Protective Effect of Vaccines in Experimental

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1 INFECTION AND IMMuNrY, June 9, p American Society for Microbiology Protective Effect of Vaccines in Experimental Mycoplasma pneumoniae Disease Vol., No. Printed in U.S.A. GERALD W. FERNALD AND WALLACE A. CLYDE, JR. Departments of Pediatrics and Bacteriology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 24 Received for publication February 9 The mechanisms of immunity to Mycoplasma pneumoniae were investigated by evaluating different vaccination procedures in an experimental animal model. Hamsters were immunized by intranasal inoculation of broth cultures or by parenteral injections of saline-suspended organisms. All vaccinees received a standardized intranasal challenge which produced pneumonia in 94% of controls. Intranasal immunization with virulent organisms produced a % reduction in pneumonia. Subcutaneous and intraperitoneal inoculation of the same organisms yielded and % reductions, respectively. Animals similarly immunized with an attenuated strain developed resistance to pneumonia only after intranasal infection. Serum antibody levels did not correlate with protection. Growth-inhibiting activity was demonstrated in bronchial washings of challenged animals, suggesting the development of local antibody in response to infection with M. pneumoniae. Crosschallenge studies were performed in animals vaccinated intranasally with virulent and avirulent variants of the same strain. The avirulent vaccine prevented pneumonia in animals challenged with homologous virulent organisms but not in those receiving an unrelated strain; the virulent vaccine provided protection to both homologous and heterologous challenge. These studies indicate that organism strain variation as well as vaccination technique are important determinants of the immune response to M. pneumoniae. Investigation of the pathogenesis of respiratory infections due to Mycoplasma pneumoniae has been facilitated by the availability of a suitable animal model, the Syrian hamster (Mesocricetus auratus; ). Previous reports from this laboratory have described the pulmonary lesions (2) and the immune response () produced by intranasal inoculation of live M. pneumoniae organisms. Infection by the nasal route was demonstrated to provide a significant degree of resistance to reinfection and to prevent the development of pneumonia in all animals. Intraperitoneal inoculation with killed vaccine failed to induce immunity to intranasal infection, and all challenged animals developed pneumonia (). These preliminary experiments suggested that parenterally administered M. pneumoniae vaccines were immunogenically inferior to direct infection of the respiratory tract. Since only two M. pneumoniae strains were employed and each was inoculated by a single route, the possibility that variations in these factors might affect immunogenicity was not resolved. The present studies were designed to examine the influence of route of administration and virulence of mycoplasma strains on induction of immunity to intranasal challenge. Results indicate that intranasal infection with virulent or attenuated organisms produced the most protection, although significant immunity was conferred by parenteral injection of virulent organisms. MATERIALS AND METHODS Animals. Young adult, male Syrian hamsters were used for all experiments. The techniques of infecting animals and collecting materials for analysis have been detailed elsewhere (). M. pneumoniae strains. All organisms were cultivated in standard media (2). Viability counts were established by counting colony-forming units (U) in serial plate dilutions. The standard challenge in all experiments was the sixth broth passage of strain PI 4, obtained from R. R. Gutekunst, Parris Island, S.C. Its virulence [defined as ability to produce histological pneumonia consisting of peribronchial round cell infiltration with or without intraluminal polymorphonuclear exudation (2,, ) in 8% or more of hamsters on the 4th day of infection] is documented in Table. By means of the Reed-Muench method, the % infectious dose (D) was determined to 9

2 FERNALD AND CLYDE INFEC. IMMUN. TABLE. Pathogenicity ofm. pneumoniae PI 4 (standard challenge strain) in hamsters Logio U' Infected/inoculatedb Pneumonia! inoculatedc. / / 4. 