Bases for the early immune response after rechallenge or component vaccination in an animal model of acute Mycoplasma pneumoniae pneumonitis

Vaccine, Vol. 13, No. 3, pp. 305-309, 1995 Coovriaht cc? 1995 Elsevier Science Ltd Printed’ ;n Great Britain. All rights reserved 0264-410)(195 $10.cm+0.00

Bases for the early immune response after rechallenge or component vaccination in an animal model of acute Mycoplasma pneumoniae pneumonitis Nevio Cimolai”,

Diana G. Mah, Glenn P. Taylor

and Brenda J. Morrison

The pathology of Mycoplasma pneumoniae pulmonary infection for a hamster model was examined after whole bacterium rechallenge or component vaccination. Animals which, qfter an initial infection, were rechallenged with either live or heat-killed M. pneumoniae inocula developed severe early recall lesions in thefirst 3 days. In contrast, animals infected once develop maximum histopathology at approximately IO-14 days. A severe perivascular inflammatory cellular injltrate developed in the rechallenged groups, and pulmonary pathology could also be elicited by rechallenge with bacterial growth medium components. Component vaccination with protein PI did not reduce disease in comparison to once-infected controls, and vaccination promoted an early immune recall response as well. We conclude that an earl-y immune response needs to be sought in all future experiments of challenge/rechallenge or vaccination. Vaccine studies will require an understanding of both protective and harmful immunogens. Keywords: M_vcoplasma pneumoniae; pneumonia;

pathology

Mycoplasma pneumoniae is a common cause of community-acquired respiratory disease. The lymphocytic peribronchiolar infiltrate, bronchiolar epithelial metaplasia, peribronchiolar septal widening, and intrabronchial luminal exudate serve to explain the diverse clinical and radiographic findings in human disease’. Since the discovery of the bacterium three decades ago, several experimental models of pulmonary infection have been developedzp4 and similarities are evident for human and model disease. As the understanding of the frequency and potential severity of human disease increased, the desire to define strategies for disease prevention increased, as exemplified by the attempts at vaccine development. In the context of emerging advances in viral vaccines, M. pneumoniae vaccines were utilized in human trials as early as the 1960~~~~. These studies were terminated for several reasons; the most compelling concern was the experience of some immunized volunteers who developed more severe illness after experimental challenge with live Division of Medical Microbiology, Department of Pathology, and Department of Health Care and Epidemiology, The University of British Columbia, Vancouver, Canada. *To whom correspondence should be addressed at: Program of Microbiology, Department of Pathology, British Columbia’s Children’s Hospital, 4460 Oak Street, Vancouver, British Columbia, Canada V6H 3V4. (Received IO February 1994; revised 30 June 1994; accepted 12 July 1994)

bacterium. Although the latter adverse event has never been fully explained, the hypothesis, which proposes that prior sensitization to the bacterium may subsequently lead to an accentuated immune response, has been utilized to explain the severity of community-acquired infection in adulthood. In essence, the delayed-type hypersensitivity might recruit a cell-mediated immune response which is responsible in large part for the pulmonary pathology. In animals, an accentuated immune response after rechallenge was documented previously*. Such findings were also believed to validate further the hypothesis that delayed hypersensitivity is a major factor in the development of acquired immunity’. Subsequent studies have reaffirmed an enhanced disease after reinfection3 and, furthermore, other investigations also support these findings by proposing that some component vaccines may also sensitize the host3,10. In our current investigations, we attempt to identify further the basis for accentuated immune responses in the animal host. We examined the histopathological response in challenge/rechallenge experiments and in component vaccination with protein Pl.

METHODS Bacterium and growth media M. pneumoniae strain Ml85 was the sole isolate which was utilized for all experiments where either live or

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Early immune response to M. pneumoniae: N. Cimolai et al.

heat-killed bacteria were required. The bacterium was cultivated in Hayflick’s medium as previously described1 ’ and the serum base of this medium contained either horse serum (Gibco Laboratories, OH) or rabbit serum (Sigma, MO) depending upon the experiment. The broth base, yeast dialysate, and serum for each inoculum medium were of the same lot number. Bacterial inocula were adjusted so as to approximate 1 x 104-1 x lo5 c.f.u./lOOpl. Heat-killed bacterial inocula were prepared by heating the organism in its growth medium to 70°C for 25 min. The bactericidal effect of heat was confirmed by the inability to cultivate the bacterium after 6 weeks incubation on both solid and liquid media.

