EXPERIMENTAL VIABLE VACCINE AGAINST PULMONARYCOCCIDIOIDOMYCOSIS IN MONKEYS1

JOHN L. CONVERSE, MERIDA W. CASTLEBERRY,2 AND ERNEST M. SNYDERU.S. Army Biological Laboratories, Fort Detrick, Frederick, Maryland

Received for publication 21 June 1963

ABSTRACTCONVERSE, JOHN L. (U.S. Army Biological

Laboratories, Fort Detrick, Frederick, Md.),MERIDA W. CASTLEBERRY, AND ERNEST M. SNY-DER. Experimental viable vaccine against pul-monary coccidioidomycosis in monkeys. J.Bacteriol. 86:1041-1051. 1963.-Monkeys (Ma-caca mulatta) vaccinated by subcutaneous injec-tion in the forearm with from 10 to 108 viableCoccidioides immitis arthrospores were protectedagainst respiratory challenge with approximately7000 viable arthrospores administered 6 monthsafter vaccination. Protection was evident from:the healthy appearance throughout 4 monthsafter respiratory challenge; negative chest X raysat 15, 30, 60, and 120 days; and only very minorhistopathological pulmonary changes on autopsyat 120 days, with negative lung cultures in 80%of the animals. This was in striking contrast tothe outward clinical appearance of control mon-keys that were unvaccinated or had receivednonviable arthrospore vaccines. These monkeysshowed severe disease (loss of weight, acceleratedrespiration, severe coughing, general debilita-tion), positive X rays, massive pulmonary de-struction, positive lung cultures, and death of fiveof nine animals. The appearance of spherules(very few in number, accompanied by very minorpathological changes) in the lungs of some of the"dissemination controls" (subcutaneous viablevaccination without respiratory challenge) indi-cated possible dissemination from the primarycutaneous infection, although oral transmissionfrom the cutaneous lesions could not be ruled out.

The apparent protection afforded by primary1 Animals maintained in compliance with the

"Principles of Laboratory Animal Care" as

promulgated by the National Society for MedicalResearch, 1961, Bio-Medical Purview 1:14.

2 Present address: Research and NutritionLaboratory, Fitzsimmons U.S. Army GeneralHospital, Denver, Colo.

pulmonary coccidioidomycosis against secondary,exogenous reinfection, and the absence of re-ported cases of systemic dissemination of Coc-cidioides immitis from foci of primary cutaneousinfections suggested the subcutaneous inoculationof this organism as a viable vaccine to establishimmunity against the disease.

Smith (1957), in several very definitive epi-demiological studies involving follow-ups ofthousands of human pulmonary Coccidioidesinfections, found no evidence of second infectionswith the fungus.Documented primary cutaneous infections

with C. immitis are few, and are limited almostentirely to personnel working with the organism.Among these are Wilson, Smith, and Plunkett's(1953) report of a mortician's contracting alesion on the finger from embalming the body ofa fatal case of coccidioidal granuloma; Guy andJacobs' (1926) description of the infection in apatient inoculated by the prick of a cactus thorn;and several unpublished laboratory accidents(self-inoculation). Winn (1961) and Meis (1961)have treated an additional 12 cases of primarycutaneous coccidioidomycosis. In the majority ofthese patients, infection was mild and limited tofocal lesions at or near the site of inoculation.In no instance was dissemination of the organismnoted beyond the regional lymph nodes of theaffected appendage.There have been numerous experimental stud-

ies of primary cutaneous coccidioidomycosis inanimals: the dog and the rabbit (Rixford, 1894a,b; Ophuls, 1905); the guinea pig (Davis, 1924;Chipman and Templeton, 1930; Guy and Jacobs,1927); and mice (Pappagianis et al., 1959). Inthese studies, systemic dissemination was ex-tremely rare. Moreover, the few visceral lesionsnoted were small focal areas, unaccompanied byany clinical evidence of illness.

Several investigators [Pappagianis et al. (1959)with subcutaneous vaccination with a highlyvirulent strain of C. immitis, and Converse et al.(1962a) with intraperitoneal vaccination with a

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strain of low virulence] have studied the resistanceof mice, previously inoculated with the viableorganism, to a second infection (via the intra-peritoneal route) with a virulent strain. Sub-stantial resistance to the challenge dose wasindicated in both of these studies, by histopatho-logical evidence as well as by mortality rates.Pappagianis et al. (1960) reported a similar re-sistance to second infection in preliminary studieswith cynomolgus monkeys (M1acaca irus).

