Br. J. exp. Path. (1975), 56, 17
PATHOLOGICAL ASPECTS OF IMMUNIZATION OF MICE AGAINST INFLUENZA VIRUS INFECTION A. BASKERVILLE, G. THOMAS* AND S. PEACOCK From the Microbiological Research Establishment, Porton Down, Salisbury, Wilts.
Received foI publication 19 August 1974
Summary.-Groups of mice were immunized against influenza Ao/NWS virus by a single intranasal administration of inactivated homologous virus, by 2 intranasal doses of vaccine separated by an interval of 2 weeks, or by 2 intraperitoneal doses of the same vaccine. When subjected 2 weeks later to a standard challenge of 6 x 105 egg infecting units Ao/NWS virus instilled intranasally, mortality fell significantly from 64% in unimmunized mice to 39% in mice given a single intranasal dose of vaccine and to 29% in animals which received double intranasal vaccine. The best protection was conferred by double intraperitoneal immunization, after which mortality was 10%. Immunity waned with time, since the mortality of mice doubly immunized by the respiratory route and challenged 30 weeks later was 49o%. Intrapulmonary lymphoid tissue developed in large amounts in a proportion of mice immunized by all methods and challenged after an interval of 2 weeks. Attention is drawn to this reaction as a possible unfavourable consequence of vaccination. There were no lesions in the lungs or central nervous system after immunization without subsequent challenge. The importance of histopathology in vaccine trials in experimental animals is emphasized by the consistently higher detection rate of lesions in lungs by histological examination than by visual inspection alone. IN RECENT YEARS attempts have been made to utilize the local defence mechanisms of the respiratory tract for immunization against viral respiratory disease, in particular influenza. Respiratory immunization has now been extended from studies on experimental animals (de St Groth and Donnelley, 1950) to numerous clinical trials on human volunteers (Waldman et al., 1968; Van Kirk, Mills and Chanock, 1971; Downie, 1973; Thomas, 1973) and inactivated influenza vaccines are now commercially available for use in man. However, relatively little attention has been paid to the changes which may be induced by such vaccines in the lungs or in the central nervous system, which can also be damaged in a small proportion of human cases of natural influenza infection (Hoult and Flewett, 1960). To investigate this risk, a series of experi*
ments was undertaken with mice using the
NWS strain of influenza/AO virus to study not only the protection afforded by respiratory vaccination but also the longterm effects on the lungs and brain of single and double respiratory immunization alone and with subsequent challenge. In addition, the study was used to assess the value of histological examination of organs in trials of vaccine efficacy and to compare this with the standard methods of recording survival and the presence of visible lesions after challenge. MATERIALS AND METHODS Female outbred Porton albino specific pathogen-free mice 3-6 weeks old were used in a series of experiments. A mouse-adapted strain of influenza AO/NWS virus was employed in all the experiments and a pool of this virus was prepared by inoculating it into the allantoic cavity of 11-day old embryonated eggs and harvesting
Pr'esent address: Essex Chemie AG, Lutcerne, Switzerland1.
18
A. BASKERVILLE, G. THOMAS AND S. PEACOCK
the fluid after 3 days' incubation at 37°. This stock virus was stored in aliquots of 10 ml at - 700 and had a titre of 6 x 105 egg infecting units (EIU) per 005 ml. Mice were infected by instilling 0-05 ml of virus pool suspension into the nostrils under light ether anaesthesia, care being taken to ensure that the entire volume of the drops was inhaled and retained. Two groups of animals (Groups J and K) were infected by exposure to an aerosol of influenza virus in a Henderson apparatus (Henderson, 1952). The calculated retention dose of virus per mouse in this experiment was 9 04 x 102 EIU, based on the lung retention in this apparatus measured by Harper and Hood (1962). For use in the vaccine experiments, the NWS strain virus pool was inactivated by incubation with formalin and treated as recommended in the British Pharmacopoeia (1968). 0 05 ml of this vaccine was administered to anaesthetized mice either intranasally (i.n.) or intraperitoneally (i.p.). A further 50 mice were inoculated i.n. with 0-05 ml of sterile physiological saline. The mice were used in groups as follows: Group A.-300 to provide data on mortality in unimmunized mice and 100 for sequential histopathology of lungs and brain and immunofluorescence studies of lungs after i.n. infection with 6 x 105 EIU NWS influenza virus. Group B.-100 for sequential histopathology after a single i.n. dose of inactivated NWS vaccine. Group C.-100 for histopathology after 2 i.n. doses of inactivated NWS vaccine, with an interval between doses of 2 weeks. Group D.-100 for mortality studies and 100 for histopathology after a single i.n. dose of inactivated NWS vaccine followed by i.n. challenge with 6 x 105 EIU NWS influenza virus 2 weeks later. Group E.-150 for mortality and 100 for histopathology and immunofluorescence after 2 i.n. doses of inactivated NWS vaccine 2 weeks apart, challenged i.n. 2 weeks after the second dose with 6 x 105 EIU NWS influenza virus. Group F.-80 for mortality and histopathology after 2 i.n. doses of inactivated NWS vaccine, followed by i.n. challenge with 6 x 105 EIU NWS influenza virus 30 weeks later. Group G.-50 mice for histopathology after i.p. injection of 2 doses of inactivated NWS vaccine 2 weeks apart. Group H.-50 for mortality studies and 50 for sequential histopathology after double i.p immunization followed by i.n. challenge with 6 x 105 EIU NWS influenza virus 2 weeks later. Group J.-40 unimmunized mice for mortality studies and histopathology after aerosol infection (calculated retention dose of 9 04 x 102 EIU NWS influenza virus). Group K.-81 for mortality studies and histopathology after double i.n. immuinization
and aerosol challenge (calculated retention dose of 9 04 x 102 EIU NWS influenza virus) 2 weeks later. A large number of infected mice died but long-term survivors of the mortality experiments were also used for histopathology. Mice were killed with ether at each of the following stages of infection: 24 h daily to 10 days, at 14 and 17 days, at weekly intervals from 3 to 8 weeks, and then at monthly intervals to 6 months. Some survivors from Groups A and E were killed 8, 9 and 10 months after infection. At necropsy, the lungs and brain were removed from each animal and after visual inspection for lesions were fixed by immersion in 5% formol saline and processed by standard histological methods. The intact lungs were blocked out in such a way that sections cut from the ventral surface included portions of each lobe. Paraffin sections cut at 5 ,um were stained with haematoxylin and eosin, and selected sections were also stained by Gordon and Sweet's method for reticulin fibres. An attempt was made to examine by immunofluorescence whether the intrapulmonary plasma cell population which developed was producing specific antibody to AO/NWS virus. For these immunofluorescence studies, NWS influenza virus antiserum produced in rabbits was labelled with fluorescein isothiocyanate by standard methods. Mouse lungs were rapidly frozen immediately after killing and 6 ,gm thick cryostat sections of them were cut. The labelled NWS influenza antiserum was then used with these sections in an indirect staining method. Unfixed frozen lung sections were incubated for either 1 or 2 h with live NWS virus, washed with phosphate buffered saline and then further incubated for 1 h with diluted fluorescein-labelled tNWS antiserum. Mice from Groups A and E, killed at 2 weeks, 2 months and 3 months after infection, and also from uninfected controls, were used for immunofluorescence. RESULTS
Histopathological findings The prevalence of histological lesions in the lungs and brains in the different experimental groups is summarized in Tables I and II. Group A: Unimmunized mice Lungs.-The changes which occurred in the acute stage of the infection from 24 h to the 7th day have been described previously (Baskerville et al., 1974) and were typical of infection with influenza A
19
IMMUNIZATION OF MICE AGAINST INFLUENZA VIRUS INFECTIO1N
TABLE I. Prevalence of Pulmonary Lesions Double i.p. Uniinmmuiiize(I Douible i.n. Double i.n. Unjininunized Single i.n. mice inmunization. mice. immunization. immunization. immunization.
