Immunoprotective Activity of Ribosomes from

Transcription

1 INPECTION AND IMMUNITY, Feb. 1977, p Copyright 1977 Americn Society for Microbiology Vol. 15, No. 2 Printed in U.S.A. Immunoprotective Activity of Ribosomes from Hemophilus influenze MELVYN LYNN,' RAM P. TEWARI,* AND MORRIS SOLOTOROVSKY Deprtment of Microbiology, Rutgers University, New Brunswick, New Jersey 08903, nd Deprtment of Medicl Sciences, Southern Illinois University, School of Medicine, Springfield, Illinois 62708* Received for publiction 23 August 1976 Immuniztion with ribosoml preprtions from Hemophilus influenz type b elicited protective immunity in mice. Ribosomes from disrupted cells were isolted by differentil centrifugtion using sodium dodecyl sulfte. The wshed ribosomes contined 25% protein nd 75% ribonucleic cid nd sedimented s single pek on sucrose density grdient nlysis with sedimenttion coefficient of 67S, using Escherichi coli ribosomes s 70S mrker. Immunodiffusion tests with ntipolyribose phosphte serum showed tht the ribosomes were free from cpsulr mteril. Mice immunized subcutneously with ribosomes, with or without djuvnt, were chllenged intrperitonelly with 100 to 1,000 men lethl doses of H. influenze type b suspended in gstric mucin. Significnt protection ws induced by ribosomes nd ws comprble to tht obtined fter sublethl infection with live cells. The protection ws gretly enhnced fter incorportion of ribosomes into djuvnts. Mximum protection (90 to 95%) ws observed t 1 to 2 weeks fter immuniztion. Ribosomes from nonencpsulted strin of H. influenze were s immunogenic s those from the encpsulted strin, demonstrting tht the cpsulr mteril is not responsible for immunogenicity of Hemophilus ribosomes. Hemophilus influenze type b is the most common gent of bcteril meningitis in children between the ges of 3 months nd 6 yers (22, 29, 30, 42). Despite the vilbility of brod-spectrum ntibiotics, the mortlity rte hs remined t 5 to 10% while 30 to 50% of the survivors develop chronic neurologicl complictions such s blindness, defness, convulsions, mentl retrdtion, nd hydrocephlus (19, 28, 29). There hs been significnt rise in the incidence of Hemophilus meningitis during the pst 25 yers (18, 30, 39). Since present chemotherpy is not dequte to prevent the morbidity nd mortlity, immunoprophylxis might provide n lterntive wy to control Hemophilus infections. Polyribose phosphte, the cpsulr mteril of H. influenze type b, hs been utilized in vccine ginst H. influenze infections (1, 2, 10, 22, 24, 26). Although ntibody titers cn be induced by immuniztion with cpsulr mteril, unequivocl evidence for immunoprotection hs not been demonstrted. Immuniztion with killed whole-cell vccine lso hs been unsuccessful in preventing respirtory infections (4, 8). 'Present ddress: Deprtment of Microbiology, College of Medicine nd Dentistry of New Jersey, Newrk, NJ The pioneering work of Youmns nd Youmns (45-47) hs introduced new concept in immuniztion by inducing significnt protection in mice with ribosoml preprtions from Mycobcterium tuberculosis. Similr ribosoml vccines hve been prepred from Slmonell typhimurium (13, 14, 31, 40, 41), Stphylococcus ureus (44), Pseudomons eruginos (43), Diplococcus pneumonie (33, 37), Neisseri meningitidis (35), Vibrio cholere (12), Listeri monocytogenes (32), Frncisell tulrensis (3), Streptococcus pyogenes (25), Histoplsm cpsultum (9, 34), Cndid lbicns (E. S. Sunders, M. Solotorovsky, nd R. P. Tewri, Abstr. Annu. Meet. Am. Soc. Microbiol. 1975, p. 89, F22), nd Leishmni enriettii (20). In the present investigtion, the immunoprotective ctivity of ribosomes from encpsulted nd nonencpsulted strins of H. influenze ws studied. Mice were immunized subcutneously (s.c.) with different ribosoml preprtions, nd protection ws ssessed fter intrperitonel (i.p.) chllenge. In ddition, physicl nd chemicl chrcteristics of ribosomes were studied. (This pper ws presented in prt t the 59th Annul Meeting of the Federtion of Americn Societies for Experimentl Biology, April 1975, Atlntic City, N.J.) 453

