Isolation, Structure, and Immunogenicity of Pseudomonas

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1 INFCTION AND IMMUNITY, Feb. 1989, p /89/2426-6$2./ Copyright 1989, American Society for Microbiology Vol. 57, No. 2 Isolation, Structure, and Immunogenicity of Pseudomonas aeruginosa Immunotype 4 High-Molecular-Weight Polysaccharide GRALD B. PIR'* AND MATTHW POLLACK2 Channing Laboratory, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 2115,' and Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland 2142 Received 28 July 1988/Accepted 22 October 1988 A high-molecular-weight, immunogenic form of the lipopolysaccharide side chain of Pseudomonas aeruginosa Fisher immunotype 4 (type 4, International Antigenic Typing System 1, Lanyi :6) was isolated and characterized. Analysis by nuclear magnetic resonance spectroscopy confirmed the structural similarity of this high-molecular-weight polysaccharide and the type 4 side chain. The polysaccharide was immunogenic in rabbits and mice, eliciting opsonophagocytic killing antibodies. Immunization with the polysaccharide produced significant protection against homologous challenge in both burned and granulocytopenic mice. Naturally acquired opsonic killing antibodies to type 4 polysaccharide were present in sera from unimmunized normal adults at levels comparable to postimmunization levels achieved after immunization with other type-specific polysaccharides. The specificity of the naturally occurring antibodies for the side chain was documented by immunoblot analysis and inhibition studies. Naturally occurring polysaccharide-specific antibodies were comparable in their protective activity against live challenge in neutropenic animals to immunization-induced murine antibodies with similar specificity. These data suggest that naturally occurring serum antibody to P. aeruginosa type 4 lipopolysaccharide side chains in most adults is not distinguishable in quantity or quality from immunization-induced antibodies in mice; evaluation of type 4-specific vaccines in humans may be complicated by this finding. The serotypes of Pseudomonas aeruginosa are distinguished by chemical and immunologic differences among the -specific polysaccharide (PS) side chains of the lipopolysaccharide (LPS). Structures of the side chains for most of the clinically important serotypes have been determined (reviewed in reference 8). The serotype designated in the Fisher-Devlin scheme as type 4 (International Antigenic Typing Scheme 1, Lanyi :6 and Homma I) is a homogenous one (5). No variant or related side-chain structures have been described for this serotype, as have been for most other P. aeruginosa serotypes (8). Since immunity to P. aeruginosa infection in immunocompromised hosts is principally (but not exclusively) mediated by antibody to the side chains (2, 24), vaccines that elicit serotype-specific immunity could be important in developing active and passive immunotherapies for this disease. One approach to producing these vaccines has been the development of high-molecular-weight PS variants of the side-chain structure (12, 14). High-molecular-weight PS from Fisher types 2 and 3 have been described and confirmed by nuclear magnetic resonance (NMR) spectroscopy to be structurally identical to their corresponding side chains (12, 14), as has the high-molecular-weight PS from type 1 (unpublished observation). The large size of the high-molecular-weight PS is critical for immunogenicity, and the lack of toxicity of high-molecular-weight PS is likely due to undetectable levels of lipid A components. We have now produced high-molecular-weight PS from a Fisher type 4 strain of P. aeruginosa and determined its chemical constituents and structure by NMR. In addition, we have evaluated the ability of this PS to induce in rabbits and mice type-specific opsonic killing antibody as well as protection against bacterial challenge. Moreover, an analysis of normal human serum has indicated a high level of naturally occurring * Corresponding author. 426 opsonic killing antibody to the type 4 PS, a finding not observed in the case of heterologous serotype antigens (1, 12). MATRIALS AND MTHODS Bacterial strains. A clinical isolate from the blood of a patient with P. aeruginosa bacteremia, strain JR, was used for the isolation of high-molecular-weight PS antigen and for live bacterial challenges of experimental mice. This strain was serologically identical to the Fisher type 4 prototype strain, ATCC Additional clinical isolates of type 1, 4, and 6 P. aeruginosa were used in the opsonophagocytosis assays. Serotype was determined by agglutination with type-specific antisera. Antigen preparation. High-molecular-weight PS was prepared in Columbia broth passed through membranes (1,- molecular-weight cutoff) before use. A Biolaffite 2-liter fermentor was used containing approximately 16 liters of media. After 72 h of growth the ph of the fermentor culture was adjusted to 5.1 with glacial acetic acid and then heated at 91 C for 18 h. Purification then followed previously described protocols, except that the acetic acid hydrolysis step was omitted (1, 15, 18). LPS was prepared from strain JR as described previously (15). Most of the LPS was recovered from the phenol layer of the phenol-water extract, as was the case with type 2 LPS (16). High-molecular-weight PS intrinsically labeled with 14C was prepared as described previously (11). Chemical and structural determinations. Chemical analyses of the protein, nucleic acid, lipid, 2-keto-3-deoxyoctonoate, and sugar constituents of the high-molecular-weight PS and LPS were performed as described previously (13). Monosaccharide constituents were identified and quantified by gas-liquid chromatography of the trimethyl silyl derivatives (13) with two different columns (SP233, Supelco, Inc., Bellafonte, Pa; RSL-31, Alltech Associates, Deerfield, Ill.).