No. of mice wvith lung lesionis
6 x 105 EIU 6 x 105 EIU 6 x 105 EIU 6 x 105 EIU challenge challenge infection challenge S 7 W (72 %) 1- (62 %G) (58) (87 % )
Aerosol infection 34 (85 /)
Aerosol challenge 48 (570
TABLE II. Prevalence of Brain Lesions Double i.n. Double i.p. Unimmunized Double i.n. tlJimmtuized Single i.n. mice. mice, immunization. immunization. immunization. immunizatioin. Aerosol 6 x 105 EIU 6 x 105 EIU 6 x 105 EIU 6 x 105 EIU Aerosol challenge infection challenge infection challenge challenige
No. of inice with brain lesions
-1 (a
( 15%)
z-T
(-,4 (
virus in experimental animals and in man (Straub, 1937; Loosli, 1949; Hers, 1955). They consisted of hyperaemia, exudation of oedema fluid and fibrin into alveoli, widespread necrosis of bronchial and bronchiolar epithelium, destruction of some interalveolar septa and localized infiltration of septa by polymorphonuclear leucocytes. Repair commenced in some areas on the 7th day and was brought about by renewal of the epithelium of airways and by the activity of macrophages, which removed cellular debris, and of fibroblasts which proliferated and migrated into zones of damage, forming collagen fibres. Fibrosis and alveolar epithelialization persisted in some areas for as long as 9 months after infection. Acute and healing lesions were present in the lungs of 87 of 100 mice in this group. From 10 days to 3 months groups of large and small lymphocytes and plasma cells were present in isolated foci in regions of tissue damage, and in the lungs of 11 mice during the same period lymphoid aggregations up to 5 cells deep were also found in the walls of large bronchioli but they did not form a complete layer. In lungs with a peribronchiolar lymphoid infiltration, approximately 50%0 of all bronchioli seen in sections were affected and occasionally lymphoid cells were also present in the adventitia of accompanying blood vessels. Lymphoid infiltrations were found less commonly in the walls of bronchi. The lymphoid cell infiltration disappeared in
%)
,)4(
( 10%/)
-11 (1 %)
the later stages of infection and was present in only 10 mice killed between 4 and 5 months after infection. The lungs of all mice killed after this stage contained no lymphoid aggregates. Brain. Lesions of encephalitis were present in 15 of 100 brains examined from day 1 to 9 months after infection, though these occurred only in mice killed from the 5th day to the 4th week. The lesions consisted of endothelial swelling, a small number of perivascular lymphocytic cuffs, occasional areas of neuron degeneration and small foci of microgliosis. These changes occurred in all regions of the brain in different animals and there appeared to be no predilection site. Lesions were not found in any brain after the 4th week of infection. The results of virus isolation studies are summarized in Fig. 1.
Group B: Single i.n. immunization, unchallenged Lungs. Focal accuimulations of up to 50 lymphocytes were present in the walls of a small number of bronchioli in the lungs of 1 of 100 mice at 3 weeks after inoculation. Brain. No lesions were present in the brain of any animal.
Group C: D)ouble i.n. inirntunization, unchallenged Lungs. -Small peribronchiolar lym-
2)0
A. BASKERVILLE, G. THOMAS AND S. PEACOCK
LL
uuyL
-
FIG. 1.-Influeinza virus titres in lungs and brain after i.n. infection. (EIU)/g of lutng, EIU/g of brain.
egg iinfectiing uinits
=
phoid aggregates comprising up to 50 cells were found in the lungs of 4 of 100 mice, in animals killed at 3 and 4 weeks. Brain.-No lesions were detected in animals in this group.
acute response began at the same time as in unimmunized animals and was succeeded in the same way by removal of debris and by fibrosis. However, the striking feature of repair in this group was the development of large accumulations of lymphoreticular tissue in the Group D: Single i.n. immunization + i.n. walls of bronchi and bronchioli and, more challenge 2 weeks later variably, in the tunica adventitia of pulLungs. Lung lesions were recognized monary blood vessels (Fig. 3). A high by visual inspection at necropsy in 48 of proportion of the cells constituting the 100 exposed mice at all stages. However, lymphoid masses were plasma cells. A when these lungs were examined histo- small number of cells with the morphology logically 72 had lesions. Thus, 24% of of blast cells was recognized in all the mice had pulmonary lesions which were lymphoid aggregates, and occasionally not detectable grossly. cells undergoing mitosis were seen among In the acute stage up to the 7th day the lymphoid population. The lymphoid after infection, the lesions were identical hyperplasia first appeared 10 days after in nature and extent with those found in challenge and was present at all stages unimmunized mice. Regression of the through to 9 months. The lymphoid
IMMUNIZATION OF MICE AGAINST INFLUENZA VIRUS INFECTION
accumulations consisted of cellular masses 10-20 cells deep in one or more lobes of the lungs of 20 mice, and was 5-10 cells deep in a further 14 mice. The large lymphoid aggregates partially invaded the mucosa of the bronchioli, extending in some airways to the basement membrane of the epithelium, and they also obliterated surrounding alveoli by infiltration or displaced them by compression (Fig. 3).
The result was therefore a considerable reduction in functional alveolar surface area in affected lungs. Brain.-The brains of all animals were normal. Group E: Double i.n. immunization + i.n. challenge 2 weeks later Lungs.-Lesions were visible grossly in 36 of 100 lungs but when subjected to
FIG. 2.-Lung of Group C mouse. There is no lymphoid tissue in the bronchiolar wall. H. and E. x 95.
21
FIG. 3. Lung of Group E mouse, showing peribronchiolar and perivascular lymphoid hyperplasia. H. and E. x 130.
A. BASKERVILLE, G. THOMAS AND S. PEACOCK
histological examination lesions were found in 58 of 100. Thus, 22% of the mice had pulmonary damage which was not detectable by visual examination. The acute exudative changes were not so extensive as in the mice of Groups A and D, and fibrosis and diffuse infiltration of interalveolar septa by macrophages and lymphocytes occurred 2-3 days earlier as the predominant reaction. As in the single immunization group, focal lymphoid hyperplasia was present in the walls of bronchi, bronchioli and blood vessels from the 10th day. Lymphoid masses 10-20 cells deep were found in the lungs of 9 mice and the aggregates were from 5 to 10 cells deep in another 17 animals. The lymphoid tissue contained mitotic figures and cells of blast type. The lymphoid hyperplasia reached a maximum between 2 and 4 months after challenge and persisted to the late stages. At 9 months, however, the sectional area of lymphoid cells had decreased to approximately half the maximum area occupied earlier. Brain.-In 1 brain out of 100 examined there were mild localized changes of encephalitis, consisting of 2 perivascular cuffs in the medulla. This animal was killed 2 weeks after challenge. All other brains were normal.
type seen in the other groups was found in 6 of 80 mice.
Group G: Double i.p. immunization, un-
challenged No changes were detected in the lungs or brains of mice immunized by this route. There was no detectable increase in intrapulmonary lymphoid tissue.