2 454 LYNN, TEWARI, AND SOLOTOROVSKY MATERIALS AND METHODS Animls. White, mle CF1 mice weighing 12 to 14 g were obtined from Chrles River Co., Wilmington, Mss. The mice were divided rndomly into groups of 10, housed in metl cges, nd given feed nd wter d lib. For ech dose of vccine, 20 mice were used nd 20 nonvccinted mice served s controls. Orgnisms. H. influenze type b, encpsulted (H-1), ws obtined from subculture of primry isolte from the cerebrospinl fluid of child with meningitis. The orgnism ws clssified s H. influenze type b with typing serum (Difco) nd ws pssged through mice severl times to mintin its virulence. The orgnism ws reisolted from the brins of mice, subcultured once on Difco brin hert infusion (BHI) gr pltes enriched with 5% Fildes supplement, nd stored in lyophilized stte. The men lethl dose (LD50) of the strin for mice ws 2 x 104 orgnisms suspended in gstric mucin given i.p. The virulent strin of H. influenze (H- U) ws supplied by Grce Leidy, Columbi Presbyterin Hospitl, New York. The virulent strin did not possess the cpsulr mteril nd ws not typble with ntiserum prepred from the virulent strin. Escherichi coli ws obtined from our stock culture collection nd ws mintined in lyophilized stte. Cultivtion of orgnisms. Hemophilus cells were grown in BHI broth supplemented with 0.001% nicotinmide denine dinucleotide (Sigm Chemicl Co., St. Louis, Mo.), 0.002% hemoglobin, nd 0.16% glucose in 1-liter Erlenmeyer flsks contining 250 ml of medium. The flsks were inoculted with 2.5 ml of 10-h culture. The erly-log-phse cells of E. coli were lso grown in BHI broth. All flsks were incubted t 370C in gyrtory shker (New Brunswick Scientific Co., New Brunswick, N.J.) with shking speed of 150 rpm. Preprtion of ribosomes. Ribosomes were prepred by modifiction of the procedure ofyoumns nd Youmns (47). The erly-log-phse cells (8 h) of H. influenze were hrvested by centrifugtion nd wshed twice in 0.01 M phosphte buffer (ph 7.0). All steps, unless otherwise specified, were performed t 40C. Wshed cells were resuspended in equl volumes of phosphte buffer (ph 7.0) contining 0.44 M sucrose, 0.25% sodium dodecyl sulfte (SDS), nd 3 x 10-2 M MgC12, nd were trnsferred to Brun (MSK) homogenizer glss bottles contining cid-wshed glss beds (0.17 to 0.18 mm) equl to the wet weight of the cells. The cells were disrupted in the Brun homogenizer using three 2-min breking periods, nd the chmber ws kept cool with liquid CO2. The homogenized mixture ws centrifuged successively t 27,000 nd 47,000 x g for 10 min ech to sediment intct cells nd cellulr debris. The upper four-fifths of the superntnt ws pipetted nd centrifuged in Beckmn ultrcentrifuge (L2-65B) t 270,000 x g for 65 min, using 65 rotor. The pellet, designted s prticulte frction, ws resuspended in 0.01 M phosphte buffer contining 10-2 M MgC12 to concentrtion of 100 mg (wet weight) per ml. INFECT. IMMUN. The prticulte suspension ws dded to flsk contining n equl volume of 0.5% SDS in the phosphte buffer t room temperture. The flsk ws gently gitted by hnd for 10 min. The suspension ws trnsferred to 25-ml Corex centrifuge tube nd refrigerted t 40C overnight in n ice bth to seprte SDS by precipittion. The following morning, SDS ws sedimented by centrifugtion t 37,000 x g for 15 min. The upper two-thirds of the superntnt ws removed nd centrifuged t 270,000 x g for 65 min to obtin the ribosoml frction. The ribosoml frction ws wshed twice nd resuspended in the phosphte buffer. The ribosoml suspension ws stndrdized on the bsis of protein content. Inocultion of the ribosoml suspension on pproprite medi did not revel the presence of vible Hemophilus orgnisms. Chemicl nd physicl nlyses. Protein ws determined by the method of Lowry et l. (16), using bovine serum lbumin frction V s stndrd. Ribonucleic cid (RNA) nd deoxyribonucleic cid (DNA) were mesured by the orcinol (27) nd diphenylmine (5) methods, respectively. Ribose nd deoxyribose served s stndrds. Crbohydrte ws ssyed by the chromotropic cid (15) nd the cysteine-h2so4 (7) procedures, using glucose nd ribose, respectively, s stndrds. For density grdient nlysis, 0.1 ml of ribosoml preprtion (contining 400 u.tg of RNA) ws lyered over 5-ml, 5 to 20% liner sucrose grdient prepred in 0.02 M tris(hydroxymethyl)minomethne buffer (ph 7.8) contining 10-2 or 10-4 M MgC12. The grdient ws centrifuged in n SW50L rotor t 45,000 rpm for 70 min t 40C. The frctions were collected by upwrd displcement fter piercing the bottom of the