. For passive protection studies, one day prior to induction of aspiration, mice were injected intraperitoneally with either 500 μ l saline or 300 μ l plus 200 μ l of heat inactivated (56°C for 30 minutes) serum obtained from rabbits immunized withaluminum hydroxide with or without WCA as described below. All animal studies wereapproved by our local animal ethics committees. Rabbit immunization and toxicology studies All rabbit immunizations were performed at MPI (Mattawan, MI). Female New ZealandWhite rabbits in groups of three were given 0.5 ml injections of saline, Al(OH) 3  alone(containing 0.6 mg of Al), Al(OH) 3 -adsorbed WCB at doses of 50, 500 or 5000 μ g or awhole cell Diphtheria-Tetanus-Pertussis whole cell (DTwP) vaccine (clinical product fromInstituto Butantan) intramuscularly on day 1, 15, 29 and 43. Sera were obtained before eachimmunization and at a terminal bleed on day 45 and shipped frozen to Children’s Hospital Lu et al.Page 3 Vaccine . Author manuscript; available in PMC 2011 November 3. N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t    Boston for measurement of antibodies. Observations for morbidity, mortality, clinical signs,body temperature and food and water consumption were conducted on a regular basis for allanimals. Dermal irritation scores were evaluated prior to each dose and daily for 3consecutive days following each dose (with the exception of the last dose). Clinicalpathology was performed at baseline, on day 2 and at termination. At study termination (twodays post last dose), macroscopic examinations were performed, organ weights wererecorded, and the injection sites were microscopically examined. Enzyme-linked immunosorbent assay (ELISA) Assays for murine antibodies to WCA were done in Immulon 2 HB 96-microwell plates(Thermo Scientific, Waltham, MA) coated with WCA 100 μ g of protein per ml in PBS.Plates were blocked with 1% BSA in PBS. Antibody diluted in PBS-T was added andincubated at room temperature for 2 hours. Plates were washed with PBS-T, and secondaryHRP-conjugated antibody to mouse immunoglobulin G, G1 or G2 (all from Sigma) wasadded and incubated at room temperature for one hour. The plates were washed anddeveloped with SureBlue TMB Microwell Peroxidase Substrate (KPL, Gaithersburg, MD). Assay of IL-17A production in whole blood samples Fifty μ l of heparinized blood was added to 450 μ l DMEM (BioWhittaker, Walkersville,MD) containing 10% low-endotoxin defined FBS (Hyclone, Logan, UT), 50 μ M 2-mercaptoethanol (Sigma) and ciprofloxacin (10 μ g/ml, Cellgro, Manassas, VA). Except forthe nonstimulated control, the cultures were incubated at 37°C for 6 days with 10 7  cells of pneumococcal WCA. Supernatants were collected following centrifugation and stored at − 80°C until analyzed by ELISA for IL-17A concentration (R&D Systems, Minneapolis,MN). Surface killing assay Neutrophil surface killing assays were performed as described previously [8,12]. Briefly,type 6B strain 0603 [5] was grown to mid-log phase and frozen in THY/10% glycerol at − 80°. On the day of the experiment, bacteria were thawed and diluted to 100 CFU/  μ L inRPMI supplemented with 10% FBS and opsonized with normal rabbit serum or serum fromrabbits that had been immunized three times with WCV as described above for 30 minutes at37°C. In all cases, rabbit sera were heated for 30 min at 52°C to inactivate complement.Polymorphonuclear leukocytes were purified from the peripheral blood of human volunteersusing a Histopaque 1077, 1119 gradient (Sigma-Aldrich, St. Louis, MO) according to themanufacturers instructions and used immediately. Five μ l of the opsonized bacterialsuspension, diluted to contain 30 cfu, was spotted at room temperature on trypticase soyagar with 5% defibrinated sheep blood (TSA II) (BD) with 5 replicates per plate, and thefluid was allowed to absorb, requiring about 15 minutes. Ten microliters of neutrophils(3×10 6  cells/mL; resulting in a bacterial:neutrophil ratio of approximately 1:60) were thenoverlaid, allowed to absorb, and incubated overnight at 37° with 5% CO 2.  