a The protein array pionee
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`iic'iii;8éi.:?acnigged
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`firejournal qr
`Immunologic Impairment in
`n 1
`Mice Treated Intravenously
`Immu O ogy with Killed Coccidioides
`Immitis Spherules: Suppressed
`ThIS information IS current as Res onse t0 Intramuscular
`of September 19, 2016.
`DOSES
`
`H. B. Levine and Yi-Chi M. Kong
`
`J Immunol 1966; 97:297—305; ;
`http ://WWw.j immunol . org/content/97/3 /29
`7
`
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`9103‘61loqutoldagno1san3ziqxfilomunumnl'mmgflduqu101_1papnoplmoq
`
`The Journal of Imrmnol ogy is published twice each month by
`The American Association of Immunologists, Inc,
`9650 Rockville Pike, Bethesda, MD 20814-3 994.
`Copyright © 1966- by The American Association of
`Immunologists, Inc. All rights reserved.
`Print ISSN: 0022-1767 Online ISSN: 1550-6606.
`
`
`
`AstraZeneca Exhibit 2130 p. l
`InnoPharma Licensing LLC v. AstraZeneca AB
`IPR2017-009‘04
`
`Fresenius-Kabi USA LLC v. AstraZeneca AB
`
`IPR2017-01910
`
`

`

`THE JOURNAL on IMMUNOLOGY
`Copyright © 1966 by The Williams & Wilkins Co.
`
`Vol. 97, No. 3
`Printed in USA.
`
`IMMUNOLOGIC IMPAIRMENT IN MICE TREATED INTRAVENOUSLY
`WITH KILLED COCCIDIOIDES IMMITIS SPHERULES: SUPPRESSED
`RESPONSE TO INTRAMUSCULAR DOSES1
`
`H. B. LEVINE AND YI-CHI M. KONG
`
`From the Naval Biological Laboratory, School of Public Health, University of California,
`Berkeley, California
`
`Received for publication March 28, 1966
`
`In earlier studies (1, 2), formalin—killed spher-
`ule vaccines of Coccidfoides immitt’s were found
`
`to be very eflficacious in mice and monkeys when
`administered by the subcutaneous and intra—
`muscular (i.m.) routes. We recently investigated
`intravenous (i.v.) vaccination because Larson
`et al. (3*) observed that it was strikingly effective
`with particulate tubercular antigens (in oil) and
`also because it had been used on occasion to test
`
`other fungal vaccines.
`i.v.
`immitis,
`In the present study with C.
`administered spherules not only immunized mice
`poorly, but prevented the development of strong
`immunity from i.m. doses given up to 35 days
`earlier. Animals so treated succumbed to chal—
`
`in contrast,
`lenge with approximately 5 LD50;
`those vaccinated by the i.m. route alone survived
`11p to 200 LDso.
`The attributes of this deficient response were
`of concern for several reasons: with the possible
`exception of Cryptococcus (4), the reaction had not
`generally been reported for fungal antigens and,
`inasmuch as it was demonstrable primarily by iv.
`injection, the possibility was raised that a similar
`reaction might have been a factor in poor immunity
`to numerous other fungi following i.v. vaccina-
`tion and/0r challenge (5). Finally, impaired im—
`munity was markedly pronounced in our model
`which differed from others demonstrating unre-
`sponsiveness in that the challenge was adminis-
`tered intranasally, producing primarily pulmo-
`nary disease (6), and that the particulate vaccines
`producing impaired immunity were comprised of
`structures degraded very slowly in vivo (7).
`
`1 This work was sponsored by the Office of Naval
`Research and the Bureau of Medicine and Surgery,
`United States Navy, under a contract between the
`Office of Naval Research and the Regents of the
`University of California. Reproduction in whole
`or in part is permitted for any purpose of the
`Unite d States Government.
`
`Impaired immunity to Coccidioides was medi-
`ated by immunospecific substances, but heat-
`killed preparations of reduced immunogenicity
`were still effective. With formalin—killed prepara-
`tions, there was no adverse effect on existing im-
`munity when the i.v. dose followed the i.rn. dose
`by 1% to 3 months; i.v. treatment at 3 months
`actually potentiated immunity. Although the
`mechanisms of the deficient response are obscure,
`its major features are similar to those described
`for
`immunologic unresponsiveness induced by
`soluble bacterial and other antigens (8, 9).
`
`METHOD S
`
`The Silveira strain of C.
`
`immitis was used
`
`throughout. Mice were vaccinated with spherules
`or endospores cultured and formalin-killed as de—
`scribed earlier (1, 10). The animals were later
`challenged intranasally (11) with the arthrospore
`phase of the fungus. The vaccine and challenge
`doses, and the intervals between them, were
`varied and are detailed in conjunction with in-
`dividual experiments. Census of fungal numbers
`in the lungs of mice was made as described
`earlier (6).
`Spherule walls were isolated and purified from
`mechanically disrupted, formalin-killed spherules
`as described by Kong et al. (12) with the modifi-
`cation that a Braun Model MSK homogenizer
`(Brownwill Scientific, Rochester, N. Y.) was used;
`the soluble protoplasmic material was separated,
`filtered (12) and investigated for its influence on
`immunity and suppression of immunity. Simi—
`larly, the effect of iv. injection of intact Crypto—
`coccus neoformans or Saccharomyces cercvist'ae on
`both responses was studied with formalin-killed
`organisms. C’ryptococcus was grown in heart
`infusion broth (Difco) for 48 hr with shaking at
`35°C and commercial baker’s yeast cake was the
`source of Saecharomyoes. In one experiment non—
`formalinized, autoclaved (121°C, 18 hr) spherules
`
`297
`
`
`
`
`
`9103‘61requioidegnoisonfiziqxfiiowunumnf'mat/[duqwonpopcoplmoq
`
`AstraZeneca Exhibit 2130 p. 2
`
`