4/4 /4. / 2/ 2. / /. / / a Colony-forming units in. ml of intranasal inoculum. bfifty per cent infectious dose (ID) = 2. U. c Fifty per cent pneumonia-producing dose (PD) = 4. U. be 2. U, and the % pneumonia-producing dose (PDo) was " U (4). All animals were challenged with approximately U in. ml (or PDI). Strain Mac M, in high passage (egg, 8; agar, 2; broth, 2), was found previously to be avirulent (no pneumonia) when given in a dose of. ml containing U (). Strain Mac H, a strain which again became virulent after eight serial passages of Mac organisms in the hamster, provided the opportunity to study a homologous pair of virulent and avirulent organisms (). Immunization procedures. Intranasal inoculation was performed under sodium pentobarbital anesthesia with. ml of culture containing approximately U. Intraperitoneal (ip) and subcutaneous (sc) injections of. or.2 ml of saline-washed, concentrated organisms contained an estimated 8 U per ml; total protein determined by the Lowry method was 4 mg/ml. Material for footpad injections was prepared by emulsification of equal parts of vaccine and HRa complete adjuvant (Difco);. ml of the mixture was given in each hind footpad. Microbiological and histological studies. On the 4th day after challenge inoculation, animals were exsanguinated under anesthesia and lungs were removed for quantitative culture and histological examination as previously detailed (2). In some experiments, bronchial lavage with 2 ml of saline was performed in situ (). Bronchial washings were heated at C for min and were stored at -2 C prior to analysis. Lung lesions were graded according to severity of the histological reaction: + = single or minimal lesions; 2+ = two or more definite infiltrates; + = all bronchi affected. The degree of pneumonia in affected animals was averaged and expressed as a numerical "score" for each experimental group. Serological determinations. Complement-fixing () and growth-inhibiting antibodies in serum and bronchial washings were determined by microtitration techniques (). titers were expressed as -log2 dilutions. Growth-inhibiting antibody was expressed in units, unit being that amount of activity required to produce a two-fold reduction in organism dilution end point compared with the control (9). RESULTS Effect of route of administration on antigenicity of M. pneumoniae. To determine the effect of vaccine route on the antigenicity of M. pneumoniae, hamsters were inoculated by a variety of methods. Live cultures of strains PI 4 and Mac M were inoculated intranasally, and saline suspensions of the same strains were injected ip and sc. Footpad injections with PI 4 in complete adjuvant were included because of the efficiency of this method in raising antibody with small amounts of antigen, and because of the possibility that this method would induce delayed hypersensitivity. Two to three months after immunization, all vaccinees were bled by cardiac puncture. The serological responses of animals in several experiments are presented in Table 2. Differences between antibody titers raised by the various immunization schedules were not great. In both PI and Mac vaccinees, the intranasal route elicited relatively higher growth-inhibiting levels than did sc or footpad inoculation. On the other hand, parenteral inoculation appeared to yield the highest titers. These data suggested that both intranasal infection and parenteral immunization were effective in stimulating serum antibody production. Effect of route of administration on immunogenicity of M. pneumoniae. The immunogenic TABLE 2. Effect of various routes of administration on the antigenicity of M. pneumoniae vaccines in hamsters Serum antibodyb Strain and routea hao.mos (-log2) (units) PI 4 Intranasal..... Intraperitoneal..... Subcutaneous Footpad MAC M Intranasal Intraperitoneal Subcutaneous Uninoculated a Intranasal inoculum = U; intraperitoneal =. ml of saline suspension (at U) five times; subcutaneous =.2 ml of saline suspension three times; footpad =. ml with complete adjuvant in each hind foot. bmean complement-fixing () and growthinhibiting () antibody determined 2 to months after vaccination.

3 VOL., 9 MYCOPLASMA PNEUMONIAE VACCINES TABLE. Effect of various routes of aci'ministration per lung section. The lesion score in intranasal on the immunogenicity of M. pneumoniae vac- Mac vaccinees with pneumonia was of the same cines in hamsters order as the similar strain PI group, suggesting a challengeb Immunization' strain and route ) n Results of ( Infection No. Per cent No. protective effect by this route only. In all experiments, the number of animals with Pneumonia positive lung cultures was greater than the number with pneumonia. This suggested that Per Scorec immunization affected the infectivity of the cent challenge strain less than its pathogenicity. _ However, since the challenge dose was designed PI 4 to contain PDSO, it also contained nearly, Intranasal... 2 Intraperitoneal >2 'DEO (Table ). Such an overwhelming infectious Subcutaneous challenge easily could have overcome any resist- Footpad ance attained in this model. Reduction in infectivity therefore appeared to be much more MAC M Intranasal difficult to achieve and was convincingly demon- Intraperitoneal strated only in the intranasal PI 4 vaccinees. Subcutaneous.. 