Group H: Double i.p. immunization + i.n. challenge 2 weeks later Lungs. When examined grossly the lungs of 16 of 50 mice had lesions. However, when the lungs were examined histologically a total of 31 had lesions, i.e. 30% of the mice had pulmonary lesions which would have been missed by gross inspection alone. The nature and extent of the histological changes in the lungs in the acute and repair phases were similar to those following double i.n. vaccination and occurred over the same period of time. In the chronic stage peribronchiolar and perivascular lymphoid cell populations 10-20 or more cells deep had developed in 10 mice and lymphoid aggregations 5-10 cells deep were present in a further 10. In many instances perivascular lymphoid tissue merged with that of the related airway. All the lymphoid areas contained Groutp F: Double i.n. inmmunization + i.n. large numbers of plasma cells. Mitotic figures and blast cells were observed in challenge 30 weeks later Lungs. Histological lesions were many of the lymphoreticular nodules. Brain.-Lesions were not found in the present in the lungs of 66 of 80 mice, including those which died after infection. brains of any mice in this group. The pneumonic changes of the acute disease were the same as those of Groups A and Group J: Aerosol infection of unimmunized D mice. In the chronic stage between 2 mice weeks and 3 months after challenge, The acute and chronic lesions produced aggregates of lymphoid tissue developed in the lungs and brains of mice exposed in in the walls of bronchi and bronchioli as a Henderson apparatus to an aerosol the only pathological change in 16 lungs, having a calculated retention dose per but the degree of lymphoid hyperplasia mouse of 9 04 x 102 EIU of NWS inwas appreciably less than that in Groups fluenza virus were of the same type as D, E and H. As in these groups, plasma those resulting from infection with intracells constituted a high proportion of the nasal drops. Histological lesions were lymphoreticular cell population. present in 34 of 40 lungs and in 4 of 40 Brain. Localized encephalitis of the brains. The lungs and brains of mice
IMMUNIZATION OF MICE AGAINST INFLUENZA VIRUS INFECTION
which died were also examined histologically. Group K: Double i.n. immunization + aerosol challenge 2 weeks later Lesions were found histologically in the lungs of 46 of 81 mice which were sacrificed or died. The exudative and necrotizing lesions of the acute stage were identical with those following aerosol infection of unimmunized mice but involved smaller areas of the lung lobes. Peribronchiolar and perivascular aggregations of lymphoid tissue up to 10 cells deep appeared during the repair phase, though these were consistently less extensive than those produced by intranasal drop challenge. As in the other groups, the lymphoreticular tissue contained a high proportion of plasma cells. The changes of encephalitis in the one brain affected out of 81 were similar to those
seen in mice exposed to intranasal infec-
tion. Control lungs.-Abnormalities were not detected in the lungs or brains of mice used as uninfected controls.
Assessment of immunity to challenge A. Mortality The degree of protection conferred by immunization in the different groups, as measured by differences in mortality to a standard challenge, is summarized in Fig. 4 and 5. Intransasal administration of a single dose of killed NWS vaccine reduced the total mortality after 6 x 105 EIU challenge from 192/300 (64%) to 39/100 (39/). Two intranasal immunizations with inactivated vaccine 2 weeks apart further reduced mortality to 58/200 (29%). Much better immunity, as measured by a fall in mortality, was provided by 2 i.p. injections of the same dose as that used
n% ,\
0 L.. 0 o
i'
4
:
5
Days Fie(.. 4.-Comparison of mortality after i.n. challenge with influenza virus of mice immunized by (lifferent methods. = deaths after i.n. infection of unimmunized mice, deaths =
after i.n. challenge of mice given a single dose of vaccine i.n.. . . . . = deaths after i.n. challenge of mice given 2 doses of vaccine i.n., -- -00 = deaths after i.n. challenge of mice given 2 doses of
v-accine
i.p.
23
24
A. BASKERVILLE, G. THOMAS AND S. PEACOCK
0
I.6. -
0
D ays Fie(,. 5.-Comnparison of imortality after aerosol infection with influenza virus of unimmunized mice and mice immunize(d i.n. = deaths after aerosol infection of unimmunized mice, deaths after aerosol challenge of mice given 2 doses of vaccine i.n.
intranasally, separated by an interval of 2 weeks. The mortality in challenged mice was then 5/50 (10%). The fall in mortality resulting from each vaccination schedule is statistically highly significant (P < 0-001%). When mice immunized by 2 i.n. doses of vaccine were subjected to the same challenge after an interval of 30 weeks instead of 2 weeks, the mortality was 39/80 (49%), indicating a substantial and significant (P < 1 %) loss of immunity with time. Exposure of mice to an aerosol cloud of NWS influenza virus produced a mortality of2l out of 40 (520%). This is ll*50°lower than infection by i.n. drops. The calculated lung retention dose in this experiment was 9 04 x 102 EIU/mouse. Aerosol challenge with this dose of virus of mice double immunized by i.n. drops resulted in a mortality of 25%. This is only slightly less than the mortality (29%) after i.n. challenge with a much larger dose (6 x 105
- -
EIU), a difference which is not statistically significant. The effectiveness of the aerosol is probably due to the penetration to distal pulmonary tissue of the small infecting particles, 9000 of which are 1 micron or less in size. B. Histoloyical lesions Protection against the challenge infection of 6 x 105 EIU NWS influenza virus was measured in terms of the prevalence of pathological changes in the lungs (Table I) and brain (Table II) of animals in the different groups compared with that in unimmunized mice. Statistical analysis of the results obtained showed that a single i.n. immunization afforded a significant protection against the development of pulmonary lesions (P < I%) and that double i.n. and double i.p. vaccination produced a highly to resistance lesions significant (P < 0 001%). The difference between
IMMUNIZATION OF MICE AGAINST INFLUENZA VIRUS INFECTION
25
the (legree of protection against pneu- pulmonary plasma cells were similarly monia provided by double i.n. immuniza- unsuccessful. tion and that by double i.p. immunization, The reason for the massive hyperplasia however, was not statistically significant. of intrapulmonary lymphoid tissue in some animals is not clear. Intranasal and i.p. administration of inactivated inImnaunofluorescent findings fluenza vaccine without subsequent inThe indirect staining method using fection did not produce comparable fluorescein labelled NWS antiserum yielded changes, nor did influenza infection of no fluorescence in either infected lungs or non-immune animals. The reaction would in those of uninfected controls. It was therefore seem to be an excessive secontherefore not possible to demonstrate dary response by a population of sensitized conclusively that the increased population cells to invasion by the antigen. The of intrapulmonary plasma cells which lymphoid hyperplasia corroborates the developed after challenge of immunized findings of in vitro studies, that lymphoanimals was producing specific antibody cytes harvested from animals presensito AO/NWS virus. tized to an antigen undergo proliferation when confronted later with the same antigen, a reaction which appears to DISCUSSION involve both T and B lymphocytes Peribronchiolar and perivascular lym- (Oppenheim, 1968). We had postulated phoid aggregates developed in the lungs initially that if development of lymphoid of a proportion of animals immunized by masses around bronchioli was due solely all routes and subsequently exposed to to stimulation of locally situated sensitized infection. The lymphoid masses persisted cell populations, then i.n. administration for several months and encroached upon of vaccine should provoke a greater large areas of respiratory tissue. The lymphoid reaction after challenge than formationi of intrapulmonary lymphoid i.p. This was not the case since the tissue is not a feature of unmodified reaction which developed in mice iminfluenza virus infection in mice and it was munized i.p. was equally extensive. The not found in the lungs of control mice or of implication is therefore that a systemic mice vaccinated but not challenged. In response is also involved at some stage of view of the widespread use of inactivated the process, and this is further suggested influenza vaccines in man, attention is by the fact that in most mice lymphoid drawn to this potentially unfavourable cells were also present to some degree in reaction, which requires further investiga- the adventitia of pulmonary blood vessels. tion. It is generally accepted that such Jericho (1966) noted the similarity between intrapulmonary lymphoid cell populations perivascular lymphoid tissue in the lung containing a high proportion of plasma and the lymphoid sheath which surrounds cells develop as a result of local antigenic small arterioles in the white pulp of the stimulation, and it has been demonstrated spleen. The perivascular lymphoid popuin a number of diseases caused by bacteria lation in the lung may produce antiand mycoplasms (Eveland, 1970) that bodies in response to antigenic stimutheir function is to produce for local use lation in the same way as its counterpart specific antibody to the infecting organism. in the spleen. The interval between For technical reasons the presence of vaccination and the challenge infection specific antibody in these cells has never played an important role in the response, been demonstrated in diseases caused by for mice challenged 30 weeks after imviruses, and our attempts to detect by munization developed appreciably less immunofluorescence specific antibody to intrapulmonary lymphoid tissue than AO/NWS influenza virus in the intra- those challenged only 2 weeks later.