`

`298
`
`H. B. LEVINE AND YI-CHI M. KONG
`
`[von 97
`
`TABLE I
`
`Mortality in mice challenged inti'anasally with
`Coccidioides
`immitis after vaccination by
`difierent routes with formalin—
`killed spherules
`Dead/Total (30 Days Postchallenge)
`Challeng~
`ing£059,
`Vaccinated“
`12mg
`
`(o.A h -
`532:5
`Control its lie 23%;? ”5“.
`arm:
`i.v.
`
`
`2/9
`40
`7/10
`60
`7/10
`110
`6/10 3/10
`1/3
`10/10 0/10 0/10
`275
`9/10 8/10
`2/5
`0/10 0/10
`480
`10/10 7/9
`5/5
`1/10 1/10
`2040
`
`
`
`
`2/9 1/10 ——°4200 10/10 6/10
`
`were also studied for the effects mentioned above.
`
`All preparations for i.v. use were sedimented
`repeatedly by centrifugation and resuspended in
`pyrogen—free saline (Cutter Laboratories, Berke-
`ley, Calif.); the final suspensions were adjusted
`to permit later specified doses to be given in a
`volume of 0.5 ml. When the suspensions were to
`be used over a period of several days or weeks,
`1 :10,000 Merthiolate (final concentration) was
`added.
`Female NAMRU albino mice, penbred in our
`laboratory, were employed in these experiments.
`The animals were 6 to 12 Weeks old at the incep-
`tion of different experiments, but in any one ex-
`periment their age varied no more than 2 weeks.
`RESULTS
`
`“ With % indicated dosage on days 0, 7 and 14;
`challenged on day 42.
`" I.m. = intramuscularly; i.v. = intravenously.
`0 No data.
`
`The weak immunologic response of mice vac-
`cinated i.V. with killed spherules and the mark-
`edly impaired response in mice given vaccine by
`both the i.v. and i.m. routes are shown in Table I.
`
`TABLE II
`
`The influence of spherule dose given intravenously (11.2).) on immunity to Coccidioides immitis induced by
`intramuscular (i.m.) injection of vaccine
`% Mortalityb (30 Days Postchallenge)
`Mg Spherules per Dose“
`Experiment
`and Route (5)
`Challenging dose (no. arthrospores):
`24
`43
`89
`250
`350
`465
`740
`115041220 2400—3050
`
`
`None
`0.4 i.v.
`0.4 i.m.
`0.8 i.m.
`0.4 i.m. -|- 0.04 i.V.
`0.4 i.m. + 0.2 i.v.
`0.4i.1’n. + 0.4 i.v.
`0.4 i.m. + 0.6 i.V.
`
`100
`94
`10
`11
`39
`61
`44
`38
`
`100
`94
`0
`0
`45
`70
`61
`31
`
`100
`89
`22
`0
`70
`65
`83
`50
`
`100
`100
`50
`50
`47
`79
`82
`66
`
`44
`
`22
`
`78
`
`None
`0.4 i.V.
`0.4 i.m.
`0.8 i.m.
`0.41.111. -- 0.012 i.V.
`0.41.11). + 0.025 i.V.
`0.4 i.m. -- 0.05 i.V.
`0.4 i.m. —— 0.1 i.v.
`0.4 i.m. —— 0.2 i.v.
`0.4 i.m. —— 0.4 i.v.
`
`90
`89
`0
`0
`8
`8
`8
`33
`33
`33
`
`100
`0
`22
`25
`8
`8
`42
`83
`67
`
`100
`50
`0
`9
`55
`45
`42
`75
`58
`
`100
`30
`14
`80
`40
`67
`33
`80
`75
`
`
`
`1
`
`2
`
`
`
`
`
`
`
`9103‘61loqmo‘idogno15mgAqxfiiomunumnf'mm/[duq11101}popnoplmoq
`
`‘1 Indicated doses given on days 0, 7 and 14; mice challenged intranasally on day 49 (Exp. 1) or on
`day 46 (Exp. 2).
`5 From 7 to 20 mice per group; mice, treated by both i.m. + i.v. routes, were composed of 10 to 20
`per group.
`
`AstraZeneca Exhibit 2130 p. 3
`
`