2 VOL. 57, 1989 P. ARUGINOSA TYP 4 HIGH-MOLCULAR-WIGHT PS 427 The columns were injected simultaneously with identical samples. ach column gave rise to separate retention times and peak areas associated with the individual peaks of different monosaccharides (the monosaccharide fingerprint) allowing unequivocal identification of individual sugars. "C NMR spectra were determined on a Bruker AM 3 instrument in D2 at 5 C with methanol as an internal standard. Toxicity studies. Standard Food and Drug Administration assays for toxicity in mice and guinea pigs were carried out as described previously (15). Limulus lysate gelation assays were performed according to the instructions of the manufacturer (Cape Cod Associates, Woods Hole, Mass.). Pyrogenicity was evaluated in rabbits according to Food and Drug Administration guidelines (12). Serologic analysis. Immunodiffusion analysis was performed by the Ouchterlony technique (9). Sera were obtained from rabbits immunized with the high-molecularweight PS as described previously (15), from mice given two 1-,ug doses of PS intraperitoneally (i.p.) 5 days apart, and from normal adults (generally laboratory workers and medical students without documented exposure to type 4 P. aeruginosa). Quantitation of antibody levels in sera was performed by using a radioactive antigen-binding assay (11). Immunoglobulin isotypes specific for serotype 4 antigen were determined by using a radioimmunoprecipitin test (18). Opsonic killing titers were measured in an opsonophagocytosis assay. Heat-inactivated sera were used as the opsonin source, and fresh normal human serum (1% concentration) was used as a complement source after adsorption with 1 mg of Formalin-fixed and lyophilized strain JR organisms per ml at 4 C for 3 min. Human leukocytes obtained by dextran sedimentation (18) were used as the phagocytic cell source. Samples (.1 ml) containing the following reagents were added to sterile microfuge tubes: heat-inactivated antiserum, 1% adsorbed fresh human serum, 2 x 16 bacteria, and 2 x 16 leukocytes. A 25-ptl sample was removed from selected tubes at the start of the experiment, diluted, and plated in duplicate onto Trypticase soy agar plates for enumeration of bacteria. The tubes were tumbled end over end for 9 min at 37 C; 25-pld samples were then removed, diluted, and plated onto agar for bacterial enumeration. The percent kill was calculated as follows: percent kill = 1 - [(number of CFU surviving at 9 min/number of CFU determined at min) x 1]. Titers were defined as the reciprocal of the serum dilution yielding -7% killing of the input inoculum. Immunoblot analysis. The type 4 LPS was analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and by immunoblotting after transfer to nitrocellulose (22). These immunoblots were then probed with 1:5 dilutions of normal human serum and visualized as described by Blake et al. (1). Animal protection studies. Burn infections were produced in mice by the method of Stieritz and Holder (21) after immunization with two 5-,ug doses of high-molecularweight PS given 5 days apart. Challenge was 7 days after the second dose of PS. The neutropenic mouse model of Cryz et al. (4) was modified as follows. Mice were given cyclophosphamide (2 mg/kg i.p.) every other day for 5 days and challenged i.p. 2 days after the third dose of drug. Deaths were recorded for 1 days. Strain JR had a 5% lethal dose (LD5) of log1o 3.2 (95% confidence interval, Log1o 2.64 to 4.63) as determined by probit analysis (6) with five mice per dose and five- and 1-fold dilutions of bacteria. Active immunization of neutropenic mice was accomplished by giving two 5-Rg doses of high-molecular-weight PS or two, A FIG. 1. Immunodiffusion analysis of the type 4 high-molecularweight PS. Wells: center, antisera to whole cells of type 4 P. aeruginosa; A, fraction 1 of high-molecular-weight PS; B, acetic acid hydrolyzed LPS; C, fraction 2 of high-molecular-weight PS; D, intact LPS. 1-,ug doses of LPS 5 days apart then giving the cyclophosphamide regimen 5 days after the second dose of antigen. Passive immunization with