tube. The bsorbnce of the grdient t 260 nm ws recorded by using Gilford spectrophotometer equipped with flow cell nd recorder. The opticl density of ech frction t 235 nd 280 nm ws lso determined. The sedimenttion coefficient ofh. influenze ribosomes ws determined by the method of Mrtin nd Ames (17), using ribosomes from E. coli, prepred by the method of Trub et l. (38), s 70S mrker. Immuniztion of mice. Mice were immunized s.c. with ribosoml preprtion with or without incorportion into Freund incomplete djuvnt (FIA). One prt ofthe vccine ws mixed with three prts of the djuvnt. In some experiments, Hillemn djuvnt- 65 (11) nd Bordetell pertussis vccine (Difco) were lso used s djuvnts. Adjuvnt-65 ws prepred by emulsifying 0.4 g of luminum monosterte in 8.6 g of penut oil nd 1 g of mnnide monoolete (Arlcel A). These djuvnts were mixed with equl volumes of ribosomes. To obtin stble suspension, the mixture of ribosomes nd djuvnt ws pssed severl times through syringe. The finl volume of vccine for ech mouse ws 0.4 ml. For positive vccine controls, formlin-killed nd livecell vccines were lso used. A formlin-killed cell vccine ws prepred by suspending 6-h growth from Fildes-enriched BHI gr culture in 0.01 M phosphte buffer contining 0.85% NCl nd 0.5%

3 VOL. 15, 1977 formldehyde. The suspension ws djusted turbidimetriclly to contin 15 x 108 cells/ml. Live-cell vccine consisted of sublethl dose of the orgnisms (5 x 103). Both live- nd killed-cell vccines were emulsified with FIA before immuniztion. Groups of 20 mice were used for ech dose of vccine, nd 20 unvccinted mice served s controls. Controls were injected s.c. with 0.4 ml of djuvnt or buffer. Evlution of immune response. At specified times fter immuniztion, ll mice were chllenged i.p. with lethl dose of H. influenze type b suspension in 1 ml of5% gstric mucin (ph 7.0) contining 1% glucose. The bcteril suspension for chllenge ws prepred from n 18-h Fildes gr plte culture. The growth ws suspended in buffered sline nd stndrdized turbidimetriclly to contin the desired number of vible cells. The immune response ws ssessed by 7-dy survivl time nd isoltion of Hemophilus from internl orgns of surviving nimls. Animls were utopsied, nd portions of spleen, liver, lung, brin, nd hert blood were cultured on BHI-Fildes gr pltes for the isoltion of vible orgnisms. Sttisticl comprison between groups of mice ws done by the chisqure test. Chemicls. All chemicls used were of regent grde or of the highest purity vilble commercilly. Bovine pncretic trypsin, Pronse, deoxyribonuclese, nd bovine pncretic ribonuclese were purchsed from Worthington Biochemicls Corp., Freehold, N.J.; ribose, deoxyribose, nd crystlline bovine serum lbumin frction V from Nutritionl Biochemicls Corp., Clevelnd, Ohio; mnnide monoolete (Arlcel A) from Atls Chemicls, Wilmington, Del.; nd luminum monosterte from Witco Chemicl Co., New York, N.Y. RESULTS Chrcteristics of ribosomes. The ribosoml preprtions of H. influenze contined pproximtely 75% RNA nd 25% protein but did not contin ny detectble DNA or crbohydrte. When centrifuged over sucrose density grdient, the ribosoml preprtions sedimented s single frction (Fig. 1). The rtios between the bsorbnces t 260 nd 280 nm nd t 260 nd 235 nm were 1.96 nd 1.73, respectively. The sedimenttion pttern ws similr to tht of E. coli ribosomes prepred under similr conditions (Fig. 2). The sedimenttion coefficient of Hemophilus ribosomes ws 67S s determined by the method of Mrtin nd Ames (17), using E. coli ribosomes s the 70S mrker. The ribosomes dissocited into 30S nd 50S subunits fter dilysis for 18 h ginst 0.01 M phosphte buffer contining 10-4 MgCl2 (Fig. 3). Immunogenicity of ribosomes. Mice were immunized with vrying doses of ribosomes with or without incorportion into FIA. As positive controls, mice were immunized s.c. with HAEMOPHILUS RIBOSOMAL VACCINE 455, cj co m6 0.0) 0 Top I Frction Number FIG. 1. Absorbnce pttern of 5 to 20% sucrose density grdient frctions ofhemophilus ribosomes fter centrifugtion t 45,000 rpm for 70 min t 4 C. 0) o A, T Top Frction Number FIG. 2. Absorbnce pttern of 5 to 20% sucrose density grdient frctions of E. coli ribosomes fter centrifugtion t 45,000 rpm for 70 min t 4 C. I -20 0) co -15 O 0cn -10 ) U) C.) -10 co _.