This report describes an extensive study of theimmunogenic response of rhesus monkeys (Ma-caca mulatta) to subcutaneous vaccination withviable C. immitis arthrospores, and the patho-genesis of primary cutaneous infections in themonkey.

MATERIALS AND METHODS

Organism (Friedman et al., 1956). C. immitisstrain Silveira (isolated from a recovered primarypulmonary human infection), strain Cash (froman extrapulmonary disseminated nonfatal infec-tion), and strain M-1 1 (a rodent isolate fromArizona) were used as viable vaccines. StrainCash, killed with 0.5% formalin, was used as thenonviable vaccine, and strain Silveira was usedas the respiratory challenge organism. StrainsCash and M-11 arthrospores were harvestedfrom 14-day submerged growth (34 C with shak-ing) in the liquid synthetic medium of Roessleret al. (1946). Strain Silveira was harvested byvacuum as dry arthrospores from 5- to 8-weekgrowth on modified (0.1% yeast extract) Sa-bouraud agar, after desiccation of the medium.

Immunization. Monkeys (Mllacaca mulatta) ofboth sexes, weighing approximately 3 kg, wereimmunized by subcutaneous injections of viableor nonviable arthrospores (0.5 ml of a salinesuspension) in the medial surface of the rightforearm. They were housed with two monkeysper cage.

Respiratory exposure. Respiratory challenge ofthe animals was obtained by inhalation of a drycloud of arthrospores aerosolized in a 4800-literexposure chamber (head exposure only) by meansof compressed air. The exposure dose was calcu-lated from the average volume of monkey lungs,the respiration rate, the exposure time, and thecloud density.

Pathogenesis. The course of the infections(primary cutaneous as well as pulmonary) was

followed by: clinical observation, coccidioidinskin-hypersensitivity tests, determination ofprecipitin (Ppn) and complement-fixation (CF)titers, frontal chest X rays, and gross and histo-pathological studies and mycological culture ofautopsy material at death, or upon sacrifice at4 months after respiratory challenge.

Tissues for microscopic study were fixed in10% formalin, impregnated with paraffin, sec-tioned, and stained with Giemsa or the Gomorisilver methenamine stains. The fluorescent-anti-body technique was used as a further check onthe presence of the fungus in the tissues.

RESULTS

Monkeys, in groups of four animals each, weregiven either a single injection of 10, 100, or 1000viable strain Silveira arthrospores, or three in-jections (30 days apart) of formalin-killed ar-throspores (total dose 109 spores). At 6 monthsafter vaccination, these four groups of animals,together with a group of nonvaccinated controlmonkeys, were challenged by the respiratoryroute with a calculated inhaled dose of approxi-mately 7000 viable strain Silveira arthrospores.An additional group of monkeys, receiving theviable vaccine (10 to 108 spores in tenfold incre-ments) but not challenged by the respiratoryroute, was maintained as "dissemination con-trols."

Vaccination with viable arthrospores resultedin subcutaneous lesions 1 to 3 cm in diameter.Draining lesions (Fig. 1) at the vaccination siteand enlargement of the right axillary lymph nodes(Fig. 2) were noted in approximately 50% of theanimals. The open lesions were healed, and themajority of the axillary lymph nodes had re-turned to normal size 6 months after vaccination(Fig. 3). At this time, all monkeys which receivedthe viable vaccine exhibited skin hypersensitivityto coccidioidin (Table 1); the reactions of thosevaccinated with the killed product remaineddoubtful (some erythema, but no induration).With one exception, the Ppn and CF titers of allvaccinated animals either had become negativeor were at a low level (1:4 to 1:64). The animalin question was shown, on autopsy, to havefour active subcutaneous head lesions, whichprobably were responsible for the high titers(CF, 1:256; Ppn, 1:512).

After respiratory exposure, the nonvaccinated

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FIG. 1. Vaccination site on right forearm of a monkey receiving 108 viable Coccidioides immitis strainSilveira arthrospores (14 days postimmunization).

control monkeys and those vaccinated with theformalin-killed product exhibited extreme debilita-tion (Table 2), suffering from loss of appetite,emaciation, and pronounced accelerated respira-tion, accompanied by coughing. Widespread,wispy infiltration throughout all lobes of thelungs and visible consolidation in some areaswere noted in X rays of these animals 15 days

after respiratory exposure (Fig. 4). The majorityof the unvaccinated controls showed a markedrise in both CF and Ppn titers during the 4-month holding period. Approximately half ofthe unvaccinated control group and of the for-malin-killed vaccine group died from pulmonarycoccidioidomycosis within 4 months after respira-tory exposure.