immune animal and normal human sera was performed by giving.2 to.5 ml of serum (depending on the antibody quantity determined in the radioactive antigen-binding assay) i.p. 2 days after the final cyclophosphamide dose and 2 h before i.p. bacterial challenge. Statistical analysis. The total antibody levels and opsonic killing titers in rabbit and mouse sera pre- and postimmunization were compared by using a t test. Analysis of the protective efficacy of the vaccines in mice was performed by using either a Spearman-Karber analysis of the differences in LD5s between immune and nonimmune groups (6) or by a Mann-Whitney U test on the length of time of survival (6). RSULTS Characterization of high-molecular-weight PS. Isolation of the type 4 high-molecular-weight PS was initially hampered by low yields of the material with previously described methods (15). We then determined that after growth of the culture for 72 h at 37 C with vigorous aeration (.5 liter per min) we could enhance recovery of high-molecular-weight PS by lowering the ph of the entire fermentor culture to 5.1 and then heating the culture to 91 C for 18 h. This resulted in recovery of >7 mg of antigen with an approximate molecular size of 2 kilodaltons (fraction 1), and >8 mg of antigen with an approximate molecular size of 1 kilodaltons (fraction 2). Presumably heating the culture at an acidic ph after growth enhanced the extraction and/or solubilization of the PS material before further purification. Immunodiffusion analysis of these two PS fractions resulted in a single precipitin line reacting with rabbit antisera to whole bacterial cells (Fig. 1). These precipitin lines gave reactions of identity with LPS side chains obtained by acetic acid hydrolysis of intact LPS. Chemical analysis of the two PS fractions (Table 1) indicated that they contained little protein, nucleic acid, lipid, 2-keto-3-deoxyoctonoate, or neutral sugars. Amino sugar analysis accounted for only 22 to 25% of the material. However, gas-liquid chromatography of the trimethylsilyl derivatives (performed simultaneously on two different col-

3 428 PIR AND POLLACK TABL 1. Chemical analysis of type 4 high-molecular-weight PS Weight % Component Method of analysis Fraction Fraction 1 2 Protein Dye assay Nucleic acid Spectrophotometry.5.4 Neutral sugar Colorimetry Amino sugar Colorimetry Keto-3-deoxyoctonoate Colorimetry <.1a <.1a Lipids Gas chromatography <.1a <.1a Carbohydrate Gas chromatography a Lower limit of detection. umns) identified 2-acetamido-2-deoxy galactose (GalNAc), 2-acetamido-2,6-dideoxy glucose (QuiNAc), and 2-acetamido-2,6-dideoxy galactose (FucNAc) based on retention times identical to those of authentic standards. A fourth series of peaks was present in a different configuration after reduction of the high-molecular-weight PS with carbodiimide (23). Based upon the analysis by Dmitriev et al. (5) this fourth component was assumed to be 2,3-diacetamido-2,3- dideoxy glucuronic acid [Glc(NAc)2A]. Confirmation of the presence of these four sugars was obtained by using 13C NMR spectroscopy (Fig. 2). The spectrum determined for fraction 1 (>2 kilodaltons) of the high-molecular-weight PS was identical to that of the much smaller (<2-kilodalton) LPS side-chain PS previously reported by Dmitriev et al. (5) for the Lanyi :6 strain of P. aeruginosa. The side chain from this strain is serologically identical to that of the Fisher type 4 strain. From this analysis it appears that the high-molecular-weight PS from type 4 P. aeruginosa has the following structure (see abbreviations above):-*4)dgalnac(ot->4)dglc(nac)2a(,l-*3) DFucNAc(otl-*3)DQuiNAc(otl--. INFCT. IMMUN. Toxicity studies. A 25-,ug dose of type 4 high-molecularweight PS produced no apparent toxic reactions or alterations in weight gain after i.p. injection into 36 to 37-g guinea pigs or 2-g mice. A 1.4-[xg/kg dose of PS produced a.2 C temperature rise over 3 h in two rabbits and no temperature rise in a third rabbit. The aggregate rise of.4 C was within the Food and Drug Administration designated limits for pyrogenic activity. One hundredfold