26
A. BASKERVILLE, G. THOMAS AND S. PEACOCK
Immunity waned during this time, as shown by increased susceptibility to pulmonary lesions and death, and it would seem that the population of lymphoid cells capable of responding both by multiplying at these sites and by migrating to them had diminished. The changes in intrapulmonary lymphoid tissue in the immune mice suggest that these cells are playing some part in defence against influenza infection. Although all the mechanisms which operate in immunity to influenza are not yet completely understood, recent studies on human volunteers (Jurgensen et al., 1973) have indicated that cell mediated immunity may be important, in addition to that conferred by circulating and secretory antibody. Our results have revealed a further set of circumstances in which cellular responses in immune animals are operative. It was recently shown by Potter, Jennings and MeLaren (1973) that immunization against influenza of ferrets previously infected with the same virus is more successful than of controls, and they postulated that prior infection potentiates in some way the response to immunization, although the mechanism by which this occurs is not clear and the effects were not correlated with changes in the lungs. The phenomenon of excessive secondary lymphoid response described in the present work may be another facet of this effect, though in this case the sequence of infection-vaccination is reversed. Two of the criteria commonly used in animal experiments to assess the degree of protection to challenge conferred by a vaccine are a reduction in mortality and in the prevalence and severity of lesions. In the experiments reported here, a single dose of inactivated influenza vaccine given i.n. reduced mortality, compared with that of unimmunized animals, from 64% to 39% and double vaccination further reduced it to 29%. The best protection was given by double i.p. vaccination, after which mortality was 10%. The immunity waned with time,
however, since the mortality in animals challenged 30 weeks later rose from 29% to 4900. Although all the immunization methods produced a very highly significant protection against the development of pulmonary lesions there was no significant difference between the immunity to pulmonary lesions conferred by double i.n. and that by double i.p. immunization. Ginsberg (1954) and Loosli, Hertweck and Hockwald (1970) have also reported that parenteral vaccination of mice with inactivated homologous influenza virus did not give complete protection against pulmonary lesions following i.n. or aerosol challenge, although the resulting lesions were milder and less extensive. In the present work, necrosis of bronchial and bronchiolar epithelium still occurred in many immunized mice challenged by i.n. drops or by aerosol. This is in contrast to the findings of Denk and Kovac (1966), who recorded complete protection of bronchial epithelium by i.n. vaccination to an i.n. challenge. However, although these authors used a challenge of similar magnitude to that in the present work, the important difference was that their vaccine virus, influenza A2/Asia, was living. This emphasizes the higher immunogenicity of live virus vaccine compared with inactivated. Aerosol challenge was carried out in one group of mice to compare the lesions produced by small infective particles with those produced by the relatively large volume of 0 05 ml placed in the nasal cavity. It has been shown (de St GrQt-h and Donnelley, 1950) that 15-20% of the volume of a liquid adininistered i.n. to mice does not remain in the upper respiratory tract but reaches the lungs. This is advantageous when applying vaccine i.n. to mice, since a large surface area of the lung has contact with the fluid. It is a disadvantage, however, for challenge with live virus because the virus infection may overwhelm the lung, producing an unnaturally heavy challenge. For this reason a comparison was made between the mortality of unimmunized mice when
IMMUNIZATION OF MICE AGAINST INFLUENZA VIRUS INFECTION
exposed to an aerosol challenge and that of mice infected by intranasal drops. No significant difference in mortality was found, nor was there a significant difference in the prevalence of lung lesions produced by aerosol (84% and that following intranasal infection (87)0). A comparable number of lesions was also obtained when the aerosol and i.n. drop methods were used for challenge of mice doubly immunized i.n. (57 % and 58 % respectively). Comparisons therefore can be drawn in these circumstances between the results ofthe two modes of infection. It seems probable that the deeper penetration of the aerosol particles is offset by the lower dose of virus administered by this means. There was no difference in the nature of the lesions in the brain which occurred in non-immune and immune animals, and there was no evidence of any long-term harmful effects on the central nervous system attributable to immunization. The present work emphasizes the importance of histopathological examination of the organs in vaccine trials in experimental animals. In all groups of mice there was consistently a great difference, ranging from 22% to 30,0o between the number of animals with lung lesions recognized on visual inspection and those with lesions which, though severe and extensive, were detectable only by histopathology. In the case of the central nervous system, histological examination is the only method by which damage can be detected. Spuriously favourable results can thus be ascribed to vaccination if histological examination is not a part of the experimental programme. REFERENCES BASKERVILLE, A., THOMAS, G., WOOD, M. & HARRIS, W. J. (1974) Histology and Ultrastructure of Metaplasia of Alveolar Epithelium following Infection of Mice and Hamsters with Influenza Virus. Br. J. exp. Path., 55, 130. DENK, H. & KOVAC, W. (1966) Immunofluoreszenzuntersuchungen uber den Ablauf einer intranasalen Grippeinfektion bei immunisierten Mausen. Arch. Virusforsch., 18, 294. DOWNIE, J. C. (1973) The Sequential Appearance of Antibody and Immunoglobulins in Nasal Secretions after Immunization of Volunteers with Live
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and Inactivated Influenza B Virus Vaccines. J. Hyg., Ca-nb., 71, 433. EVELAND, W. C. (1970) Fluorescent Staining with Labelled Mycoplasma Antigen. Direct Reaction with Antibody-producing Cells. Archs envir. Hlth, 21, 397. GINSBERG, H. S. (1954) Production of Pulmonary Lesions by Influenza Viruses in Immunized Mice. J. Immun., 72, 24. HARPER, G. J. & HOOD, A. M. (1962) Lung Retention in Mice Exposed to Airborne Micro-organisms. Nature, Lond., 196, 598. HENDERSON, D. W. (1952) An Apparatus for the Study of Airborne Infection. J. Hyg., Camb., 50, 53. HERS, J. F. PH. (1955) The Histopathology of the Respiratory T7ract in Human Influenza. Leiden: H. E. Steufert Kroese. p. 77. HOULT, J. G. & FLEWETT, T. H. (1960) Influenzal Encephalopathy and Post-influenzal Encephalitis: Histological and Other Observations. Br. med. J., i, 1847. JERICHO, K. W. (1966) Intra-pulmonary Lymphoid Tissue in Pigs. T'et. Bull., 36, 687. JURGENSEN, P. F., OLSEN, G. N., JOHNSON, J. E., SWENSON, E. W., AYOUB, E. M., HENNEY, C. S. & WALDMAN, R. H. (1973) Immune Response of the Human Respiratory Tract. II. Cell-mediated Immunity in the Lower Respiratory Tract to Tuberculin, Mumps and Influenza Viruses. J. infect. Dis., 128, 730. VAN KIRK, J. E., MILLS, J. & CHANOCK, R. M. (1971) Evaluation of Low-temperature Grown Influenza A2/Hong Kong Virus in Volunteers. Proc. Soc. exp. Biol. Med., 136, 34. LoOSLI, C. G. (1949) The Pathogenesis and Pathology of Experimental Air-borne Influenza Virus A Infections in Mice. J. infect. Dis., 84, 153. LoOSLI, C. G., HERTWECK, M. S. & HOCKWALD, R. S. (1970) Airborne Influenza PR8-A Virus Infections in Actively Immunized Mfice. Arch8. envir. Hlth, 21, 332. OPPENHEIM, J. J. (1968) Relationship of in vitro Lymphocyte Transformation to Delayed Hypersensitivity in Guinea-pigs and Man. Fedn Proc., 27, 21. POTTER, C. W., JENNINGS, R. & MCLAREN, C. (1973) Immunity to Influenza in Ferrets. VI. Immunization with Adjuvanted Vaccines. Arch. Virusforsch., 42, 285. DE ST GROTH, S. F. & DONNELLEY, M. (1950) Studies in Experimental Immunology of Influenza. IV. The Protective Value of Active Immunization. Aust. J. exp. Biol. ned. Sci., 28, 62. STRAUB, M. (1937) The Microscopical Changes in the Lungs of Mice Infected with Influenza Virus. J. Path. Bact., 45, 75. THOMAS, G. (1973) Respiratory Immunization with Inactivated Influenza Aerosol Vaccine in Man followed by Live Challenge. IVth Internat. Symp. Aerobiology. Ed. J. F. Ph. Hers and K. C. Winkler. Utrecht: Oosthoek Publishing Co. p. 328. WALDMAN, R. H., KASEL, J. A., FULK, R. V., ToGo, Y., HORNICK, R. B., HEINER, G. G., DAWKINS, A. T. & MANN, J. J. (1968) Influenza Antibody in Human Respiratory Secretions after Subcutaneovs or Respiratory Immunization with Inactivated Virus. Nature, Lond., 218, 594.