2. The failure of the Mac M vaccine to protect against PI 4 in the foregoing experiments Uninoculated _4 raised the possibility that immunity induced by a See footnote one a, Table 2. M. pneumoniae strain might be directed b Intranasal challenge with IJ of virulent only against a homologous challenge organism. PI 4; sacrificed on day 4. To examine this possibility, the protective effect c Mean degree ( to -+) of pneur monia in af- of infection with virulent organisms was tested fected animals. against homologous and heterologous challenge strains in the experiment shown in Table 4. effect of various vaccine routes was determined Twenty-four hamsters immunized by intranasal by administering a standard intranassal challenge inoculation of PI 4 were divided into four of U of strain PI 4 to each of the groups of six, each with an equal number of animals shown in Table 2. Mean reesults of lung matched, unimmunized controls. After a -month cultures and histological examinatiion of lung convalescence period, the surviving animals in sections for each group of vaccineaes are pre- each group were challenged with one of four sented in Table. In the nonimmunized controls, virulent strains, including PI 4. All but 94% of animals were infected by tlhe challenge one of the controls developed pneumonia, inoculum and the same number deve monia. Examination of the data for lopped pneu- whereas only one or none of each group of in PI 4 vaccinees revealed a lovv attack rate vaccinees sustained lesions. It was concluded in intranasal (2 %) ip ( %), and sc (8r%) that no observable difference existed between recipients. In contrast to this, to % of hamsters immunized with the Mac vaccine, by TABLE 4. Resistance of intranasally infected ham- sters to subsequent challenge with homologous the same three routes, developed pneiumonia. For purposes of statistical analysiss, intranasal, and heterologous M. pneumoniae strains ip, and sc vaccinees for each strairi were comintrols. The Pneumonia in bined and matched with the cc challenged hamsters difference in pneumonia observed in strain PI Challenge strain (source)a vaccinees and controls was highl y significant PI (x2 = 2.9; P = <.). The Mac vaccine was 4b Unimmunized vaccinees controls less effective in preventing pneumornia; as com- this group Bru (Chapel Hill, 9). /4c / pared with the controls, protection in was of borderline significance (> zc2 = 4.4; C (Chapel Hill, 94).. / / P = <.). The pneumonia rate in strain PI M29 (Chapel Hill, 9).. / / vaccinees was significantly less thean in strain PI 4 (Parris Island, Mac vaccinees (x2 =.; P = <I.2). 9)... / / The histological scoring of lunig lesions in Totals... 2/ 22/2 affected animals further supports thesse differences (Table ). Animals in the protected groups a Each contained to U in. ml. showed few areas of infiltrate, whereeas those with b Infected months previously. high pneumonia attack rates had several lesions c Number with pneumonia/number challenged.

4 2 FERNALD AND CLYDE INFEC. IMMUN. TABLE. M. pneumoniae antibody levels in hyperimmunized hamsters (results of intranasal challenge) Animala no. I Means > Prechallenge antibodiesb Antibodies Postchallenge Serum Bronchial Serum Bronchial > > 9 > < 4 S. Infection (U/g of lung), -9 Pneumonia (score) c a Immunized with. ml of adjuvant-treated vaccine in footpad, followed after 2 months by. ml of booster ip 2 and weeks prior to challenge. I See footnote b, Table 2; 2 ml of bronchial washings heat-inactivated but not concentrated before testing. Extent of lesions as in Table. animals challenged with the homologous or heterologous organisms tested. Effect of hyperimmunization on resistance to challenge. The foregoing experiments lent further support to the concept that humoral antibody per se does not confer resistance to M. pneumoniae. Although this suggested the presence of local immune factors within the respiratory tract, higher levels of serum antibody might have afforded protection. In a test of these alternatives, hamsters were hyperimmunized by footpad and ip inoculation and were examined individually (Table ). Prior to challenge, serum was collected from all animals, and in four animals bronchial lavage was performed via tracheostomy. At termination of the experiment, all animals were bled and subjected to bronchial lavage, after which the lungs were removed for culture and sectioning. and growth-inhibition tests were performed on the unconcentrated bronchial washings. Examination of the data in Table reveals that high serum levels of antibody were stimulated by the vaccine schedule employed; the mean growth-inhibiting titer was not significantly different from that produced by the vaccine schedules shown in Table 2. Neither type of antibody was detected in bronchial washings from immunized animals before challenge. After challenge, there was a tendency for serum antibody levels to rise. This was apparently in 2. response to proliferation of organisms within the lung. No protection was afforded by the elevated serum antibody, as evidenced by the presence of pneumonia in six of eight animals. The presence of growth-inhibiting antibody in terminal bronchial washings was of interest, because it was not demonstrated prior to challenge in a sample of four hyperimmunized animals. In contrast, activity was detected in the bronchial secretions of only one animal, despite titers in serum in the :2 range. These data suggested a qualitative difference between the antibody appearing in the bronchial secretions and in serum. Relation