more highmolecular-weight PS was required compared with type 4 LPS to gel a Limulus lysate (1, versus 1 ng/ml). The scherichia coli endotoxin control was positive at.2 ng/ml. Immune responses in animals. Rabbits and mice immunized with fraction 1 of the type 4 high-molecular-weight PS responded with significant (P <.1) increases in the level of binding antibodies (Fig. 3). The specificity of these antibodies for the type 4 determinant was shown by inhibition studies, wherein cold type 4 PS, LPS, and side chains inhibited antibody binding to the 14C-labeled PS, whereas similar preparations from types 1 and 6 P. aeruginosa did not. These antisera also had opsonic killing titers against strain JR of.4 (Fig. 3), whereas preimmune sera from rabbits and mice failed to mediate killing when used undiluted in the opsonophagocytosis assay. We also noted opsonic killing of three heterologous type 4 strains of P. aeruginosa by these antisera and no killing of type 1 and 6 strains (data not shown). Type 4 high-molecular-weight PS and LPS inhibited opsonic killing of these immune animal sera, whereas type 1 and 6 PS and LPS did not (data not shown). nzyme-linked immunosorbent assay of the mouse immune sera indicated responses in both the immunoglobulin M (IgM) and IgG isotypes. Antibodies in normal human sera. Analysis of human sera from normal adults revealed high levels of antibody to the type 4 high-molecular-weight PS in all individuals examined (Fig. 4). The antibodies were roughly equally distributed ). PPM FIG C NMR spectrum of the type 4 high-molecular-weight PS.

4 VOL. 57, 1989 P. ARUGINOSA TYP 4 HIGH-MOLCULAR-WIGHT PS 429 A B 12 7._ Pro-immune Post-immune.2 U , I / IL 2 /l... / / /-d Serum dilution Serum FIG. 3. Animal immune response to type 4 high-molecular-weight PS. (A) Total antibody responses from three rabbits (1) and 1 mice (H). (B) Opsonic killing activity in indicated serum dilution found in pooled sera from 3 rabbits (H) and 1 mice (U). Bars represent means, and error bars represent the standard deviations. among the IgG, IgM, and IgA classes (Fig. 4). These naturally acquired antibodies were also opsonic in the phagocytic killing assay at titers comparable to those observed after immunization with type 1 and 2 high-molecularweight PS (Fig. 4) (1, 12). Specificity of the opsonic killing antibody for the type 4 antigen was demonstrated in inhibition studies with purified PS and LPS antigens (Fig. 4). When the type 4 LPS was separated by polyacrylamide gel electrophoresis and transferred to nitrocellulose, normal human sera reacted with the ladderlike side-chain bands (Fig. 5). The specificity of these antibodies for the side- a 3 - a 2 U m 1 A o 1 28 chain structure identical to that of the high-molecular-weight PS was shown by the ability of PS antigen to inhibit binding in the immunoblot (data not shown). Normal human sera used at a dilution of 1:5 failed to react with polyacrylamide separated and blotted LPS from the other six Fisher types of P._aeruginosa (data not shown). Animal studies. Immunization of mice with type 4 highmolecular-weight PS resulted in an increased LD5 after homologous type 4 challenge in the burn infection model (Table 2). Similarly, immunization before induction of neutropenia with cyclophosphamide resulted in resistance to Y D O _ C 4 A B I ol ol 1 ol C _ 3 o.. cn a,.2 Intact +T-4.T4 *T-1 +T-1 PS LPS PS LPS Human Sera Preparations FIG. 4. Human serum antibody levels and activities of type 4 (T-4) high-molecular-weight PS and organisms. (A) Total antibody specific for type 4 high-molecular-weight PS in 19 normal human sera, and percentage of antigen-specific IgG, IgM and IgA (percentage of total antibody). Bars represent means, and lines represent 95% confidence intervals for total antibody and range for the percentages of the different isotypes. (B) Opsonic titer (log2) of 19 normal human serum against type 4 P. aeruginosa. Serum preparations were either intact or inhibited by type 1 or 4 high-molecular-weight PS or LPS as indicated. Bars represent mean opsonic titers, and lines represent the titer ranges.