Rechallenge experiments One set of experiments was designed to examine the basis for early immune recall during either reinfection or secondary medium component challenge after initial infection. Table 1 describes an overview of these experiments and Table2 details the inocula and timing of animal harvest. Five animals were included in each group except for group II(iii) which had six animals. All hamsters were initially infected with live M. pneumoniae inocula which had been prepared in a horse serum-based liquid Hayflick’s medium. Animals were then rechallenged after 6 weeks with live bacterium (grown in horse or rabbit serum-based media), heat-killed bacterium (rabbit serum-based medium), uninoculated horse serum-based

Pl antigen preparation Pl, a major immunogenic protein of 16&170 kDa, is recognized as being part of an adhesion complex, and is strongly recognized in the acute humoral response12. Pl was purified by electroelution13. For the latter, whole cells were lysed after boiling in sample buffer”. The sample was separated on 5% sodium dodecyl sulfatepolyacrylamide resolving gels. Portions of a gel were stained with Coomassie blue so as to localize Pl and then the gel containing Pl was excised and reduced to cubes of side 3-5 mm. The cubes were electroeluted at 60 mA for 6 h (BioRad ElectroEluter). The product was proven to retain immunological reactivity by Western blotting. The Western blotting was performed with alkaline phosphatase-labelled anti-human IgG and a positive human serum as previously described14.

Female golden hamsters were placed on an inclining headboard after gaseous anaesthesia. Each animal received both intranasal and intratracheal inocula (100 ~1). After resuscitation, animals were separately housed in containment. Lungs were harvested after 1, 3 or 10 days of inoculation depending upon the experiment. The latter hamsters were killed with an excess dose of pentobarbital, and sagittal sections of right and left lungs were obtained for histopathological review. Pulmonary tissue was placed into 10% buffered formalin for fixation and paraffin-embedded sections were stained with haematoxylin and eosin.

Histopathological scoring Our previously described pulmonary pathology scoring scheme was used to assess lung sectionslo. This method is based on the description of pathology related to peribronchiolar and peribronchial cellular infiltrates, bronchiolar and bronchial luminal exudates, perivascular cellular infiltrates, and parenchymal pneumonia. A score per animal, in the range O-26, is derived by averaging the score from each lung. The overall score is weighted with emphasis on the bronchial and bronchiolar disease since these sites manifest the majority of pathology at all gradations of infection. The set of microscopy slides from each animal were scored simultaneously although the animal number and study source were unknown to the observer.

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Synopses of experiment protocols

A. Challenge-rechallenge 6 weeks (a) Infection -

Rechallenge Harvest at 1, 3 or 10 days same live inocula l live in alternative serum . heat-killed l complete broth medium . complete broth without medium

l

Harvest at 10 days

initial infection -

(b) Control uninfected 8. Pl immffnization

2 weeks (a) Pl immunized -

2 weeks

Pl immunized -

Infection --+

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Table 2 protocol

Detailed hamster challenge

Group

Challenge

l(i) l(ii) l(iii) II(i) ll(ii) ll(iii) Ill(i) lll(ii) IV(i) IV(ii) V VI

Live Live Live Live Live Live Live Live Live Live Live Nil

Mp Mp Mp Mp Mp Mp Mp Mp Mp Mp Mp

1 hs* hs hs hs hs hs hs hs hs hs hs

Harvest at 3 or 10 days

tlnfection -

Harvest at 10 days

and rechallenge

experimental

(b) Control unimmunized

Hamster model

306

Table 1

Challenge

28

Live Mp hs Live Mp hs Live Mp hs Live Mp rsc Live Mp rs Live Mp rs Killed Mp rsd Killed Mp rs Liquid medium hse Liquid medium hs Base medium‘ Live Mp hs

Day of lung harvest after challenge 2 1 3 10 1 3 10 3 10 3 10 3 10

‘Challenge 2 was given 6 weeks after challenge 1 %ombined intranasal and intratracheal challenge with lo“-10’ colony-forming units (c.f.u.) of live M. pneumoniae which were suspended in horse serum-based medium ‘Combined intranasal and intratracheal challenge with 104-lo5 c.f.u. of live M. pneumoniae which were suspended in rabbit serum-based medium dCombined intranasal and intratracheal challenge with l@-105 c.f.u. equivalent of heat-killed M. pneumoniae which were suspended in rabbit serum-based medium *Combined intranasal and intratracheal challenge with an equivalent volume of uninoculated horse serum-based Hayflick’s liquid growth medium ‘Combined intranasal and intratracheal challenge with an equal volume of uninoculated serum-free Hayflick’s liquid growth medium