PATHOLOGICAL ASPECTS OF IMMUNIZATION OF MICE AGAINST INFLUENZA VIRUS INFECTION A. BASKERVILLE, G. THOMAS* AND S. PEACOCK From the Microbiological Research Establishment, Porton Down, Salisbury, Wilts.
Received foI publication 19 August 1974
Summary.-Groups of mice were immunized against influenza Ao/NWS virus by a single intranasal administration of inactivated homologous virus, by 2 intranasal doses of vaccine separated by an interval of 2 weeks, or by 2 intraperitoneal doses of the same vaccine. When subjected 2 weeks later to a standard challenge of 6 x 105 egg infecting units Ao/NWS virus instilled intranasally, mortality fell significantly from 64% in unimmunized mice to 39% in mice given a single intranasal dose of vaccine and to 29% in animals which received double intranasal vaccine. The best protection was conferred by double intraperitoneal immunization, after which mortality was 10%. Immunity waned with time, since the mortality of mice doubly immunized by the respiratory route and challenged 30 weeks later was 49o%. Intrapulmonary lymphoid tissue developed in large amounts in a proportion of mice immunized by all methods and challenged after an interval of 2 weeks. Attention is drawn to this reaction as a possible unfavourable consequence of vaccination. There were no lesions in the lungs or central nervous system after immunization without subsequent challenge. The importance of histopathology in vaccine trials in experimental animals is emphasized by the consistently higher detection rate of lesions in lungs by histological examination than by visual inspection alone. IN RECENT YEARS attempts have been made to utilize the local defence mechanisms of the respiratory tract for immunization against viral respiratory disease, in particular influenza. Respiratory immunization has now been extended from studies on experimental animals (de St Groth and Donnelley, 1950) to numerous clinical trials on human volunteers (Waldman et al., 1968; Van Kirk, Mills and Chanock, 1971; Downie, 1973; Thomas, 1973) and inactivated influenza vaccines are now commercially available for use in man. However, relatively little attention has been paid to the changes which may be induced by such vaccines in the lungs or in the central nervous system, which can also be damaged in a small proportion of human cases of natural influenza infection (Hoult and Flewett, 1960). To investigate this risk, a series of experi*
ments was undertaken with mice using the
NWS strain of influenza/AO virus to study not only the protection afforded by respiratory vaccination but also the longterm effects on the lungs and brain of single and double respiratory immunization alone and with subsequent challenge. In addition, the study was used to assess the value of histological examination of organs in trials of vaccine efficacy and to compare this with the standard methods of recording survival and the presence of visible lesions after challenge. MATERIALS AND METHODS Female outbred Porton albino specific pathogen-free mice 3-6 weeks old were used in a series of experiments. A mouse-adapted strain of influenza AO/NWS virus was employed in all the experiments and a pool of this virus was prepared by inoculating it into the allantoic cavity of 11-day old embryonated eggs and harvesting
Pr'esent address: Essex Chemie AG, Lutcerne, Switzerland1.
18
A. BASKERVILLE, G. THOMAS AND S. PEACOCK
the fluid after 3 days' incubation at 37°. This stock virus was stored in aliquots of 10 ml at - 700 and had a titre of 6 x 105 egg infecting units (EIU) per 005 ml. Mice were infected by instilling 0-05 ml of virus pool suspension into the nostrils under light ether anaesthesia, care being taken to ensure that the entire volume of the drops was inhaled and retained. Two groups of animals (Groups J and K) were infected by exposure to an aerosol of influenza virus in a Henderson apparatus (Henderson, 1952). The calculated retention dose of virus per mouse in this experiment was 9 04 x 102 EIU, based on the lung retention in this apparatus measured by Harper and Hood (1962). For use in the vaccine experiments, the NWS strain virus pool was inactivated by incubation with formalin and treated as recommended in the British Pharmacopoeia (1968). 0 05 ml of this vaccine was administered to anaesthetized mice either intranasally (i.n.) or intraperitoneally (i.p.). A further 50 mice were inoculated i.n. with 0-05 ml of sterile physiological saline. The mice were used in groups as follows: Group A.-300 to provide data on mortality in unimmunized mice and 100 for sequential histopathology of lungs and brain and immunofluorescence studies of lungs after i.n. infection with 6 x 105 EIU NWS influenza virus. Group B.-100 for sequential histopathology after a single i.n. dose of inactivated NWS vaccine. Group C.-100 for histopathology after 2 i.n. doses of inactivated NWS vaccine, with an interval between doses of 2 weeks. Group D.-100 for mortality studies and 100 for histopathology after a single i.n. dose of inactivated NWS vaccine followed by i.n. challenge with 6 x 105 EIU NWS influenza virus 2 weeks later. Group E.-150 for mortality and 100 for histopathology and immunofluorescence after 2 i.n. doses of inactivated NWS vaccine 2 weeks apart, challenged i.n. 2 weeks after the second dose with 6 x 105 EIU NWS influenza virus. Group F.-80 for mortality and histopathology after 2 i.n. doses of inactivated NWS vaccine, followed by i.n. challenge with 6 x 105 EIU NWS influenza virus 30 weeks later. Group G.-50 mice for histopathology after i.p. injection of 2 doses of inactivated NWS vaccine 2 weeks apart. Group H.-50 for mortality studies and 50 for sequential histopathology after double i.p immunization followed by i.n. challenge with 6 x 105 EIU NWS influenza virus 2 weeks later. Group J.-40 unimmunized mice for mortality studies and histopathology after aerosol infection (calculated retention dose of 9 04 x 102 EIU NWS influenza virus). Group K.-81 for mortality studies and histopathology after double i.n. immuinization
and aerosol challenge (calculated retention dose of 9 04 x 102 EIU NWS influenza virus) 2 weeks later. A large number of infected mice died but long-term survivors of the mortality experiments were also used for histopathology. Mice were killed with ether at each of the following stages of infection: 24 h daily to 10 days, at 14 and 17 days, at weekly intervals from 3 to 8 weeks, and then at monthly intervals to 6 months. Some survivors from Groups A and E were killed 8, 9 and 10 months after infection. At necropsy, the lungs and brain were removed from each animal and after visual inspection for lesions were fixed by immersion in 5% formol saline and processed by standard histological methods. The intact lungs were blocked out in such a way that sections cut from the ventral surface included portions of each lobe. Paraffin sections cut at 5 ,um were stained with haematoxylin and eosin, and selected sections were also stained by Gordon and Sweet's method for reticulin fibres. An attempt was made to examine by immunofluorescence whether the intrapulmonary plasma cell population which developed was producing specific antibody to AO/NWS virus. For these immunofluorescence studies, NWS influenza virus antiserum produced in rabbits was labelled with fluorescein isothiocyanate by standard methods. Mouse lungs were rapidly frozen immediately after killing and 6 ,gm thick cryostat sections of them were cut. The labelled NWS influenza antiserum was then used with these sections in an indirect staining method. Unfixed frozen lung sections were incubated for either 1 or 2 h with live NWS virus, washed with phosphate buffered saline and then further incubated for 1 h with diluted fluorescein-labelled tNWS antiserum. Mice from Groups A and E, killed at 2 weeks, 2 months and 3 months after infection, and also from uninfected controls, were used for immunofluorescence. RESULTS
Histopathological findings The prevalence of histological lesions in the lungs and brains in the different experimental groups is summarized in Tables I and II. Group A: Unimmunized mice Lungs.-The changes which occurred in the acute stage of the infection from 24 h to the 7th day have been described previously (Baskerville et al., 1974) and were typical of infection with influenza A
19
IMMUNIZATION OF MICE AGAINST INFLUENZA VIRUS INFECTIO1N
TABLE I. Prevalence of Pulmonary Lesions Double i.p. Uniinmmuiiize(I Douible i.n. Double i.n. Unjininunized Single i.n. mice inmunization. mice. immunization. immunization. immunization.