5 43 PIR AND POLLACK INFCT. IMMUN. FIG. 5. Immunoblot analysis of antibodies in normal human sera with immunotype 4 LPS antigen. Lanes show reaction of 1:5 dilutions of six normal human sera with blotted LPS. challenge with a dose of 5 x i4 bacteria per mouse, equivalent to 43 LD5s (95% confidence interval, 1.2 to 114 LD5s). This dose was lethal to 1% of nonimmune mice. Immunization of neutropenic mice with 1,ug of type 4 LPS also was protective, whereas immunization with 1,ug of a heterologous P. aeruginosa LPS (type 1) was not. This later control assured us that nonspecific activation of the immune system due to endotoxin contamination of the PS preparation was not responsible for the observed results, since the PS could have maximally contained only.5 p.g of endotoxin based on the Limulus gelation results. Passive transfer to neutropenic mice of.2 ml of type 4 high-molecular-weight PS-specific rabbit antisera also protected against bacterial challenge, as did 2,ug of antibody ih normal human sera (Table 2). Adsorption of the normal human serum with type 4 organisms removed the protective activity of this serum, whereas adsorption with two other serotypes of P. aeruginosa had no effect (Table 2). TABL 2. Active and passive protection of mice with type 4 (T-4) high-molecular weight PS, LPS, and sera Model Immunogena (>jg) LD5 aliveb control Burn infection T-4 PS (5) 8.8 x 17 Saline 1.4 x 17 <.5 Neutropenic T-4 PS (5) 8.7 mouse T-4 LPS (1) 1.2 T-1 LPS (1) 1 NS Saline.2 ml of IRS ml of NRS 1 2,Lg of Ab in NHS ,ug of Ab in NHS adsorbed with: T-4 cells 1 T-1 cells 9.19 T-6 cells a IRS, Immune rabbit serum; NRS, normal rabbit serum; NHS, normal human serum; Ab, antibody. b A total of 1 mice were tested. The P value for the burn infection model was determined by the method of Spearman-Karber (6) for the neutropenic mouse model determined by a Mann-Whitney U test on the length of survival up to 1 days. The controls for type 4 PS and LPS immunizations were saline-immunized animals; control for passively transferred immune rabbit serum was normal rabbit serum; the control for normal human serum and normal human serum adsorbed with type I and type 6 cells was normal human serum adsorbed with type 4 cells. DISCUSSION We have described the isolation and characterization of a high-molecular-weight form of the P. aeruginosa type 4 LPS O side chain, analogous to similarly obtained materials isolated from types 1 through 3 (13-16). This PS was composed mostly of carbohydrate, was serologically and structurally identical to the type 4 side chain, was nontoxic, and elicited binding and opsonic antibodies in immunized rabbits and mnice. Increased yields of type 4 highmolecular-weight PS were obtained by a change in methodology used to extract and purify the PS without affecting size, toxicity, or immunogenicity. Structural analyses confirmed that the stationary cultures of P. aeruginosa used to isolate high-molecular-weight PS were, in fact, producing high-molecular-weight species of the LPS side chain. We believe that these antigens are a result of the synthesis during the stationary phase of side chains that are not attached to the LPS core. Cadieux et al. (2) have provided evidence that P. aeruginosa continues synthesis of LPS components after the organisms enter the stationary phase. Our results suggest that side chains produced during the stationary phase are much larger in size than those found on the LPS from younger (18-h) cultures. Antibodies to the type 4 high-molecular-weight PS protected burned or granulocytopenic mice against lethal bacterial challenges. High levels of naturally occurring antibody to the type 4 high-molecular-weight PS were also detected in 19 normal human sera. These antibodies mediated killing in an opsonophagocytosis assay and bound to the side-chain component of the type 4 LPS in an immunoblot. The levels of naturally occurring type 4-specific serum antibody were comparable to those observed in humans immunized with other high-molecular-weight PS preparations. Interestingly, type 4 P. aeruginosa is a relatively common serotype isolated from the blood of infected patients. In a study by Pier and Thomas (17) type 4 strains accounted for 41 (14.6%) of 281 bacteremic isolates obtained in four hospitals over a 7-year period. It was the third most common serotype in this