of virulence to immunogenicity. If direct local infection of the respiratory tract is necessary for effective immunization with M. pneumoniae, then development of vaccine for human use would be feasible only if virulence could be attenuated while immunogenicity was preserved. The Mac strain, originally recovered from a fatal case of atypical pneumonia (), became avirulent after multiple passage on artificial media; however, the immunogenicity of this strain was also decreased. Direct passage of Mac strain-infected lung suspension in hamsters resulted in recovery of virulence; i.e., % of animals developed pneumonia (). Whether this strain with renewed virulence (Mac H) also had regained its immunogenicity was tested in an experiment comparing the protective effect

5 VOL., 9 TABLE. Relative immunogenicity of virulent and avirulent homologous strains of M. pneumoniae No. of No. Pneumonia Vaccinea ham- Challengea instrain sters organism fected No. Score Mac Mb Mac H 9 - Mac M PI Mac H Mac H. Mac H PI 4. Control Mac H 8. Control PI a Live M. pneumoniae, ' U given intranasally. b Produces no pneumonia in hamsters given. ml ( U) intranasally. of Mac M and H vaccines against homologous and heterologous (PI 4) challenge. The results (Table ) show that the Mac M vaccine, which was barely immunogenic in previous experiments (Table ), produced resistance to pneumonia from homologous challenge in all of animals tested. H vaccine was equally effective against both homologous and heterologous challenge. Approximately % of challenged animals in each category developed pneumonia, compared with 8% of controls (X2 =.8; P = <.); scoring of these lesions averaged.4 compared with 2. for controls. Thus, Mac M vaccine was protective in reference to its virulent counterpart, but not against a heterologous virulent strain. This suggests that strain variation may be an important consideration in selecting an attenuated vaccine candidate. DISCUSSION In these studies, two methods of immunization were found to promote resistance to M. pneumoniae: intranasal inoculation of live organisms, and injection of killed mycoplasmas. When challenged with a standard inoculum, intranasally vaccinated hamsters developed less pneumonia than animals given injections of inactivated vaccine. Attenuation diminished, but did not eradicate, immunogenicity of the live vaccine strain. Conversely, it was observed that the effectiveness of an inactivated vaccine was related directly to the degree of virulence of the strain from which it was prepared. Human volunteer studies have shown that naturally acquired M. pneumoniae infection provides resistance to reinfection (). Attempts to develop an attenuated live vaccine by repeated MYCOPLASMA PNEUMONIAE VACCINES passage of the organism on artificial media resulted in a decrease of virulence concomitant with a decrease in antigenicity and infectivity for man (, ). Although these findings discouraged further development of a live vaccine, the present study suggests that the method is feasible provided the properties of the organism can be standardized and controlled. In this regard, the hamster model could be used for all but the final stages of testing. Inactivated M. pneumoniae vaccines have been prepared and evaluated by several investigators. Most preparations have been sufficiently antigenic to induce various serological responses (, 2), but only one was tested for protective effect in a large field trial (). In this latter study, although over % of the vaccinees developed growth-inhibiting antibodies for M. pneumoniae, the pneumonia attack rate was reduced only 4 %. A potential hazard to further evaluation of inactivated vaccines was suggested by a volunteer study in which challenge inoculation produced more severe disease in some vaccinees than in unimmunized control subjects (, ). The mechanisms of this anomalous effect is unknown, although the problem occurred among vaccinees who failed to develop detectable serological responses (). In view of the results obtained in the hamster model, it would seem that only M. pneumoniae strains of high virulence should be employed in developing future killed vaccines. An apparent correlation of serum growthinhibiting antibody with resistance to natural reinfection () and to challenge with artificially propagated strains of M. pneumoniae () has been reported. However, the validity of assuming that serum antibody necessarily equates with protection has been questioned by the same investigators (). Studies of M. pneumoniae infection in hamsters have shown that serum antibodies are not direct mediators of resistance, although they regularly accompany it. For example, previously infected animals with minimal serum growth-inhibiting levels were more resistant to challenge than parenterally immunized animals with high serum titers (). Such observations suggest that the antigenicity of M. pneumoniae strains (i.e., the property of stimulating antibody) is not the same as their immunogenicity (i.e., the ability to provoke specific resistance). The present study provides further evidence that immunity to M. pneumoniae in the hamster is most effectively induced by infection with virulent organisms, implying the importance of local immune phenomena. Attempts to measure antibody in bronchial washings of immune