No. of mice wvith lung lesionis
6 x 105 EIU 6 x 105 EIU 6 x 105 EIU 6 x 105 EIU challenge challenge infection challenge S 7 W (72 %) 1- (62 %G) (58) (87 % )
Aerosol infection 34 (85 /)
Aerosol challenge 48 (570
TABLE II. Prevalence of Brain Lesions Double i.n. Double i.p. Unimmunized Double i.n. tlJimmtuized Single i.n. mice. mice, immunization. immunization. immunization. immunizatioin. Aerosol 6 x 105 EIU 6 x 105 EIU 6 x 105 EIU 6 x 105 EIU Aerosol challenge infection challenge infection challenge challenige
No. of inice with brain lesions
-1 (a
( 15%)
z-T
(-,4 (
virus in experimental animals and in man (Straub, 1937; Loosli, 1949; Hers, 1955). They consisted of hyperaemia, exudation of oedema fluid and fibrin into alveoli, widespread necrosis of bronchial and bronchiolar epithelium, destruction of some interalveolar septa and localized infiltration of septa by polymorphonuclear leucocytes. Repair commenced in some areas on the 7th day and was brought about by renewal of the epithelium of airways and by the activity of macrophages, which removed cellular debris, and of fibroblasts which proliferated and migrated into zones of damage, forming collagen fibres. Fibrosis and alveolar epithelialization persisted in some areas for as long as 9 months after infection. Acute and healing lesions were present in the lungs of 87 of 100 mice in this group. From 10 days to 3 months groups of large and small lymphocytes and plasma cells were present in isolated foci in regions of tissue damage, and in the lungs of 11 mice during the same period lymphoid aggregations up to 5 cells deep were also found in the walls of large bronchioli but they did not form a complete layer. In lungs with a peribronchiolar lymphoid infiltration, approximately 50%0 of all bronchioli seen in sections were affected and occasionally lymphoid cells were also present in the adventitia of accompanying blood vessels. Lymphoid infiltrations were found less commonly in the walls of bronchi. The lymphoid cell infiltration disappeared in
%)
,)4(
( 10%/)
-11 (1 %)
the later stages of infection and was present in only 10 mice killed between 4 and 5 months after infection. The lungs of all mice killed after this stage contained no lymphoid aggregates. Brain. Lesions of encephalitis were present in 15 of 100 brains examined from day 1 to 9 months after infection, though these occurred only in mice killed from the 5th day to the 4th week. The lesions consisted of endothelial swelling, a small number of perivascular lymphocytic cuffs, occasional areas of neuron degeneration and small foci of microgliosis. These changes occurred in all regions of the brain in different animals and there appeared to be no predilection site. Lesions were not found in any brain after the 4th week of infection. The results of virus isolation studies are summarized in Fig. 1.
Group B: Single i.n. immunization, unchallenged Lungs. Focal accuimulations of up to 50 lymphocytes were present in the walls of a small number of bronchioli in the lungs of 1 of 100 mice at 3 weeks after inoculation. Brain. No lesions were present in the brain of any animal.
Group C: D)ouble i.n. inirntunization, unchallenged Lungs. -Small peribronchiolar lym-
2)0
A. BASKERVILLE, G. THOMAS AND S. PEACOCK
LL
uuyL
-
FIG. 1.-Influeinza virus titres in lungs and brain after i.n. infection. (EIU)/g of lutng, EIU/g of brain.
egg iinfectiing uinits
=
phoid aggregates comprising up to 50 cells were found in the lungs of 4 of 100 mice, in animals killed at 3 and 4 weeks. Brain.-No lesions were detected in animals in this group.
acute response began at the same time as in unimmunized animals and was succeeded in the same way by removal of debris and by fibrosis. However, the striking feature of repair in this group was the development of large accumulations of lymphoreticular tissue in the Group D: Single i.n. immunization + i.n. walls of bronchi and bronchioli and, more challenge 2 weeks later variably, in the tunica adventitia of pulLungs. Lung lesions were recognized monary blood vessels (Fig. 3). A high by visual inspection at necropsy in 48 of proportion of the cells constituting the 100 exposed mice at all stages. However, lymphoid masses were plasma cells. A when these lungs were examined histo- small number of cells with the morphology logically 72 had lesions. Thus, 24% of of blast cells was recognized in all the mice had pulmonary lesions which were lymphoid aggregates, and occasionally not detectable grossly. cells undergoing mitosis were seen among In the acute stage up to the 7th day the lymphoid population. The lymphoid after infection, the lesions were identical hyperplasia first appeared 10 days after in nature and extent with those found in challenge and was present at all stages unimmunized mice. Regression of the through to 9 months. The lymphoid
IMMUNIZATION OF MICE AGAINST INFLUENZA VIRUS INFECTION
accumulations consisted of cellular masses 10-20 cells deep in one or more lobes of the lungs of 20 mice, and was 5-10 cells deep in a further 14 mice. The large lymphoid aggregates partially invaded the mucosa of the bronchioli, extending in some airways to the basement membrane of the epithelium, and they also obliterated surrounding alveoli by infiltration or displaced them by compression (Fig. 3).
The result was therefore a considerable reduction in functional alveolar surface area in affected lungs. Brain.-The brains of all animals were normal. Group E: Double i.n. immunization + i.n. challenge 2 weeks later Lungs.-Lesions were visible grossly in 36 of 100 lungs but when subjected to
FIG. 2.-Lung of Group C mouse. There is no lymphoid tissue in the bronchiolar wall. H. and E. x 95.
21
FIG. 3. Lung of Group E mouse, showing peribronchiolar and perivascular lymphoid hyperplasia. H. and E. x 130.
A. BASKERVILLE, G. THOMAS AND S. PEACOCK
histological examination lesions were found in 58 of 100. Thus, 22% of the mice had pulmonary damage which was not detectable by visual examination. The acute exudative changes were not so extensive as in the mice of Groups A and D, and fibrosis and diffuse infiltration of interalveolar septa by macrophages and lymphocytes occurred 2-3 days earlier as the predominant reaction. As in the single immunization group, focal lymphoid hyperplasia was present in the walls of bronchi, bronchioli and blood vessels from the 10th day. Lymphoid masses 10-20 cells deep were found in the lungs of 9 mice and the aggregates were from 5 to 10 cells deep in another 17 animals. The lymphoid tissue contained mitotic figures and cells of blast type. The lymphoid hyperplasia reached a maximum between 2 and 4 months after challenge and persisted to the late stages. At 9 months, however, the sectional area of lymphoid cells had decreased to approximately half the maximum area occupied earlier. Brain.-In 1 brain out of 100 examined there were mild localized changes of encephalitis, consisting of 2 perivascular cuffs in the medulla. This animal was killed 2 weeks after challenge. All other brains were normal.
type seen in the other groups was found in 6 of 80 mice.
Group G: Double i.p. immunization, un-
challenged No changes were detected in the lungs or brains of mice immunized by this route. There was no detectable increase in intrapulmonary lymphoid tissue.