4 456 LYNN, TEWARI, AND SOLOTOROVSKY E c 0 Cuj G) 0co on T Top 30S 50S 70 S Frction Number FIG. 3. Absorbnce pttern of 5 to 20% sucrose density grdient frctions ofhemophilus ribosomes (dilyzed ginst 1O-4 M MgCl2 buffer) fter centrifugtion t 45,000 rpm for 70 min t 40C. either 5 x 103 live cells or 1.5 x 108 formlinized cells of H. influenze (H-i) suspended in djuvnt. Nonimmunized controls received sline or sline plus djuvnt. Three weeks lter, ll nimls were chllenged i.p. with lethl dose of H. influenze type b suspended in gstric mucin. The results of such n experiment re shown in Tble 1. Ribosoml preprtions contining 100, 50, 25, 10, nd 5 jig ofprotein, fter incorportion into djuvnt, protected 60, 80, 60, 25, nd 10% of the immunized mice, respectively. The sme mounts of ribosomes without djuvnt provided lower levels of protection (P < 0.01). The degree of immunity ws dose dependent from 5 to 50 jug of ribosomes. At the 100-,jg level, however, the protection ws lower thn tht with 50 jig of ribosomes (P < 0.01). The protection induced by immuniztion with ribosomes ws comprble to tht obtined with live cells nd ws superior to tht elicited with killed cells (P < 0.01). Blood, spleen, liver, lungs, nd brin of the surviving nimls were free from vible cells ofh. influenze. To determine the optiml intervl between immuniztion nd chllenge, mice were inoculted with live cells or with ribosoml vccines contining 50 jig of protein with or without INFECT. IMMUJN. incorportion into djuvnt. Twenty mice from ech group were chllenged t 1, 2, 3, 4, nd 6 weeks fter immuniztion (Tble 2). Both live cells nd ribosoml vccines produced high degree of protection ginst chllenge with 100 LDO of H. influenze from 1 to 6 weeks fter immuniztion. The live-cell vccine elicited 90% protection t 1 week fter immuniztion; this protection grdully declined to 70% fter 6 weeks. Similrly, protection induced by ribosoml preprtions reched mximum of 95% t 1 week post-immuniztion; this declined to 60% fter 4 weeks. No orgnism ws isolted from the internl orgns of the surviving nimls. Similr results were obtined in two dditionl trils. Effect of multiple immuniztion. The effect of multiple immuniztion ws evluted by s.c. inocultions of one, two, or three doses of ribosomes contining 50 Rg of protein. The first dose of ribosoml preprtion ws incorported into djuvnt; subsequent doses were given without djuvnt t weekly intervls. Controls included mice receiving 5 x 103 live cells ofh. influenze, sline, or djuvnt. Three weeks lter ll nimls were chllenged with 100 LD50 TABLE 1. Survivl of immunized mice fter chllenge with 100 LDO ofh. influenze type b 7-Dy survivl (%) Immunogen With Without djuvnt b djuvnt Live cells, 5 x Killed cells, 1.5 x Ribosomes, 100 l jgc Ribosomes, 50 jig Ribosomes, 25 jug Ribosomes, 10 l g Ribosomes, 5 jug 10 0 Controls 0 0 Ech group consisted of 20 mice. b Freund incomplete djuvnt. c Expressed s protein content of ribosomes. TABLE 2. Survivl of immunized mice fter chllenge with 100 LD50 of H. influenz type b t different intervls fter immuniztions 7-Dy survivl (%) fter chllenge t: Immunogen week weeks weeks weeks weeks Live cells, x 103 Ribosomes, jgb Controls Ech group consisted of 20 mice. b Expressed s protein content of ribosomes.