Early immune response to M. pneumoniae:

media, or serum-free Hayflick’s broth base. A control group of animals, who were not initially infected, received their first live challenge at the same time that other animals were rechallenged. Animal lungs were then obtained at 1, 3 or 10 days after the final challenge depending upon the experimental group. All animals, regardless of group, were of the same age. Samples of right and left lung from some animal groups were cultured for M. pneumoniae on both solid and liquid Hayflick’s medium. Pl immunization

and challenge

Table 1 summarizes the protocol. Hamsters were immunized twice intraperitonally with 2Opg of Pl (dialysed against isotonic saline) and an equal volume (100~1) of complete Freund’s adjuvant. The immunizations were administered 14 days apart, and 14 days after the last injection hamster sera were assayed for IgG anti-P1 by immunoblotting. The immunoblotting substrate was developed as described above. Hamster sera were diluted 1: 100 and an alkaline phosphatase-labelled goat anti-hamster antibody (Jackson Immunoresearch, PA), which was diluted 1:500, was used as the secondary antibody. Of the 14 animals so immunized, only eight were considered to have a significant IgG anti-P1 response demonstrable. The eight immunized animals were challenged the next day (essentially 2 weeks after the last vaccine) with live bacteria via both the intranasal and intratracheal routes as for our other experiments. A control unimmunized group of five hamsters was similarly infected on the same day. Five of the eight immunized and infected animals were harvested on day 10 postinfection, as were the controls. The remaining three immunized and infected animals were killed on the third day. Both histological assessment and culture were performed on right and left lungs. Statistical

scores

for

animals

from

Cimolai et al.

infection

and

rechallenge experiments

Group

Mean score

Standard deviation

I(i)

16.0 15.6 6.7 la.7 17.3 a.7 14.6 7.3 7.1 4.0 4.0 15.8

1.98 3.80 1.07 0.67 4.06 I .a5 4.40 1.86 1.89 0.39 0.81 2.86

I(ii) I(iii) II(i) Il(ii) ll(iii) Ill(i) lll(ii) IV(i) IV(ii) V VI

Comparisons’ a,d.m a,e,l,n a-l,0 b,f,m b,g,l,n b,j,o c,h,l.m c,k,o q

q d,e.f,g,h,i,j,k

“Comparisons: a, p
analysis

The Mann-Whitney rank tests were used to assess the probability that a significant difference existed between animal groups.

RESULTS Phenomena

Table 3 Histopathology

N.

of rechallenge

The mean pulmonary histopathology scores for animal groups in the infection and rechallenge study are listed in Table3. The details of the histopathology were analogous to those we have previously describedlO (see discussion of histopathological scoring in Methods). A common feature of the histopathology is periluminal cellular infiltrate and intraluminal inflammation. A representative example of severe peri- and intraluminal disease is shown in Figure 1. Animals who were initially infected and later rechallenged with either live or killed M. pneumoniae inoculum developed significantly greater pathology within the first 3 days of rechallenge as compared with the disease at day 10 (~~0.05 for all comparisons of day 10 scores to day 1 or day 3 within groups I, II and III). Scores for lungs harvested at day 1 or 3 from groups I, II and III were either slightly worse or not significantly different from comparably aged animals who were infected once (group VI) but examined

Figure 1 Representation of pulmonary histopathology in the hamster model. Photomicrograph illustrates typical peribronchiolar infiltrate and intraluminal exudate. Haematoxylin and eosin stained, bar represents 4-Oum

at day 10. Significantly less disease was apparent from day 10 tissue of groups I, II and III than group VI (p < 0.05 for day 10 comparisons of groups I, II and III to group VI). A striking feature of the early pathology after rechallenge was the marked degree of perivascular cellular infiltrate (Figure 2). The perivascular changes were relatively minor in animals infected once (group VI) despite the overall high cumulative scores [pO.lO for same-day intergroup comparisons) would support the notion that the severe early recall pathology is not a function of organism viability. In addition to equivalent mean scores, these three groups also shared elevated perivascular scores. Although sensitization to horse serum had been proposed as a detriment to vaccine development in the past5 when horse-based media were utilized to grow M. pneumoniae,