6 4 FERNALD AND CLYDE INFEC. IMMUN. animals were unsuccessful; however, growthinhibiting antibody was detected in pulmonary secretions of these animals after challenge inoculation (Table ). It is unlikely that this result reflected exudation of serum, since the activity accompanying serum growth-inhibiting levels was absent from the bronchial washings. Earlier studies collating human immunoglobulin classes with M. pneumoniae serological activity revealed that growth-inhibiting antibody comprised IgM, IgA, and IgG, while activity was mediated by IgM and IgG (8). In the hamster, IgM contributes very little to either or growth-inhibiting antibody, nearly all of the activity distributing in IgG-containing serum fractions (). The presence of growth-inhibiting activity and the absence of activity in hamster bronchial secretions thus may indicate accumulation of IgA during the postchallenge period, although this could not be determined directly owing to insufficient material for analysis. In an earlier experiment, where both and growth-inhibiting activity were found in bronchial washings, most of the antibody was identified as IgG (). In retrospect, it is apparent that some of these animals may have been dead or dying when bronchial lavage was performed, therefore allowing serum IgG to enter the bronchial lumen (8). In the present experiment, care was taken to use light anesthesia during the lavage procedure: the lack of activity in washings suggests that the normal barrier to serum antibody was not compromised. In addition to humoral and secretory antibody, cellular immunity also could mediate acquired resistance to M. pneumoniae. Although use of adjuvants may foster delayed hypersensitivity, hamsters immunized with adjuvant-treated vaccine failed to resist challenge inoculation. Furthermore, intradermal skin testing of these animals with M. pneumoniae antigens and tuberculin (purified protein derivative) revealed no cutaneous reactivity (G. W. Fernald, unpublished data). Preliminary results obtained by use of a similar procedure in guinea pigs indicate that delayed hypersensitivity can be stimulated by M. pneumoniae antigens. Further work is needed to assess this immune mechanism in relation to experimental and natural disease. The apparent importance of local immune phenomena in M. pneumoniae disease has a counterpart in contagious bovine pleuropneumonia. This infection, caused by M. mycoides, is contracted by the respiratory route and produces a severe systemic, often fatal, disease. Serum antibodies are generated as a result of natural infection or vaccination, but they do not relate to resistance; neither does convalescent serum confer immunity (4). Examination of nasal mucus of infected animals for growthinhibiting antibodies has been negative. These observations have led to the assumption that some form of local immunity, perhaps cellmediated, is responsible for resistance to M. mycoides in cattle (). Further insight into host immune mechanisms as well as factors of organism pathogenicity are germane to development of effective immunoprophylaxis against M. pneumoniae. Implications from the data presented are (i) that local immune factors are of primary importance in mediating resistance, (ii) that means must be found to preserve immunogenicity of live attenuated strains, and (iii) that killed vaccines should be prepared from fully virulent organisms. ACKNOWLEDGMENTS This work was conducted under sponsorship of the Commission on Acute Respiratory Diseases, Armed Forces Epidemiological Board, and was supported by the U.S. Army Medical Research and Development Command, Department of the Army (contract DA-49-9-MD-289). G. W. Fernald is the recipient of Research Career Development Award l-k4-ai-4244-, National Institute of Allergy and Infectious Diseases. W. A. Clyde, Jr., is the recipient of Research Career Development Award - K-AI-9-, National Institute of Allergy and Infectious Diseases. We thank F. W. Denny for many helpful comments and continued interest throughout these studies. The technical assistance of Ruth Roghelia is also gratefully acknowledged. LITERATURE CITED. Couch, R. B., T. R. Cate, and R. M. Chanock. 94. Infection with artificially propagated Eaton Agent (Mycoplasma pneumoniae). J. Arner. Med. Ass. 8: Dajani, A. S., W. A. Clyde, Jr., and F. W. Denny. 9. Experimental infection with Mycoplasma pneumoniae (Eaton's Agent). J. Exp. Med. 2:-8.. Davies, G. 99. The examination of nasal mucus for antibodies to Mycoplasma mycoides. Vet. Rec., p Davies, G., and J. R. Hudson. 98. The relationship between growth-inhibition and immunity in contagious bovine pleuropneumonia. Vet. Rec., p Eaton, M. D., G. Meiklejohn, and W. Van Herick Studies on the etiology of primary atypical pneumonia. I. 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