5 VOL. 15, 1977 of H. influenz (Tble 3). Mice receiving one, two, nd three doses of ribosoml vccine showed 65, 70, nd 85% protection, respectively. The internl orgns of surviving mice were free from vible H. influenze. Effect of different chllenge doses. Mice were inoculted s.c. with live cells, ribosomes, sline, or djuvnt. Immunized nd nonimmunized mice were ech chllenged 7 dys lter with 100, 200, 500, or 1,000 LD.,, of H. influenze (Tble 4). Immuniztion with live cells or ribosomes elicited mximl protection ginst chllenge with 100 or 200 LD., ofh. influenze. There ws significnt reduction in the degree of protection when the chllenge dose ws incresed to 500 or 1,000 LD5, (P < 0.01). No vible cells ofhemophilus were isolted from internl orgns of surviving mice. Similr results were obtined in dditionl trils. Comprtive immunogenicity of ribosomes from nonencpsulted nd encpsulted strins. Mice were immunized s.c. with 25 or 50 Ag of ribosomes extrcted from the encpsulted (H-i) nd the nonencpsulted (H-U) strins ofh. influenz. Controls included mice receiving sline or djuvnt. Three weeks postimmuniztion, ll nimls were chllenged with 100 LD.,, of H. influenze (Tble 5). Ribosoml preprtions from both nonencpsulted nd encpsulted strins produced similr levels of protection: 70 to 75% with 50 1Lg nd 50 to 55% with 25 lug of ribosomes. The internl orgns of surviving mice were free from vible TABLE 3. Effect of multiple immuniztions on immunogenicity of ribosoml vccine Immunogen No. of doses 7-Dy survivl Live cells, 5 x Ribosomes, 50,4gb Controls 3 0 Ech group consisted of 20 mice. b Expressed s protein content of ribosomes. TABLE 4. Effect of different chllenge doses on the immunogenicity of Hemophilus ribosomes Immunogen 7-Dy survivl (%) fter chllenge with: ,000 LD,0 LD,0 LDso LDs, Live cells, 5 x Ribosomes, 50 jugb Controls Ech group consisted of 20 mice. b Expressed s protein content of ribosomes. HAEMOPHILUS RIBOSOMAL VACCINE 457 orgnisms. The immunity elicited by ribosomes ws not type specific nd ws not dependent upon the presence of cpsule. Effect of different djuvnts on immunogenicity of ribosomes. Severl experiments were performed to determine whether other djuvnts tht produce less inflmmtory response could be substituted for FIA in immuniztion with ribosoml vccine. Mice were inoculted with live cells, ribosomes, or ribosomes mixed with either FIA, B. pertussis ntigen, or Hillemn djuvnt. All nimls were chllenged 7 dys post-immuniztion (Tble 6). Ribosoml frction lone protected 60%, nd ribosomes fter incorportion into FIA rised the protection of immunized mice to 95% (P < 0.01). Incorportion of ribosomes into pertussis ntigen or Hillemn djuvnt provided protection equivlent to tht obtined with FIA. The internl orgns of surviving nimls were free from vible cells of H. influenze. DISCUSSION These results demonstte tht immuniztion of mice with ribosoml preprtions from H. influenze elicited significnt protection ginst chllenge with the homologous orgnism. The degree of immunity ws dose dependent from 5 to 50,ug of ribosomes. However, TABLE 5. Survivl of mice immunized with ribosomes from the nonencpsulted nd encpsulted strins fter chllenge with 100 LD50 of H. influenze type b ~~Source of 7-Dy Immunogen ribosomes survivl Ribosomes, 50,ugb Nonencpsulted 70 Ribosomes, 25,g Nonencpsulted 50 Ribosomes, 50 ptg Encpsulted 75 Ribosomes, 25,ug Encpsulted 55 Controls 0 Ech group consisted of 20 mice. b Expressed s protein content of ribosomes. TABLE 6. Effect of different djuvnts on immunogenicity of ribosoml vccine 7-Dy Immunogen Adjuvnt survivl (%) Live cells, 5 x 103 Freund 85 Ribosomes, 50 pgb None 60 Freund 95 Pertussis 85 Hillemn 90 Controls 0 Ech group consisted of 20 mice. b Expressed s protein content of ribosomes.