`

`1966]
`
`IMMUNOLOGIC IMPAIRMENT TO 0. IMMITIS
`
`299
`
`Whereas 60 or more arthrospores administered in-
`tranasally were lethal to 70 to 100% of nonvac-
`cinated mice, animals that had been immunized
`i.m. with three doses of spherules, totaling 1.2
`or 2.4 mg, were well protected against challenge
`doses of up to at least 4200 arthrospores. Neither
`vaccine dosage was very effective when given
`iv; immunity so induced was sufficient only
`to reduce mortality rates in mice challenged
`with liSO or fewer arthrospores. This was also the
`
`TABLE III
`
`Influence of spherule vaccine given intravenously.
`on mortality after challenge with Coccid'ioides
`immitis
`% Mortality“ (30 Days Postchallenge)
`Challenging dose (no. arthrospores):
`Spherule Dose
` 60 150 630
`
`
`mg
`80
`0.0
`90
`60
`0.00001
`100
`95
`0.0001
`70
`100
`0 . 001
`90
`63
`0 . 01
`90
`100
`0 . 1
`
`
`921 . O 100
`
`50
`
`" From 10 to 15 mice per group; challenged in-
`tranasally ’40 days after vaccination.
`
`case in mice vaccinated i.m. with 1.2 mg of
`vaccine if they received concurrently an addi-
`tional 1.2 mg i.v., notwithstanding the eflficacy
`of 1.2 or 2.4 mg when given by the i.m. route
`alone.
`
`The impaired response of i.m. vaccinated mice
`also treated iv with the antigenic preparation is
`again evident in Table II; those given iv. as little
`as 120 to 150 ug of intact spherules were poorly
`immunized. These dosages contained insufficient
`antigen to confer immunity by the i.m. route (10).
`Nor was immunity to 150 to 630 arthrospores in-
`duced by any i.v. dose of spherules between 0.01
`,ug and 1.0 mg (Table III). Conversely, suppressed
`immunity did not follow i.m. injection of 20 mg
`of spherules or 4.5 mg of spherule walls (Table
`IV), the major locus of immunogens (12). The
`immunogen content of 4.5 mg of walls corre-
`sponded approximately to that of 18 to 22 mg of
`intact spherules.
`Spherule walls, given by different routes, in-
`duced the same responses in mice as intact organ.
`isms: They were highly immunogenic by the i.m,
`route, poorly immunogenic by the i.v. route and-
`when administered i.v., impaired the response to
`i.m. doses (Table V). We were therefore interested
`in determining if the soluble protoplasmic moiety
`of spherules, which is poorly immunogenic except
`
`TABLE IV
`
`Influence an immunity to Coccidioides immitis of dose of spherule walls and
`intact spherules given intramuscularly (i.m.)
`% Mortality“ (30 Days Postchallenge)
`Challenging dose (no. arthrospores):
`
`Preparation
`
`Dose
`
`Spherule walls
`
`Intact spherules
`
`Me
`
`0
`0 . 0045
`0.045
`0.45
`4.5
`
`0
`2.0
`6.0
`10.0
`20.0
`
`30
`40-50
`75—100
`175
`455
`fit?)
`4,800
`$69,335
`15,000
`55,000
`
`
`30
`
`90
`
`70
`70
`10
`10
`0
`
`100
`30
`30
`0
`
`100
`11
`30
`0
`
`100
`50
`20
`0
`
`100
`60
`50
`30
`
`70
`
`40
`
`100
`
`90
`40
`20
`10
`0
`29
`20
`30
`0
`40
`20
`20
`0
`
`
`
`70600 20
`
`
`
`9103‘61leqmeldegno15mgziqxfiromunumnf'mmm/fduq11101}pepeopI/noq
`
`
`
`
`
`“ From 7 to 11 mice per group. Vaccinated With kc: indicated dosage on days 0, 7 and 14 (spherule
`walls) or % indicated dosage on days 0, 7, 14 and 20 (intact organisms); challenged intranasally 22
`or 29 days respectively later.
`
`AstraZeneca Exhibit 2130 p. 4
`
`