study. This raises questions regarding the role of naturally acquired antibodies specific for type 4 LPS in preventing or modulating P. aeruginosa bacteremia. No study has directly addressed this issue, but a study of acute-phase infection antibody levels of individuals with P. aeruginosa type 4 bacteremia may delineate whether these antibodies are decreased in these individuals (thus explaining, in part, the lack of protection by serum antibody) or at levels comparable to those reported here, which would suggest that naturally occurring antibodies afford little protection to these patients. Current interest in the prevention and treatment of P. aeruginosa infections centers on passive immunoprophylaxis and therapy, respectively. Possible vehicles for such therapies include normal IgG prepared for intravenous use (IGIV), IgG obtained from humans prescreened for high titers of preexisting antibody, immune IgG preparations obtained from immunized volunteers, and monoclonal antibodies. The presence of high levels of naturally occurring type 4-specific antibody in humans, about one-third of which is IgG, suggests that normal serum could be a rich source of these antibodies. Pollack (19) measured hemagglutinating antibody titers to the seven Fisher LPS types in 27 lots of IGIV from seven manufacturers. Data for different lots of IGIV from the same manufacturer prepared in an identical manner were averaged. In six of seven cases the titer to type 4 was higher than it was to the other six types, although

6 VOL. 57, 1989 P. ARUGINOSA TYP 4 HIGH-MOLCULAR-WIGHT PS 431 some of the titers to non-type 4 antigens were within one tube dilution of the type 4 titer. In one lot of hyperimmune IGIV prepared from plasma obtained from volunteers immunized with a hepatavalent P. aeruginosa LPS vaccine, the titers to all seven LPS were within two dilutions of each other (256 through 1,24). Whether the higher titer to type 4 LPS seen in the hyperimmune product compared with that in normal human IVIG was a result of a specific immune response was unclear, since preimmunization titers of this population were either not measured or not reported. The question of whether immunization with a type 4 antigen would boost serum antibody levels in normal adults bears directly on the development and use of type 4 vaccines. Relevant in this regard is the fact that primary human immune responses to bacterial polysaccharides are usually not augmented by subsequent immunization (7). If the naturally occurring levels of type 4-specific antibody are near the maximum response level of particular individuals, subsequent immunization with high-molecular-weight PS will have little effect. Alternatively, a polysaccharide-protein conjugate vaccine, such as the one recently reported by Cryz et al. (3), might increase type 4-specific antibody levels if high specific antibody levels were required for adequate protection. The isolation of another high-molecular-weight PS antigen from P. aeruginosa extends the general applicability of this methodology to producing immunogenic, nontoxic serotypespecific vaccines. Since the efficacy of the high-molecularweight PS as a vaccine has been demonstrated, its immunogenicity may be enhanced by conjugation to protein carriers as has been shown for other bacterial polysaccharides (4). The ultimate utility of these antigens will depend upon a thorough understanding of human immunity to P. aeruginosa and the development of reagents capable of enhancing the specific immunity of patients at risk. ACKNOWLDGMNTS This work was supported by Public Health Service grants Al (to G.B.P.) and Al 2276 (to M.P.) from the National Institutes of Health and by a Research Scholar Award to G.B.P. from the Cystic Fibrosis Foundation. We thank Nina A. Kocharova, Yuriy A. Knirel, and Alexander S. Shashkov for recording the "3C NMR spectra. LITRATUR CITD 1. Blake, M. S., K. H. Johnston, G. J. Russell-Jones, and. C. Gotschlich A rapid, sensitive method for detection of alkaline-phosphatase conjugated antiantibody on Western blots. Anal. Biochem. 136: Cadieux, J.., J. Kuzio, P. H. Milazzo, and A. M. Kropinski Spontaneous release of lipopolysaccharide by Pseudomonas aeruginosa. J. Bacteriol. 155: Cryz, S. J., Jr.,. Furer, A. S. Cross, A. Wegmann, R. Germanier, and J. C. 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