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FIG. 2. Right axillary lymph node on the monkey shown in Fig. 1 (30 days postimmunization).

In contrast, monkeys vaccinated with theviable preparations exhibited no visible clinicalsymptoms of the disease after respiratory chal-lenge. The X rays were negative throughout the4-month holding period, and the majority ofserological titers remained at a low level (1:4 to1:64). No deaths occurred in this group.Upon autopsy (Table 3), the lungs of the un-

vaccinated control group and formalin-killedvaccine group were bosselated in appearance,and were covered with surface lesions. Largepalpable consolidated areas present throughoutall lobes of the lungs were caseous and necrotic

upon section (Fig. 5). There was extensive ad-hesion of the lungs to the pleura and diaphragm.Histopathological examination revealed granu-lomas with spherules in the lungs of every ani-mal in these two groups, and extrapulmonarydissemination in more than half of them. Alllung cultures were positive for C. immitis.

In five animals of the viable vaccine groups,histopathological lung sections revealed severalself-contained, focal lesions containing spherules.This minimal involvement, because of the char-acter of the lesions and the time intervals in-volved, was attributed to the vaccine injection

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FIG. 3. Healed vaccination site and decreased swelltng of the axhllary lymph node at 6 months postim-munization. The monkey shown in Fig. 1 to 3 was an extreme case. Tissue reaction to vaccination was muchless in those receiving the lower doses (10 to 1000 spores).

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TABLE 1. Serological response of monkeys to cutaneous and pulmonary coccidioidomycosis

Prechallenge After respiratory challenge

ImmunizatiSknotstCFntier Precipitin* Maximal MaximalImmunizationSkintest CF* titer titer CF titer precipitin titer

Unvaccinated (controls) - - - 128 64- - - 256 256- - - 256 256- - - 256 512- - - 256 1024

Viable vaccine dose (spores)10 + - 8 64 16

+ - 8 8 16+ - 4 16 16+ 32 64 64 64

100 + - 4 8 16+ 16 32 32 32+ - 4 8 8+ - 2 8 8

1000 + 256 512t 512 1024+ 16 32 32 32+ - 8 128 128+ - 4 - 4

Nonviable vaccine - - 16 324-i- - 64 16

i - - 64 64i - - 8 16

* Postimmunization, 6 months.t This animal was shown to have four subcutaneous head lesions on autopsy, probably explaining

the high titers.t Questionable results (erythema but no induration).

TABLE 2. Clinical response of monkeys to pulmonary coccidioidomycosis

Immunization Clinical symptoms X ray MortalityClinicalsymptoms X ray ~~~~~~~(dead/total)

Unvaccinated (controls) Loss of appetite and en- Widespread wispy infiltrations 2/5ergy, emaciation, gen- throughout all lobes of theeral debilitation, ac- lungs by the 15th day aftercelerated respiration, respiratory challenge. Latercoughing. consolidated areas.

Viable vaccine None Negative 0/12

Nonviable vaccine FSame as unvaccinated Less involvement than con- 3/4controls. trols. Delayed development

(30 vs. 15 days).

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FIG. 4. Top row: three nonimmunized control monkeys (15 days after respiratory exposure to approxi-mately 7000 arthrospores) showing pneumonic infiltration throughout the whole lung. Note consolidatedarea in animal at top left. Bottom row: from left, animals receiving 10, 100, and 1000 viable spore vaccina-tions (15 days after respiratory exposure to approximately 7000 arthrospores). Note lack of evidence of infec-tion.

rather than the respiratory challenge. This wasfurther indicated by the increase in the numberof animals showing this condition with increasesin the viable vaccine dose. Moreover, the onlypositive lung cultures in this group occurred inanimals receiving the highest vaccine dose.Of the "dissemination controls" (vaccinated

but not challenged by the respiratory route),80% exhibited this same minimal systemic dis-semination of the organism from the cutaneousinfection to the lung (Fig. 6) but with no physical

signs of illness. In three of those receiving vaccinedoses in the 105 to 108 range dissemination wasnoted in other organs (spleen, kidney, or liver).

In these same studies, other monkeys werevaccinated with viable arthrospores of strainsCash and M-11. These animals exhibited re-sistance to pulmonary challenge similar to thatshown by the monkeys receiving viable strainSilveira vaccine, indicating that the protectiveantigen of the viable vaccine was not strain-specific.