`

`800
`
`H. B. LEVINE AND YI—CHI M. KONG
`
`[VOL. 97
`
`TABLE V
`
`Influence on immunity of Coccidioides immitis spherule fractions given intramuscularly (i.m.) and/07“
`intravenously (Lu)
`Dead/Total (33 Days Postchallenge)
`Treatment“
`
`
`Challenge
`231356 (No'
`1'
`rospores
`
`)
`
`9.3 mglwailss
`0 3
`0'5 13g 150mb
`o s
`d’
`F
`1 bl
`11
`.
`03
`03
`S
`116111]
`LI“. 1) 115
`.
`. mg W3- 5
`pro Op-aSmlC
`. mg 50 u . 6
`NW
`“an” must-5m
`1:22am?
`$123331:
`wan???“ heats-W peasant
`adjuvant 1.m.
`fraction i.v.
`
`
`1 /10
`5/10
`7/11
`
`3/ 10
`5/10
`7/10
`
`20
`40
`0/10
`0/10
`6/10
`80
`0/10
`5/9
`9/10
`0/10
`130
`3/9
`6/8
`7/8
`1/10
`475
`
`1150 3/10
`
`2/10
`4/10
`6/10
`
`0/10
`0/10
`0/10
`
`‘1 With % indicated dosage on days 0, 7, 14; challenged intranasally on day 44.
`'7 Freund’s complete adjuvant, 1:1 with either saline or soluble protoplasmic fraction.
`
`TABLE VI
`
`to mice
`Influence an immunity to Coccidioides immitis of autoclaved spherules given intravenously (131).)
`mocinaded intramuscularly (i.m.)
`Dead/Total (30 Days Postchallenge)
`
`Treatment“
`Challenging dose (no. arthrospore’s):
`so
`120
`1200
`
`
`8/10
`None
`5/10
`3/10
`1.0 mg spherules i.m.
`5/10
`0/10
`2.0 mg spherules i.m.
`8/9
`7/10
`1.0 mg autoclaved spherules i.m.
`10/10
`4/10
`2.0 mg autoclaved spherules i.m.
`6/6
`4/5
`1.0 mg autoclaved spherules i.v.
`8/8
`2/8
`1.0 mg spherules i.m. plus 1.0 mg spherules i.v.
`
`1.0 mg spherules i.m. plus 1.0 mg autoclaved spherules i.v. 6/6 2/6
`
`
`3/10
`
`“ One dose as shown on day 0; mice challenged intranasally on day 30. Spherules were killed by for-
`malin, except those designated “autoclave-d” which were killed by heating at 121 °C for 18 hr.
`
`when emulsified with Freund’s complete adj u—
`vant (12) , similarly impaired the response of mice.
`The findings in Table V show that the weakly im-
`munogenic soluble fraction, given i.V., suppressed
`only slightly the development of i.m. induced im—
`munity. The relationship between the capacities
`of a preparation to confer immunity by the i.m.
`route and to suppress it by the i.V. route was not,
`however, an uncomplicated function of immuno-
`genieity. The i.v. injection of autoclaved spher—
`ules of markedly reduced immunogenieity still
`suppressed immunity development in mice vac-
`cinated i.m. with formalinized spherules (Table
`VI).
`Formalinized endospores, somewhat
`
`less im-
`
`munogenic than formalinized spherules (10, 13)
`but more immunogenic than heated spherules
`(Table VI), also interfered with immunity devel—
`opment when given i.v. (Table VII, Exp. 1)
`to
`either spherule— or endospore—vaccinated (i.m.)
`mice. Correspondingly,
`l.V. administered spher-
`ules had a similar effect on endospore—vaccinated
`mice. Both morphologic phases showed suppres-
`sive activity by the intraperitoneal route (Table
`VII, Exp. 2), but this was less pronounced than
`that induced i.v. Since the spherules ranged from
`15 to 20 H in diameter and endospores from 1 to
`3 1.1, suppressed immunity induced by either route
`apparently was not attributable to the large size
`of injected spherules. This consideration is noted
`
`
`
`
`
`9103‘61requieideg110159113Aqxfiiomunumuf'mm/[duquioripepeoplmoq
`
`
`
`
`
`
`
`AstraZeneca Exhibit 2130 p. 5
`
`