6 458 LYNN, TEWARI, AND SOLOTOROVSKY t the 100-Iug level the protection ws lower thn tht with 50 1Lg of ribosomes, suggesting n induction of tolernce due to n excess of ntigens. The degree of protection elicited by immuniztion with ribosomes ws comprble to tht obtined with live cells nd ws fr superior to the immunity induced by killed cells. The prolonged survivl of live cells in the host tissues, providing continuing ntigenic stimulus, hs been proposed s mechnism by which live cells induce better immunity (5, 46). The superiority of ribosoml vccine my be due to the lbile nture of ribosoml ntigens, which re preserved by the procedure used in ribosoml extrction but destroyed in killedcell vccine. The incorportion ofhemophilus ribosomes into djuvnt ws necessry to obtin high degree of protection. This is in conformity with the enhncing effect of djuvnt on immunogenicity of ribosoml preprtions from Mycobcterium (47), Stphylococcus (44), Pneumococcus (33, 37), Streptococcus (25), nd Histoplsm (9). In contrst, djuvnt ws not required for immuniztion with ribosoml preprtions from Slmonell (13, 31, 40), N. meningitidis (35), V. cholere (12), nd F. tulrensis (3). The reson for the difference in the requirement of exogenous djuvnt by the ribosomes from different microorgnisms is not known t the present time. FIA produces severe inflmmtory response nd is uncceptble for humn use. However, number of other djuvnts (viz., pertussis vccine, Hillemn djuvnt-65, sodium liginte, nd dextrn) tht produce less severe inflmmtory response my be cceptble for humn immuniztion. In the present study, two of these djuvnts, pertussis vccine nd Hillemn djuvnt-65, were highly effective in enhncing the immunogenicity of Hemophilus ribosomes. Pertussis vccine is known to enhnce the immunogenicity of tetnus nd diphtheri toxoids in the DTP vccine used for immuniztion of children. The ribosomes from nonencpsulted strin of H. influenze were s immunogenic s tht of the encpsulted strin, indicting tht the cpsulr mteril (PRP) is not responsible for immunogenicity of Hemophilus ribosomes. This is further substntited by the fct tht ribosoml preprtions from both encpsulted nd nonencpsulted strins were free from PRP by chemicl tests. The immunodiffusion nd plsive hemgglutintion tests lso filed to detect the cpsulr mteril in ribosoml preprtions ofh. influenze (our unpublished dt). These results re nlogous to recent findings of two seprte groups of investigtors on immunogenicity of subcellulr frctions from Streptococcus pneumonie. Both ribo- INFECT. IMMUN. soml preprtion (33) nd n undefined subcellulr preprtion (36) ofpneumococcus tht were free from detectble cpsulr polyscchride ntigens elicited significnt protection in mice. The protection ws nonspecific since immuniztion with one serotype protected the immunized mice ginst chllenge with other serotypes of S. pneumonie. The cpsulr mteril of H. influenze type b is currently under investigtion s vccine (2, 22-24). Although injection of the polyscchride ntigen to dults nd children over 2 yers old elicited serum ntibody response (22), unequivocl evidence for immunoprotection hs not been demonstrted. Infnts up to 6 months of ge, the logicl cndidtes for Hemophilus vccine, produced little or no ntibody response fter injection with PRP (22). The testing of immunoprotective ctivity of PRP is lso hmpered by the fct tht the mteril cts s hpten in experimentl nimls (22). To overcome this problem, Anderson nd Smith isolted high-moleculr-weight polyscchride from H. influenze type b tht induced serum ntibody response in wenling rbbits (P. Anderson nd D. H. Smith, Prog. Abstr. Intersci. Conf. Antimicrob. Agents Chemother., 14th, Sn Frncisco, Clif., Abstr. 157, 1974). Robbins et l. (23) hve introduced nother concept by demonstrting the formtion of ntibody by feeding nonvirulent E. coli (K-100) tht crossrects with the cpsulr mteril of H. influenze type b. At present they re exploring the possibility of deliberte coloniztion with the nonpthogenic enteric bcteri s method of immuniztion ginst encpsulted bcteri in children. The immunogenicity of the ribosoml preprtions observed in the present study provides n lterntive pproch to immunoprophylxis of Hemophilus infections. At present, we re involved in the chrcteriztion of protective ntigen in Hemophilus ribosomes nd the mechnism of immunity elicited by immuniztion with the ribosoml preprtions. Preliminry experiments hve demonstrted tht the ribosoml protein is the mjor immunogenic moiety in Hemophilus ribosomes. The detils of these findings will be reported in subsequent publiction. ACKNOWLEDGMENTS This investigtion ws supported in prt by reserch grnt from the Chrles nd Johnn Busch Foundtion, Rutgers University, nd by Public Helth Service grnt AI from the Ntionl Institute of Allergy nd Infectious Diseses. The niml fcilities provided by the Bureu of Biologicl Reserch, Rutgers University, re grtefully cknowledged. We re lso indebted to Edwrd Wrner, Southern Illinois University School of Medicine, Springfield, for preprtion of grphs nd photogrphic work for this publiction.