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Figure 2 Prominent perivascular cellular infiltrate in rechallenge animals. This specimen was taken from an animal of group I(ii) (day 3). Haematoxylin and eosin stained, bar represents 40 pm

we found equivalent pathology when rabbit serum was chosen as an alternative. The role of the serum component as a cause of some early pathology is furthermore supported by differences in severity between groups IV(i) and V. Lung tissue from group IV(i) did exhibit perivascular infiltrates as were seen in the early pathology of rechallenge from groups I, II and III. All cultures from lung samples of animals from group VI were positive. Colour change in broth media, which is indicative of bacterial growth and pH change related to acid production from glucose, occurred by days 8 and 11 after inoculation. In contrast, only 3/22 samples were culture-positive from group I(iii) and group II(iii) (day 10) samples. The frequency of positive cultures increased to 8/20 samples from group I(ii) and group II (day 3) and to 14/20 samples for group I(i) and group II(i) (day 1). All of the latter positive cultures were associated with colour changes in broth media which occurred from 11 to 28 days postinoculation. Pl immunization

Pathology scores from unimmunized animals (mean score 17.3, s.d. 2.51) were not significantly different from PI immunized animals at day 10 (mean score 15.6, s.d. 1.70) (p = 0.25 1) but an early recall response was apparent at day 3 for Pl immunized animals (mean score 21.1, s.d. 1.57; p=O.O25 for comparison of day 3 Pl immunized to day 10 of either immunized or unimmunized). The accentuated day 3 response was accompanied by greater perivascular infiltrates (p< 0.01 for day 3 immunized versus day 10 immunized or unimmunized) and these were analogous to perivascular infiltrates of animals from the previously detailed challenge-rechallenge experiments. There was no significant difference between immunized and unimmunized animals for either frequency of positive cultures or time to colour change in broth media. DISCUSSION Our results raise concern over the definition of protection. Most studies of protection have examined disease at 14 days after infection regardless of whether animals have previously experienced challenge or passive/active immunization3*8*g~‘519. Histological examination performed only at the latter time will undoubtedly not

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capture the very severe and early events which we have detailed and which others have previously noted**‘. The majority of pathology at day 10 to 14 in those rechallenged will therefore presumably represent residual disease in convalescence from the dramatic early recall. From the clinical perspective of the animal host, however, there is likely to be as much, if not more, illness from the early lower respiratory tract involvement. The early response should be considered a failed prevention of clinical and histopathological pneumonia when the measures of disease (e.g. histopathological scoring scheme) are equivalent regardless of timing. Alternatively, previous live challenge diminishes the frequency of positive cultures as well as increasing the time for cultures to become positive. The latter findings do not imply necessarily that less disease has occurred in the animal host. The phenomenon of the early recall immune response would suggest that the host has developed an accentuated response to a previously encountered antigen. Is this the antigen of the bacterium or of the exogenous antigens that accompany it? Are there several antigens, bacteriumspecific and/or non-specific, which contribute to the same events? The avidity of growth medium components to mycoplasmas is recognized”. Clyde had previously suggested that horse serum from liquid culture media may have a sensitizing effect* and thus heterologous serum was subsequently employed in rechallenge experimentsg. Our findings lead us to hypothesize that growth medium sera do contribute to the early immune recall but that sensitization is independent of the animal source. Secondly, a recall effect is not dependent upon the viability and multiplication of the organism since the accentuated response followed rechallenge with nonviable bacterium. Despite the similarity in mean score between the usual pathology in animals infected once and recall response in those rechallenged, the two groups differed in the degree of perivascular reaction. In the challengerechallenge experiments, an intense perivascular mononuclear infiltrate was a marker of an accentuated early immune and presumably a sensitization response. Such a phenomenon had been part of the recall lesions that others have proposed as evidence for implicating delayed hypersensitivity as a major contributor to acquired immunityg. A role for delayed hypersensitivity and its consequences in the development of pulmonary disease in acute infection has been projected by some. This theory would contend that repeated infection presensitizes the human host so that a subsequent infection would include an exaggerated (i.e. early recall) immune response. The clinical manifestations would then be a result of both bacterium-induced pathology and the pathological consequences of the immune reaction to the same. Contrary to this hypothesis, however, epidemiological studies do not support an age-related increase in the severity of M. pneumoniae illnesses. As well, a similar incubation time for both children and adults argues somewhat against the notion of enhanced disease with prior exposure. Furthermore, post mortem pathology and open lung biopsy descriptions do not detail disease which we can acknowledge as a correlate of the perivascular infiltrate in the animal rechallenge studies. Experience with component vaccination for M. pneumoniae is limited. We have assessed the potential of