`

`1966]
`
`IMIVIUNOLOGIC IMPAIRMENT T0 0. IMMITIS
`
`301
`
`TABLE VII
`
`Influence of spherules and endospores given intravenously (in) or intraperitaneally (i.p.) on immunity
`to Coccidioides immitis induced intramuscularly (i.m.)
`% Mortality“ (30 Days Postchallenge)
`
`Exp.
`Treatment
`Challenging dose (no. arthrospores):
`30
`60
`120
`600
`1200
`
`
`1
`
`50
`
`None
`2.0 mg spherules i.m.
`1.0 mg spherules i.m.
`2.0 mg endospores i.m.
`1.0 mg endospores i.m.
`1.0 mg spherules i.v.
`2.0 mg endospores i.v.
`1.0 mg endospores i.v.
`1.0 mg spherules i.m. plus 1.0 mg spherules i.v.
`1.0 mg spherules i.m. plus 1.0 mg endospores i.v.
`1.0 mg endospores i.m. plus 1.0 mg endospores i.v.
`1.0 mg endospores i.m. plus 1.0 mg spherules i.v.
`78
`None
`10
`0
`1.0 mg spherules i.m.
`86
`0
`1.0 mg endospores i.p.
`30
`20
`1.0 mg spherules i.p.
`90
`60
`1.0 mg spherules i.m. plus 1.0 mg endospores i.v.
`50
`10
`1.0 mg spherules i.m. plus 1.0 mg endospores i.p.
`40
`50
`1.0 mg spherules i.m. plus 1.0 mg spherules i.v.
`
`1.0 mg spherules i.m. plus 1.0 mg spherules i.p. 15 33
`
`
`70
`0
`0
`20
`0
`50
`80
`100
`50
`20
`0
`20
`30
`
`10
`30
`30
`20
`100
`100
`100
`100
`80
`100
`100
`
`
`
` 9103‘61lequleldeg{1015mgAqxfilomunumul'mm/[duq11101}pepeoplmoq
`
`2
`
`
`
`
`
`
`
`‘1 7 to 13 mice per group; challenged intranasally 30 days after treatment.
`TABLE VIII
`
`Influence on immunity to Coccidioides immitis of formalin-killed Cryptococcus neoformans or
`Saccharomyces cerevisiae given intravenously (75.1).) in mice vaccinated intramuscularly (i.m.)
`with spherules
`% Mortality“ (30 Days Postchallenge)
`
`Challenge dose (no. arthrospores):
`50-60
`90-120
`450—660
`900
`20—30
`2400
`
`Treatment
`
`Exp.
`
`1
`
`90
`
`None
`0.4 mg spherules i.m. days 0, 7, 14
`0.8 mg spherules i.m. as above
`0.4 mg spherules i.v. as above
`0.8 mg spherules i.v. as above
`0.4 mg spherules i.m. plus 0.4 mg spherules i.v. as above
`0.4 mg Spherules i.m. plus 0.4 mg Cryptococcus neo—
`formans i.v. as above
`
`90
`
`78
`0
`10
`47
`8
`60
`0
`
`40
`6,0
`85
`42
`80
`14
`
`40
`70
`92
`67
`90
`36
`
`2
`
`20
`
`50
`
`56
`
`None
`0.4 mg spherules i.m. days 0, 7
`0.4 mg spherules i.m. day 0; 0.4 mg spherules i.v. day 7
`0.4 mg spherules i.m. day 0
`0.4 mg spherules i.m. day 0; 0.4 mg Sacchammyces
`63
`33
`11
`cerevisiae i.v. day 7
`
`0.4 mg Saccharomyces cerevisiae days 0, 7 63 89 75
`
`
`
`0
`20
`No data 78
`10
`40
`
`20
`100
`55
`
`‘1 10 to 15 mice/group (Exp. 1), 6 to 10 mice/group (Exp. 2); animals challenged intranasally 29 to 30
`days after last treatment.
`
`AstraZeneca Exhibit 2130 p. 6
`
`