7 VOL. 15, 1977 LITERATURE CITED 1. Anderson, P., R. B. Johnston, Jr., nd D. H. Smith Humn serum ctivities ginst Hemophilus influenz type b. J. Clin. Invest. 51: Anderson, P., G. Peter, R. B. Johnston, Jr., L. H. Wetterlow, nd D. H. Smith Immuniztion of humns with polyribophosphte, the cpsulr ntigen ofhemophilus influenze type b. J. Clin. Invest. 51: Andron, L. A., II, nd H. T. Eigelsbch Biochemicl nd immunologicl properties of ribonucleic cidrich extrcts from Frncisell tulrensis. Infect. Immun. 12: Brown, H. M., nd R. N. Wilson Chronic bronchitis in industry. An ccount of tril of H. influenze vccine. Br. Med. J. 1: Collins, F. M Mechnisms in ntimicrobil immunity. J. Reticuloendothel. Soc Dische, Z Color rections of nucleic cid components, p In E. Chrgoff nd J. H. Dvidson (ed.), The nucleic cids, vol. 1. Acdemic Press Inc., New York. 7. Dische, Z., L. B. Shettles, nd M. Osnos New specific color rections of hexoses nd spectrophotometric micromethods for their determintions. Arch. Biochem. 22: Edwrds, G., A. R. Buckley, E. C. Fer, G. M. Willimson, nd K. Zimmermn Adult chronic bronchitis - the infective fctor nd its tretment. Br. Med. J. 2: Feit, C., id R. P. Tewri Immunogenicity of ribosoml preprtions from yest cells of Histoplsm cpsultum. Infect. Immun.10: Hndzel, Z. T., M. Argmn, J. C. Prke, Jr., R. Schneerson, nd J. B. Robbins Heteroimmuniztion to the cpsulr polyscchride ofhemophilus influenze type b induced by enteric cross-recting bcteri. Infect. Immun. 11: Hillemn, M. R A forwrd look t virl vccines. Am. Rev. Respir. Dis. 90:6& Jensen, R., B. Gregory, J. Nylor, nd P. Actor Isoltion of protective somtic ntigen from Vibrio cholere (Ogw) ribosoml preprtions. Infect. Immun. 6: Johnson, W Ribosoml vccines. I. Immunogenicity of ribosoml frctions isolted from Slmonell typhimurium nd Yersini pests. Infect. Immun. 5: Johnson, W Ribosoml vccines. II. Specificity of the immune response to ribosoml ribonucleic cid nd protein isolted from Slmonell typhimurium. Infect. Immun. 8: Klein, B., nd M. Weissmn New color regent for determintion of hexoses. Anl. Chem. 25: Lowry, 0. H., N. J. Rosebrough, A. L. Frr, nd R. J. Rndll Protein mesurement with the Folin phenol regent. J. Biol. Chem. 193: Mrtin, R. G., nd B. N. Ames A method for determining the sedimenttion behvior of enzymes: ppliction for protein mixtures. J. Biol. Chem. 236: Michels, R. H Increse in influenz meningitis. N. Engl. J. Med. 285: Nyhn, W. L., nd F. Richrdson Complictions of meningitis. Annu. Rev. Med. 14: Preston, P. M., nd D. C. Dumonde Immunogenicity of ribosoml ntigen ofleishmni enriettii. J. R. Soc. Trop. Med. Hyg. 65: Robbins, J. B Acquisition of "nturl" nd immuniztion-induced immunity to Hemophilus influenze type b diseses, p In D. Schlesinger (ed.), Microbiology Americn Society for Microbiology, Wshington, D.C. HAEMOPHILUS RIBOSOMAL VACCINE Robbins, J. B9., R. Schneerson, M. Argmn, nd Z. T. Hndzel Hemophilus influenz type b: disese nd immunity in humns. Ann. Intern. Med. 78: Robbins, J. R., R. Schneerson, T. Y. Liu, M. S. Schieffer, G. Schiffinn, R. L. Myerowitz, G. H. Mc- Crcken, I. Orskov, nd F. Orskov Cross-recting bcteril ntigens nd immunity to disese cused by encpsulted bcteri, p In E. Neter nd F. Milgrom (ed.), The immune system nd infectious diseses (4th Lit. Convoc. mmunol.). S. Krger, Bsel. 24. Rodrigues, L., R. Schneerson, nd J. B. Robbins Immunity to Hemophilus influenz type b. I. The isoltion, nd some physiochemicl, serologic, nd biologic properties of the cpsulr polyscchride of Hemophilus influenze type b. J. Immunol. 