Early

a 43 kDa antigen for immunization” and found accentuated disease in challenged animals who were vaccinated. Such study has relevance to our current work in that, first, severe perivascular infiltrates were noted, and secondly, immunized animals developed mild pathology when challenged with sterile broth. Both of these observations support a role for non-bacterial antigen in the accentuated disease of the animal model. We did not, however, examine tissue earlier than 10 days after live challenge in these former studies. Previous studies with Pl as an antigen have not demonstrated protection3~21,22 and have also suggested that sensitization might occur. The latter studies examined pulmonary histopathology after 224 weeks in a guinea-pig model. Our findings are in part supportive of the latter but are complicated by the recognition of an accentuated early response within 3 days. The early response along with marked perivascular infiltrates again reminds us that a response to accompanying non-P1 antigens (i.e. from media) is possible. It is reasonable to expect that electroelution techniques, which extract Pl antigen from whole cell-derived profiles, will lead to contamination of product with medium or other antigens that co-migrate in the resolving gel. Component vaccine studies may require antigens of greater purity. Our studies ultimately challenge some prevailing thoughts concerning M. pneumoniae infection and immunity. Several studies have shown a protective effect of anti-M. pneumoniae polyclonal antisera even when the sources of such sera are other animal species16-“. These studies, along with others4, support a role for humoral immunity in protection. We propose that it would be prudent to exclude an early immune response in studies of passive immunity. Exclusion of such an event would strengthen the cause for further research on vaccine strategies. Is there a role for cell-mediated immunity in protection and can the hypothesis of delayed-type hypersensitivity as a cause of pulmonary disease be maintained? Our data detract from the aforementioned role and hypothesis. Future studies need consistently to address the possibility of early recall responses in challenge-rechallenge and vaccination studies. Our histopathological scoring scheme would appear to be a valuable aid in any such studies in view of the ability to both score total pathology or subscore correlates of accentuated early disease such as perivascular infiltrates.

ACKNOWLEDGEMENTS This work was supported in part by the British Columbia Health Research Foundation. Animal care approval was obtained from the University of British Columbia in accordance with the Canadian Council on Animal Care.

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Cimolai, N., Malleson, P., Thomas, E. and Middleton, P.J. Mycoplasma pneumoniae associated arthropathy: confirmation of the association by determination of the antipolypeptide IgM response. J. Rheumatol. 1989, 16, 115&l 152 15 Fernald, G.W. and Clyde, W.A. Protective effect of vaccines in experimental Mycoplasma pneumoniae disease. Infect. Immun. 1970,1, 559-565 16 Hayatsu, E. Acquired immunity to Mycoplasma pneumoniae pneumonia in hamsters. Microbial. Immunol. 1978, 22, 181-195 17 Hayatsu, E., Kawakubo, Y.. Yayoshi, M., Araake, M., Yoshioka, M. and Nishiyama, Y. Role of humoral antibodies in resistance to Mycoplasma pneumoniae pneumonia in hamsters. Microbial. Immunol. 1980, 24, 888-593 16 Hayatsu, E.. Kawakubo, Y., Yayoshi, M., Araake, M., Wakai. M., Yoshida, A. et a/. Immunological responses of hamsters in the acquired immune state to Mycoplasma pneumoniae infection. Microbial. Immunol. 1981, 25, 1255-1263 19 Barile. M.F., Chandler, D.K.F., Yoshida, H., Grabowski, M.W. and Razin. S. Hamster challenge potency assay for evaluation of Mycoplasma pneumoniae vaccines. Infect. Immun. 1988, 66, 2458-2487 20 Sugiyama, T. Medium components absorbed to mycoplasma cells. Yale J. Biol. Med. 1983, 58, 691-693 21 Jacobs, E., Stuhlert, A., Drews, M., Rock, R., Pumpe, K., Schaeffer, H.E. and Bredt, W. Cellular and humoral reactions to the adhesin of Mycoplasma pneumoniae. Zentralblatt. fur Bacterial. 1990, 2o(Suppl), 598-801 22 Jacobs, E., Stuhler-t, A., Drews, M.. Pumpe, K., Schaefer, H.E.. Kist, M. and Bredt, W. Host reactions to Mycoplasma pneumoniae infections in guinea pigs preimmunized systematically with the adhesin of this pathogen. Microb. Pathog. 1988, 5, 259-265 14

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