`

`302
`
`H. B. LEVINE AND YI—CHI M. KONG
`
`[VOL. 97
`
`
`
`
`
`
`
`9103‘61requieideguoisenfiziqxfiiomunumnf'mawfinquioripepcoplmoq
`
`shown in Table IX, a single dose of vaccine given
`iv. as late as 30 to 35 days after a single i.m. dose,
`but not 49 days later, resulted in poor immunity.
`When the i.V. dose preceded the i.m. dose by at
`least 30 (Exp. 1) or 49 (Exp. 2) days, no marked
`resistance developed. These data indicated that
`spherules given i.V. altered the host’s capacity to
`develop immunity but did not interfere with its
`capacity to express a pre—existing immunity.
`The inference that existing immunity was not
`impaired by i.V.
`treatment with antigen was
`tested further in two ways. First (Table X), mice
`were vaccinated i.m. 2 months earlier with 2.1 mg
`of spherules in three doses and were given 0.2 to
`0.8 mg spherules i.V. 4 days before challenge.
`These animals remained resistant to a challenge
`dose of 1000 arthrospores.
`(In nonvaccinated
`mice, the 4—day interval between treatment by
`various routes and challenge was insufficient to
`demonstrate unequivocal immunity development
`(10) or its suppression.) Secondly (Fig. 1), mice
`vaccinated 100 days earlier were given 0.2 or 0.02
`mg of vaccine either i.V. or i.m. 7 days before chal-
`lenge with 670 arthrospores. In these groups, im-
`
`TABLE X
`Mortalt’ty after challenge with Coccidioides tmmitis
`in spherule-vaccinatecl
`tntramuscularly (i.m.)
`and nonvacclnated mice treated with Spherules
`intravenously (iv)
`
` .
`
`Group
`
`Treatnrzleéilti151462;? before
`
`Dead/
`d'i‘gtsallagi
`challenge“
`
`Prevaccinatedb
`
`None
`0.8 mg spherules i.m.
`0.2 mg spherules i.V.
`0.4 mg spherules i.V.
`0.6 mg spherules i.V.
`0.8 mg spherules i.V.
`
`0/12
`0/13
`0/12
`0/11
`0/9
`0/8
`
`Nonvaccinated
`
`None
`0.4 mg spherules i.m.
`0.2 mg spherules i.V.
`0.4 mg spherules i.V.
`0.8 mg spherules i.V.
`0.4 mg spherules i.m.
`plus
`
`0.4 mg spherules i.V. 6/13
`
`6/12
`2/12
`5/12
`6/12
`4/12
`
`“ Prevaccinated group challenged with 1000
`arthrospores intranasally, nonvaccinated group
`with 100.
`5 Three weekly doses of 0.7 mg spherules intra~
`muscularly; last dose 8 weeks before treatment.
`
`AstraZeneca Exhibit 2130 p. 7
`
`TABLE IX
`
`Influence an immunity to Coccidioides immttis of
`spherules given intravenously (iv) at intervals
`before and after intramuscular
`(i.m.) injection
`
`% Mortality (30 Days Postchallenge)
`Vaccinated
`
`Days
`between
`Exp. Ist and Nonvac-
`D2ndsa cinated lst dosei.m.; lst dose i.m.;
`lst dose i.V.;
`056
`2nd dose i.m. 2nd dose i.V. 2nd dosei.m.
`
`
`80
`
`1 —
`0
`4
`7
`1 1
`15
`20
`30
`
`60
`60
`20
`35
`25
`15
`25
`
`70
`65
`55
`37
`2O
`55
`25
`
`70
`50”
`65
`80
`53
`45
`53
`
`2
`
`100
`
`——
`79
`94
`60
`0
`45
`53
`27
`7
`63
`75
`20
`14
`68
`31
`29
`21
`32
`47
`33
`28
`50
`50
`0
`35
`38
`29
`0
`42
`
`
`
`0 1049 50
`
`“ 0.4 mg (Exp. 1) 01‘ 1.0 mg (Exp. 2); mice chal-
`lenged intranasally with 460 arthrospores at 62
`days after first dose (Exp. 1) or with 700 arthro-
`spores at 77 days after first dose (Exp. 2).
`1' Ten mice only; 13 to 21 mice in all other
`groups.
`
`because we have observed that the i.V. injection
`of 2.0 mg of spherules or more killed some mice,
`possibly by embolism.
`The above relationships between immunity in-
`duction and its suppression by different cellular
`preparations, fractions and growth phases im-
`plicated immunospecific substances as the medi-
`ators of the impaired response. Thus, unrelated
`organisms were examined in spherule-vaccinated
`mice. Previous studies showed that such animals
`
`were unprotected against C'. neoformans (12) and
`Table VIII shows that formalin—killed crypt0«
`cocci given i.V. did not impair specific immunity
`to Coccidioides. Nor did i.v. administered S. cerevi-
`siae influence anticoccidioidal immunity (Table
`VIII).
`The effective time intervals between i.m. and
`i.V. injections for producing a deficient response
`also pointed to immunospecific relationships. As
`
`