Group H: Double i.p. immunization + i.n. challenge 2 weeks later Lungs. When examined grossly the lungs of 16 of 50 mice had lesions. However, when the lungs were examined histologically a total of 31 had lesions, i.e. 30% of the mice had pulmonary lesions which would have been missed by gross inspection alone. The nature and extent of the histological changes in the lungs in the acute and repair phases were similar to those following double i.n. vaccination and occurred over the same period of time. In the chronic stage peribronchiolar and perivascular lymphoid cell populations 10-20 or more cells deep had developed in 10 mice and lymphoid aggregations 5-10 cells deep were present in a further 10. In many instances perivascular lymphoid tissue merged with that of the related airway. All the lymphoid areas contained Groutp F: Double i.n. inmmunization + i.n. large numbers of plasma cells. Mitotic figures and blast cells were observed in challenge 30 weeks later Lungs. Histological lesions were many of the lymphoreticular nodules. Brain.-Lesions were not found in the present in the lungs of 66 of 80 mice, including those which died after infection. brains of any mice in this group. The pneumonic changes of the acute disease were the same as those of Groups A and Group J: Aerosol infection of unimmunized D mice. In the chronic stage between 2 mice weeks and 3 months after challenge, The acute and chronic lesions produced aggregates of lymphoid tissue developed in the lungs and brains of mice exposed in in the walls of bronchi and bronchioli as a Henderson apparatus to an aerosol the only pathological change in 16 lungs, having a calculated retention dose per but the degree of lymphoid hyperplasia mouse of 9 04 x 102 EIU of NWS inwas appreciably less than that in Groups fluenza virus were of the same type as D, E and H. As in these groups, plasma those resulting from infection with intracells constituted a high proportion of the nasal drops. Histological lesions were lymphoreticular cell population. present in 34 of 40 lungs and in 4 of 40 Brain. Localized encephalitis of the brains. The lungs and brains of mice
IMMUNIZATION OF MICE AGAINST INFLUENZA VIRUS INFECTION
which died were also examined histologically. Group K: Double i.n. immunization + aerosol challenge 2 weeks later Lesions were found histologically in the lungs of 46 of 81 mice which were sacrificed or died. The exudative and necrotizing lesions of the acute stage were identical with those following aerosol infection of unimmunized mice but involved smaller areas of the lung lobes. Peribronchiolar and perivascular aggregations of lymphoid tissue up to 10 cells deep appeared during the repair phase, though these were consistently less extensive than those produced by intranasal drop challenge. As in the other groups, the lymphoreticular tissue contained a high proportion of plasma cells. The changes of encephalitis in the one brain affected out of 81 were similar to those
seen in mice exposed to intranasal infec-
tion. Control lungs.-Abnormalities were not detected in the lungs or brains of mice used as uninfected controls.
Assessment of immunity to challenge A. Mortality The degree of protection conferred by immunization in the different groups, as measured by differences in mortality to a standard challenge, is summarized in Fig. 4 and 5. Intransasal administration of a single dose of killed NWS vaccine reduced the total mortality after 6 x 105 EIU challenge from 192/300 (64%) to 39/100 (39/). Two intranasal immunizations with inactivated vaccine 2 weeks apart further reduced mortality to 58/200 (29%). Much better immunity, as measured by a fall in mortality, was provided by 2 i.p. injections of the same dose as that used
n% ,\
0 L.. 0 o
i'
4
:
5
Days Fie(.. 4.-Comparison of mortality after i.n. challenge with influenza virus of mice immunized by (lifferent methods. = deaths after i.n. infection of unimmunized mice, deaths =
after i.n. challenge of mice given a single dose of vaccine i.n.. . . . . = deaths after i.n. challenge of mice given 2 doses of vaccine i.n., -- -00 = deaths after i.n. challenge of mice given 2 doses of
v-accine
i.p.
23
24
A. BASKERVILLE, G. THOMAS AND S. PEACOCK
0
I.6. -
0
D ays Fie(,. 5.-Comnparison of imortality after aerosol infection with influenza virus of unimmunized mice and mice immunize(d i.n. = deaths after aerosol infection of unimmunized mice, deaths after aerosol challenge of mice given 2 doses of vaccine i.n.
intranasally, separated by an interval of 2 weeks. The mortality in challenged mice was then 5/50 (10%). The fall in mortality resulting from each vaccination schedule is statistically highly significant (P < 0-001%). When mice immunized by 2 i.n. doses of vaccine were subjected to the same challenge after an interval of 30 weeks instead of 2 weeks, the mortality was 39/80 (49%), indicating a substantial and significant (P < 1 %) loss of immunity with time. Exposure of mice to an aerosol cloud of NWS influenza virus produced a mortality of2l out of 40 (520%). This is ll*50°lower than infection by i.n. drops. The calculated lung retention dose in this experiment was 9 04 x 102 EIU/mouse. Aerosol challenge with this dose of virus of mice double immunized by i.n. drops resulted in a mortality of 25%. This is only slightly less than the mortality (29%) after i.n. challenge with a much larger dose (6 x 105
- -
EIU), a difference which is not statistically significant. The effectiveness of the aerosol is probably due to the penetration to distal pulmonary tissue of the small infecting particles, 9000 of which are 1 micron or less in size. B. Histoloyical lesions Protection against the challenge infection of 6 x 105 EIU NWS influenza virus was measured in terms of the prevalence of pathological changes in the lungs (Table I) and brain (Table II) of animals in the different groups compared with that in unimmunized mice. Statistical analysis of the results obtained showed that a single i.n. immunization afforded a significant protection against the development of pulmonary lesions (P < I%) and that double i.n. and double i.p. vaccination produced a highly to resistance lesions significant (P < 0 001%). The difference between
IMMUNIZATION OF MICE AGAINST INFLUENZA VIRUS INFECTION
25
the (legree of protection against pneu- pulmonary plasma cells were similarly monia provided by double i.n. immuniza- unsuccessful. tion and that by double i.p. immunization, The reason for the massive hyperplasia however, was not statistically significant. of intrapulmonary lymphoid tissue in some animals is not clear. Intranasal and i.p. administration of inactivated inImnaunofluorescent findings fluenza vaccine without subsequent inThe indirect staining method using fection did not produce comparable fluorescein labelled NWS antiserum yielded changes, nor did influenza infection of no fluorescence in either infected lungs or non-immune animals. The reaction would in those of uninfected controls. It was therefore seem to be an excessive secontherefore not possible to demonstrate dary response by a population of sensitized conclusively that the increased population cells to invasion by the antigen. The of intrapulmonary plasma cells which lymphoid hyperplasia corroborates the developed after challenge of immunized findings of in vitro studies, that lymphoanimals was producing specific antibody cytes harvested from animals presensito AO/NWS virus. tized to an antigen undergo proliferation when confronted later with the same antigen, a reaction which appears to DISCUSSION involve both T and B lymphocytes Peribronchiolar and perivascular lym- (Oppenheim, 1968). We had postulated phoid aggregates developed in the lungs initially that if development of lymphoid of a proportion of animals immunized by masses around bronchioli was due solely all routes and subsequently exposed to to stimulation of locally situated sensitized infection. The lymphoid masses persisted cell populations, then i.n. administration for several months and encroached upon of vaccine should provoke a greater large areas of respiratory tissue. The lymphoid reaction after challenge than formationi of intrapulmonary lymphoid i.p. This was not the case since the tissue is not a feature of unmodified reaction which developed in mice iminfluenza virus infection in mice and it was munized i.p. was equally extensive. The not found in the lungs of control mice or of implication is therefore that a systemic mice vaccinated but not challenged. In response is also involved at some stage of view of the widespread use of inactivated the process, and this is further suggested influenza vaccines in man, attention is by the fact that in most mice lymphoid drawn to this potentially unfavourable cells were also present to some degree in reaction, which requires further investiga- the adventitia of pulmonary blood vessels. tion. It is generally accepted that such Jericho (1966) noted the similarity between intrapulmonary lymphoid cell populations perivascular lymphoid tissue in the lung containing a high proportion of plasma and the lymphoid sheath which surrounds cells develop as a result of local antigenic small arterioles in the white pulp of the stimulation, and it has been demonstrated spleen. The perivascular lymphoid popuin a number of diseases caused by bacteria lation in the lung may produce antiand mycoplasms (Eveland, 1970) that bodies in response to antigenic stimutheir function is to produce for local use lation in the same way as its counterpart specific antibody to the infecting organism. in the spleen. The interval between For technical reasons the presence of vaccination and the challenge infection specific antibody in these cells has never played an important role in the response, been demonstrated in diseases caused by for mice challenged 30 weeks after imviruses, and our attempts to detect by munization developed appreciably less immunofluorescence specific antibody to intrapulmonary lymphoid tissue than AO/NWS influenza virus in the intra- those challenged only 2 weeks later.