107: Schll, W. O., nd W. Johnson Immunogenicity of ribosoml vccines isolted from group A, type 14 Streptococcus pyogenes. Infect. Immun. 11: Schneerson, R., L. P. Rodrigues, J. C. Prke, Jr., nd J. B. Robbins Immunity to disese cused by Hemophilus influenz type b. II. Specificity nd some biologic chrcteristics of "nturl", infectioncquired, nd immuniztion-induced ntibodies to the cpsulr polyscchride ofhemophilus influenze type b. J. Immunol. 107: Schneider, W. C Phosphorous compounds in niml tissue. I. Extrction nd estimtion of deoxypentose nucleic cid nd pentose nucleic cid. J. Biol. Chem. 161: Sell, S. H. W., R. E. Merrill, E. 0. Doyne, nd E. P. Zimsky Long-term sequelle of Hemophilus influenze meningitis. Peditrics 49: Smith, A. L., D. H. Smith, D. R. Averill, Jr., J. Mrino, nd E. R. Moxon Production of Hemophilus influenze b meningitis in infnt rts by intrperitonel inocultion. Infect. Immun. 8: Smith, E. W. P., Jr., nd R. E. Hynes Chnging incidence ofhemophilus influenze meningitis. Peditrics 50: Smith, R. A., nd N. J. Bigley Ribonucleic cidprotein frctions ofvirulent Slmonell typhimurium s protective immunogens. Infect. Immun. 6: Suzuke, K Cellulr frctions oflisteri monocytogenes in reltion to the locliztion of protective ntigen nd monocytosis producing fctor. Yongo Act Med. 14: Swendsen, C. L., nd W. Johnson Humorl immunity to Streptococcus pneumonie induced by pneumococcl ribosoml protein. Infect. Immun. 14: Tewri, R. P Immuniztion ginst histoplsmosis, p In E. Neter nd F. Milgrom (ed.), The immune system nd infectious diseses (4th Int. Convoc. Immunol.). S. Krger, Bsel. 35. Thoms, D. W., nd E. Weiss Response of mice to injection of ribosoml frction from group B Neisseri meningitidis. Infect. Immun. 6: Thompson, H. C. W., nd T. K. Eisenstein Biologicl properties of n immunogenic pneumococcl subcellulr preprtion. Infect. Immun. 13: Thompson, H. C. W., nd I. S. Snyder Protection ginst pneumococcl infection by ribosoml vccine. Infect. Immun. 3: Trub, P., S. Mizushim, C. V. Lowry, nd M. Nomur Reconstitution of ribosomes from subribosoml components. Methods Enzymol Turk, D. C., nd J. P. My Hemophilus influenze: its clinicl importnce, p. 27. English University Press, Ltd., London. 40. Vennemn, M. R., nd N. J. Bigley Isoltion nd prtil chrcteriztion of n immunogenic moiety obtined from Slmonell typhimurium. J. Bcteriol.

8 460 LYNN, TEWARI, AND SOLOTOROVSKY 100: Vennemn, M. R., N. J. Bigley, nd L. J. Berry Immunogenicity of ribonucleic cid preprtions from Slmonell typhimurium. Infect. Immun. 1: Wehrle, P. F., A. W. Mthies, Jr., nd J. M. Leedom The criticlly ill child: mngement of cute bcteril meningitis. Peditrics 44: Winston, S. H., nd L. J. Berry Antibcteril immunity induced by ribosoml vccines. J. Reticuloendothel. Soc. 8: Winston, S. H., nd L. J. Berry Immunity induced by ribosoml extrcts from Stphylococcus u- INFECT. IMMUN. reuse. J. Reticuloendothel. Soc. 8: Youmns, A. S., nd G. P. Youmns Immunogenic ctivity of ribosoml frction obtined from Mycobcterium tuberculosis. J. Bcteriol. 89: Youmns, A. S., nd G. P. Youmns Preprtion nd effect of different djuvnts on the immunogenic ctivity of mycobcteril ribosoml frction. J. Bcteriol. 94: Youmns, A. S., nd G. P. Youmns Fctors ffecting immunogenic ctivity of mycobcteril ribosoml nd ribonucleic cid preprtions. J. Bcteriol. 99: Downloded from on September 26, 2018 by guest