`

`1966]
`
`IMMUNOLOGIC IMPAIRMENT TO 0. IMMITIS
`
`303
`
`NONVACCINATED I
`
`VACCINATED1
`
`NO.RECOVEREDFUNGALPARTICLES/LUNG(ALLLOBES)5dayspost
`
`
`
`
`
`
`intranasalchallengewith670arthrospores
`
`fi—l
`
`103
`
`,._. C)N
`
`»_4 C3._.
`
`>—1
`
`CO
`
`TREATMENTS
`
`Figure 1. Coccidioides immitis numbers in the lungs of mice treated with spherules before infection
`1 Vaccinated intramuscularly on days 0, 7 and 14; challenged on day 107.
`2 Intramuscular or intravenous treatment given on day 100.
`
`munity, measured by the capacity of the animals
`to restrict fungal multiplication in the lungs, was
`potentiated, not suppressed. The fungal census
`at 5 days postchallenge showed that the numbers
`of viable organisms per lung were .101~ to 106-fold
`lower in the treated, immunized mice than in im—
`munized mice not receiving the i.v. or i.m. injec-
`tion of spherules. Thus,
`in preimmunized mice,
`the i.v. dose,
`like the i.m. dose, boosted (14:)
`rather than suppressed immunity.
`
`DISCUSSION
`
`A detailed comparison of the nature of sup-
`pressed immunity by coccidioidal
`antigen(s)
`with that of numerous nonfungal systems pr0«
`ducing immunologic unresponsiveness
`is not
`feasible because of the complexity of the unpuri—
`fied fungal antigen (s). The spherule wall, the prin—
`cipal source of immunogen (12), is also the major
`cellular fraction mediating suppression of immu-
`nity (Table V). The wall is a chitin—polysaccha—
`ride-protein structure (15—18) that is catabolized
`slowly in vivo (7). As yet, we have not been able
`to extract appreciable soluble immunogenic mate—
`rial from it by physical, chemical and enzymologic
`methods (unpublished). Present evidence suggests
`that the wall encompasses either two or more im-
`
`munogens or a single immunogen having at least
`two groups of determinants. One is heat—stable,
`or,
`in its molecular arrangement,
`is protected
`from the adverse effects of heat and the other is
`labile to a temperature of 60°C (unpublished) or
`higher
`(Table VI). Optimal
`immunity results
`only in animals receiving both.
`If one speculates, however, that the stable ma-
`terial is polysaccharidal and the labile is protein-
`aceous, suppressed immunity in the coccidioidal
`system could encompass either long-lasting or
`transient features or both (19, 20) superimposed.
`Evidence that there is a long—lasting influence is
`presented in Table IX; Experiment 2 shows that
`when i.v. treatment preceded i.m. vaccination by
`49 days,
`immimity development was impaired.
`This feature directs attention to slowly catabo-
`lized moieties as possible mediators of suppressed
`immunity to C. immitis. Relatedly, it may be
`noted that the slow rate of immunity develop-
`ment after vaccination appears to be referable
`also to the slow rate at which spherules and their
`walls are catabolized (7).
`The above relationships and hypotheses are
`not incompatible with the finding (Table VI) that
`immunogenicity was reduced by heat but with-
`out a corresponding reduction in the capacity of
`
`
`
`
`
`
`
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`