26
A. BASKERVILLE, G. THOMAS AND S. PEACOCK
Immunity waned during this time, as shown by increased susceptibility to pulmonary lesions and death, and it would seem that the population of lymphoid cells capable of responding both by multiplying at these sites and by migrating to them had diminished. The changes in intrapulmonary lymphoid tissue in the immune mice suggest that these cells are playing some part in defence against influenza infection. Although all the mechanisms which operate in immunity to influenza are not yet completely understood, recent studies on human volunteers (Jurgensen et al., 1973) have indicated that cell mediated immunity may be important, in addition to that conferred by circulating and secretory antibody. Our results have revealed a further set of circumstances in which cellular responses in immune animals are operative. It was recently shown by Potter, Jennings and MeLaren (1973) that immunization against influenza of ferrets previously infected with the same virus is more successful than of controls, and they postulated that prior infection potentiates in some way the response to immunization, although the mechanism by which this occurs is not clear and the effects were not correlated with changes in the lungs. The phenomenon of excessive secondary lymphoid response described in the present work may be another facet of this effect, though in this case the sequence of infection-vaccination is reversed. Two of the criteria commonly used in animal experiments to assess the degree of protection to challenge conferred by a vaccine are a reduction in mortality and in the prevalence and severity of lesions. In the experiments reported here, a single dose of inactivated influenza vaccine given i.n. reduced mortality, compared with that of unimmunized animals, from 64% to 39% and double vaccination further reduced it to 29%. The best protection was given by double i.p. vaccination, after which mortality was 10%. The immunity waned with time,
however, since the mortality in animals challenged 30 weeks later rose from 29% to 4900. Although all the immunization methods produced a very highly significant protection against the development of pulmonary lesions there was no significant difference between the immunity to pulmonary lesions conferred by double i.n. and that by double i.p. immunization. Ginsberg (1954) and Loosli, Hertweck and Hockwald (1970) have also reported that parenteral vaccination of mice with inactivated homologous influenza virus did not give complete protection against pulmonary lesions following i.n. or aerosol challenge, although the resulting lesions were milder and less extensive. In the present work, necrosis of bronchial and bronchiolar epithelium still occurred in many immunized mice challenged by i.n. drops or by aerosol. This is in contrast to the findings of Denk and Kovac (1966), who recorded complete protection of bronchial epithelium by i.n. vaccination to an i.n. challenge. However, although these authors used a challenge of similar magnitude to that in the present work, the important difference was that their vaccine virus, influenza A2/Asia, was living. This emphasizes the higher immunogenicity of live virus vaccine compared with inactivated. Aerosol challenge was carried out in one group of mice to compare the lesions produced by small infective particles with those produced by the relatively large volume of 0 05 ml placed in the nasal cavity. It has been shown (de St GrQt-h and Donnelley, 1950) that 15-20% of the volume of a liquid adininistered i.n. to mice does not remain in the upper respiratory tract but reaches the lungs. This is advantageous when applying vaccine i.n. to mice, since a large surface area of the lung has contact with the fluid. It is a disadvantage, however, for challenge with live virus because the virus infection may overwhelm the lung, producing an unnaturally heavy challenge. For this reason a comparison was made between the mortality of unimmunized mice when
IMMUNIZATION OF MICE AGAINST INFLUENZA VIRUS INFECTION
exposed to an aerosol challenge and that of mice infected by intranasal drops. No significant difference in mortality was found, nor was there a significant difference in the prevalence of lung lesions produced by aerosol (84% and that following intranasal infection (87)0). A comparable number of lesions was also obtained when the aerosol and i.n. drop methods were used for challenge of mice doubly immunized i.n. (57 % and 58 % respectively). Comparisons therefore can be drawn in these circumstances between the results ofthe two modes of infection. It seems probable that the deeper penetration of the aerosol particles is offset by the lower dose of virus administered by this means. There was no difference in the nature of the lesions in the brain which occurred in non-immune and immune animals, and there was no evidence of any long-term harmful effects on the central nervous system attributable to immunization. The present work emphasizes the importance of histopathological examination of the organs in vaccine trials in experimental animals. In all groups of mice there was consistently a great difference, ranging from 22% to 30,0o between the number of animals with lung lesions recognized on visual inspection and those with lesions which, though severe and extensive, were detectable only by histopathology. In the case of the central nervous system, histological examination is the only method by which damage can be detected. Spuriously favourable results can thus be ascribed to vaccination if histological examination is not a part of the experimental programme. REFERENCES BASKERVILLE, A., THOMAS, G., WOOD, M. & HARRIS, W. J. (1974) Histology and Ultrastructure of Metaplasia of Alveolar Epithelium following Infection of Mice and Hamsters with Influenza Virus. Br. J. exp. Path., 55, 130. DENK, H. & KOVAC, W. (1966) Immunofluoreszenzuntersuchungen uber den Ablauf einer intranasalen Grippeinfektion bei immunisierten Mausen. Arch. Virusforsch., 18, 294. DOWNIE, J. C. (1973) The Sequential Appearance of Antibody and Immunoglobulins in Nasal Secretions after Immunization of Volunteers with Live
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and Inactivated Influenza B Virus Vaccines. J. Hyg., Ca-nb., 71, 433. EVELAND, W. C. (1970) Fluorescent Staining with Labelled Mycoplasma Antigen. Direct Reaction with Antibody-producing Cells. Archs envir. Hlth, 21, 397. GINSBERG, H. S. (1954) Production of Pulmonary Lesions by Influenza Viruses in Immunized Mice. J. Immun., 72, 24. HARPER, G. J. & HOOD, A. M. (1962) Lung Retention in Mice Exposed to Airborne Micro-organisms. Nature, Lond., 196, 598. HENDERSON, D. W. (1952) An Apparatus for the Study of Airborne Infection. J. Hyg., Camb., 50, 53. HERS, J. F. PH. (1955) The Histopathology of the Respiratory T7ract in Human Influenza. Leiden: H. E. Steufert Kroese. p. 77. HOULT, J. G. & FLEWETT, T. H. (1960) Influenzal Encephalopathy and Post-influenzal Encephalitis: Histological and Other Observations. Br. med. J., i, 1847. JERICHO, K. W. (1966) Intra-pulmonary Lymphoid Tissue in Pigs. T'et. Bull., 36, 687. JURGENSEN, P. F., OLSEN, G. N., JOHNSON, J. E., SWENSON, E. W., AYOUB, E. M., HENNEY, C. S. & WALDMAN, R. H. (1973) Immune Response of the Human Respiratory Tract. II. Cell-mediated Immunity in the Lower Respiratory Tract to Tuberculin, Mumps and Influenza Viruses. J. infect. Dis., 128, 730. VAN KIRK, J. E., MILLS, J. & CHANOCK, R. M. (1971) Evaluation of Low-temperature Grown Influenza A2/Hong Kong Virus in Volunteers. Proc. Soc. exp. Biol. Med., 136, 34. LoOSLI, C. G. (1949) The Pathogenesis and Pathology of Experimental Air-borne Influenza Virus A Infections in Mice. J. infect. Dis., 84, 153. LoOSLI, C. G., HERTWECK, M. S. & HOCKWALD, R. S. (1970) Airborne Influenza PR8-A Virus Infections in Actively Immunized Mfice. Arch8. envir. Hlth, 21, 332. OPPENHEIM, J. J. (1968) Relationship of in vitro Lymphocyte Transformation to Delayed Hypersensitivity in Guinea-pigs and Man. Fedn Proc., 27, 21. POTTER, C. W., JENNINGS, R. & MCLAREN, C. (1973) Immunity to Influenza in Ferrets. VI. Immunization with Adjuvanted Vaccines. Arch. Virusforsch., 42, 285. DE ST GROTH, S. F. & DONNELLEY, M. (1950) Studies in Experimental Immunology of Influenza. IV. The Protective Value of Active Immunization. Aust. J. exp. Biol. ned. Sci., 28, 62. STRAUB, M. (1937) The Microscopical Changes in the Lungs of Mice Infected with Influenza Virus. J. Path. Bact., 45, 75. THOMAS, G. (1973) Respiratory Immunization with Inactivated Influenza Aerosol Vaccine in Man followed by Live Challenge. IVth Internat. Symp. Aerobiology. Ed. J. F. Ph. Hers and K. C. Winkler. Utrecht: Oosthoek Publishing Co. p. 328. WALDMAN, R. H., KASEL, J. A., FULK, R. V., ToGo, Y., HORNICK, R. B., HEINER, G. G., DAWKINS, A. T. & MANN, J. J. (1968) Influenza Antibody in Human Respiratory Secretions after Subcutaneovs or Respiratory Immunization with Inactivated Virus. Nature, Lond., 218, 594.