Controls includedbacteria spotted in the absence of neutrophils or with neutrophils but no serum. Statistical analysis Antibody and IL-17A concentrations and NP colonization densities were compared by theMann-Whitney U   test using PRISM (version 4.0a, GraphPad Software, Inc). Differences insurvival were analyzed with the Kaplan-Meier test, using PRISM as well. For the toxicologystudy, all comparisons were made to the group receiving the alum adjuvant alone.Comparisons of body weight, food consumption, body temperature, hematology (exceptleukocyte counts) coagulation parameters, clinical chemistry values, and organ weights wereperformed by group pair-wise comparisons using either ANOVA or Welch’s test, with Lu et al.Page 4 Vaccine . Author manuscript; available in PMC 2011 November 3. N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t    appropriate adjustment for multiple comparisons. For leukocyte counts and urinalyses, dueto lack of normality, data were log and rank transformed, respectively, and transformed datawere analyzed as above. Erythema, eschar and edema formation were analyzed by CochranMantel Haenszel Test. RESULTS General approach Dosage of WCA is quoted in μ g of protein, of which about 85% is cellular and ca 15% issoluble [9]; 1 μ g corresponds approximately to a total dry weight of 1.7 μ g and to 10 6  CFUbefore killing. For active immunization of mice, two or three sequential injections weregiven two weeks apart, blood was taken 1 or 2 weeks after the last injection for assays of priming for IL-17A expression in vitro  and of serum IgG antibody to WCA, the animalswere challenged with pneumococcus in either the colonization model or the aspiration-sepsismodel, and the outcome in individual mice compared to the in vitro  assay values. Evaluation of aluminum adsorption by immunological assays and protection in thecolonization model Ascending doses of WCC - 1, 10, and 100 μ g - were tested in a 3-injection sequence withoutand with adsorption onto Al(OH) 3,  0.21 mg Al per dose. Without adjuvant there was nomeasurable IL-17A response (Figure 1A), while with Al(OH) 3  there was a significantresponse even to the 1 μ g dose. Without and with adsorption there were dose-dependentantibody responses, but with about 100-fold potentiation by the Al(OH) 3  (Figure 1B).Without adsorption there was no protection against experimental colonization with serotype6B strain 0603, but with Al(OH) 3  the two higher WCC doses gave significant reduction of CFU (Figure 1C). A similar requirement for Al(OH) 3  for IL-17A responses andenhancement of antibody response was noted when WCB was used (data not shown).Routinely, adsorption was done for 18–22 hours at 4°C, and the preparations were testedimmediately. To test the stability of the adsorbed antigen, Al(OH) 3  with WCC at 100 μ g perdose was incubated at 37°C for one month before testing in the above-described experiment.The IL-17A responses, antibody titers and protection results did not differ from the samedosage of freshly prepared WCC-Al(OH) 3  (Figures A, B, C, last column, indicated by the Δ superscript).Phosphate pretreatment of alum has been shown to have important effects on theimmunogenicity of aluminum-adjuvanted vaccines [13]. To evaluate this, Al(OH) 3  waspreequilibrated 7 days with sodium phosphate buffer, pH 7.5 in a range of concentrationsfrom 0–100 mM, then washed and used at a limiting Al dosage (0.085 mg) to adsorb WCCat the limiting dose of 10 μ g. Phosphate pre-treatment at 10–100 mM gave potentiations of 8–10 fold in IL-17A response (Figure 2A) and 2–3 fold increase in antibody titer (Figure2B). However, in neither assay did the phosphate treatment raise the response beyond that of the higher dosage of Al(OH) 3  (0.21 mg of Al), which was routinely used in further studies. Comparison of WCC and WCB and role of CD4+ T cells in the colonization model WCC and WCB at doses of 10 or 100 μ g, adsorbed onto Al(OH) 3  were compared. The dose-dependent IL-17A responses did not differ (Figure 3A). Clearance of serotype 6B from thenasopharynx also was indistinguishable (Figure 3B). Dependence upon the CD4+ pathwaywas evaluated in a group of WCC-immunized animals given anti-CD4+ antiserum just priorto challenge to eliminate these cells as effectors of protection; in these mice the protectionwas eliminated (2 nd  column, Figure 3B). Since beta-propiolactone is used in several otherhuman vaccines, including rabies vaccines [14], it was judged the preferred agent of killing Lu et al.Page 5 Vaccine . Author manuscript; available in PMC 2011 November 3. 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