`304
`
`H. B. LEVINE AND YI—CHI M. KONG
`
`[VOL 97
`
`heated spherules to render mice unresponsive. If
`the heat—stable moiety is chemically distinct from
`the heat—labile and is involved exclusively in sup-
`pressed immunity, its influence would not have
`been affected by the heat treatment. Alterna-
`tively,
`if
`the stable component
`is chemically
`similar to the labile but protected against heat
`denaturation, the low dosage requirement for sup-
`pression (120 to 150 pig of intact spherules, Table
`II) could account for unreduced activity. How—
`ever, inasmuch as the preparation was autoclaved
`for 18 hr, it appears that suppression is mediated,
`at least in part, by heat—stable materials distinct
`from the heat—labile components.
`Since heterologous organisms did not impair
`immunity (Table VIII) and immunogenic com—
`ponents of spherules (Table V) did,
`immuno-
`specificity was implicated. In this respect,
`it
`should be noted that the soluble protoplasmic
`fraction of spherules, virtually nonimmunogenic
`except when emulsified in Freund’s adjuvant
`(Table V), also did not suppress immunity. But
`since the dose of
`the soluble fraction in the
`presence of adjuvant was suflficient to immunize,
`its failure to induce or suppress immunity without
`adjuvant could reflect
`the host’s capacity to
`eliminate unbound antigens. Possibly, the adju-
`vant and intact spherules or walls functioned
`similarly to prolong antigenic stimulation. In sup-
`port of this, we have found that surgical removal
`of intramuscular sites at 14 days, or footpad sites
`at 32 days, postvaceination with spherules re-
`sulted in suboptimal
`immunity (7). At
`these
`times, both sites contained spherules, and residual
`immunogen was still demonstrable in footpads at
`35 days.
`Notwithstanding the undefined nature of the
`immunogens as well as the preparations producing
`an impaired immunologic response,
`there are,
`nevertheless, major parallelisms which can be
`drawn between immunity suppression in the
`coccidioidal model and immunologic unrespon-
`siveness (tolerance) in others reviewed recently
`by Smith (21) and Dvorak (8). These pertain to
`immunospecificity,
`the general unsusceptibility
`of preimmunized mice to becoming unresponsive
`(8, 22, 23), the efficacy of the i.v. route (24—26),
`the effectiveness of low doses and the duration of
`unresponsiveness (23). Additionally, as observed
`by others (8), studies in progress show that the
`delayed hypersensitivity reaction to coccidioidin
`in i.m. vaccinated mice (27) is suppressed in those
`also given spherules i.v.
`
`James
`Acknowledgments. The assistance of
`Cobb, Darryl Comstock and Edward Boerke is
`acknowledged gratefully.
`
`SUMMARY
`
`(i.m.) vaccina-
`Subsequent to intramuscular
`tion with killed Coccidioides immitis spherules,
`mice developed strong immunity and survived
`intranasal challenge doses of arthrospores in the
`magnitude of 200 LD50. This was not the case in
`mice vaccinated intravenously (i.v.); a majority
`succumbed to challenge with 5 LD50. Further-
`more, i.m. vaccinated mice also succumbed to the
`latter challenge when small amounts of antigen
`were administered i.v. either concurrently with,
`before, or up to 35 days after the i.m. doses. The
`nature of the deficient response was in general
`accord with that described for immunologic un-
`responsiveness in the adult animal and was read—
`ily demonstrable with 120 [.Lg of killed spherules.
`The i.v. administration of antigen adversely
`affected immunity development, not its expres-
`sion, because the immunity of preimmunized mice
`could not be impaired by the above—noted treat-
`ment. Immunity suppression was produced also
`with spherule walls, the major locus of the im-
`munogens. Similarly, a suppressed response fol—
`lowed the iv.
`injection of endospores in mice
`vaccinated i.m. with either the spherule or the
`endospore phase of the fungus. However, neither
`Saccharomyces cerevisiae nor Cryptococcus neo—
`former/es, given i.v., induced a deficient response
`in spherulevvaccinated mice nor did a weakly im-
`munogenic soluble cellular
`fraction impair
`it
`significantly. The data suggested that suppression
`was mediated by immunospecific substances and
`was not influenced by the size of the structures
`introduced i.v. which varied from 1 to 2 M (endo-
`spores) to 15 to 20 u (spherules).
`
`REFERENCES
`
`1. Levine, H. 13., Cobb, J. M. and Smith, C. E.,
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`J. Immun., 89: 242, 1962.
`3. Larson, C. L., Ribi, E., Wicht, W. C., List, R.
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`experi—
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`5. Levine, H. B.,
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`In
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`Thomas, Springfield, 111., 1962.
`
`
`
`9103‘61ioqureldeguoissufiziqxfiiowuuurunf'W/[duq11101}popnoprmoq
`
`
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`
`AstraZeneca Exhibit 2130 p. 9
`
`

`

`1966]
`
`IMMUNOLOGIC IMPAIRMENT TO 0. IMMITIS
`
`305
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`. Dvorak, H. F., Billote, J. B., McCarthy, J. S.
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`
`9103‘61loquloldog110159113Aqxfilomunumnf'mm/fduq11101}popcoplmoq
`
`
`
`
`
`
`
`AstraZeneca Exhibit 2130 p. 10
`
`