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TABLE 3. Histological response of monkeys to pulmonary coccidioidomycosis

HistopathologyImmunization Gross pathology, lung cLutnuge

Lung Other

Unvaccinated (controls) Bosselated appearance, scat- + NC*tered surface lesions and + Kidney, hilar lymph node +large palpable nodules + +throughout all lobes, large + Kidney +consolidated areas (caseous + +necrosis), extensive ad-hesions

Viable vaccine dose (spores)10 Few small circumscribed - _

focal lesions, 2 to 3 mm in -

diameter (generally as- - _ _cribed to mite infestations) -

100 +--

1000 Minor adhesions in 2 of the 12 + Four sc head lesions +monkeys in these groups _

+ _+. + _+

Nonviable vaccine Same as controls + Liver ++ Spleen, hilar lymph node NC+ Hilar lymph node ++ Hilar lymph node NC

* Not cultured.

DISCUSSION

It was evident from the data presented thatthe subcutaneous injection of viable C. immitisengendered an immunity in monkeys that en-abled them to resist subsequent pulmonary in-fections with the fungus. This immunity was notstrain-specific, at least with the three strainstested, including two highly virulent strains andone of very low virulence (M-11) for mice.A dose of ten viable organisms gave protection

equal to that of much higher doses, and accom-plished this with less severe primary lesions andwithout dissemination beyond the axillary lymphnode.The fact that the injection of 10 viable organ-

isms resulted in solid immunity, whereas theinjection of approximately 109 nonviable organ-isms failed to induce an immunity, indicatesthat protective antibodies are formed only in

response to multiplication of the fungus in thetissues (possibly in combination wvith some spe-cific tissue element). Although the injection of anonviable vaccine appeared to delay the progressof the disease somewhat, the end result of respira-tory challenge was the same as that noted in thenonvaccinated controls.As expected, there was a direct relationship

between skin hypersensitivity and resistance topulmonary infection. The interesting point, how-ever, was that injection of nonviable vaccinelimited the formation of complement-fixing andpolysaccharide-precipitating antibodies after re-spiratory exposure to the same extent as did theviable vaccine (Table 1), but it did not limit thespread of infection in these animals. This indicatesthat (i) the protective antibodies are distinct andseparate from those operative in the CF andPpn reactions, and (ii) that formalin treatment

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FIG. 5. Left: histological sections of three nonimmunized animals after respiratory exposure to approxi-mately 7000 arthrospores. Note the consolidated necrotic lesions in the lung sections. Right: lung sections ofthree animals vaccinated with 100 or 1000 viable arthrospores. These animals received the same respiratoryexposure as those shown on the left. The large cavitated lesion shown in the center section was shown (micro-scopically) to be a lung mite lesion.

of the arthrospore inactivates the antigen re-

sponsible for formation of protective antibodies,but has no effect on those stimulating the CFand Ppn antibodies. Thus, multiplication of theorganism in the tissues is necessary for the forma-tion of protective antibodies, but not necessary

for the organisms' action on CF and Ppn response.

A correlation can be seen (Table 3) of an in-crease in the number of animals showing positivehistopathological findings (spherule-containinggranulomas) in the lung with an increase in thenumber of viable organisms in the vaccine dose.Table 3 also indicates that the only positive lungcultures were found among the animals receivingthe higher viable-spore vaccine dose. Since all ofthe animals received approximately equal re-

spiratory challenge doses, it is our opinion that

these lesions resulted from the immunizing, ratherthan the challenge, dose. Thus, the histopatho-logical changes noted in the lungs of the vacci-nated, challenged monkeys and in the vaccinated,unchallenged dissemination controls are stronglyindicative that systemic dissemination, althoughsubelinical, can result from primary cutaneousinfection with C. immitis. Either the number oforganisms escaping from the axillary lymph nodewas so small, or the time element was such, thatsufficient immunity was developed before theorganism could exert its full virulence in thelung, consequently resulting in self-contained,subclinical infection. This may be the reasonthat systemic dissemination from primary cuta-neous coccidioidomycosis has never been recog-nized in human infections.

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FIG. 6. Coccidioides immitis spherules in tissues of dissemination control. Top (left to right): skin avaccination site and right axillary lymph node. Bottom (left to right): the lung and the spleen.

Transmission of the fungus to the lungs, viathe mouth, from the draining vaccination lesioncannot be ruled out, but this is extremely un-likely in light of results of experimental intra-venous infections in monkeys (Blundell et al.,1961), in which the character of the lung lesionsresulting from hematogenous spread of the funguswas quite different from that of lesions resultingfrom impingement, in the lung, of a fungus sporefrom an aerosol. The lung lesions noted in thepresent study were very similar to those reportedby Blundell as resulting from hematogenousspread of the fungus. However, there was nodoubt that the head lesions of one monkey (notedin Table 3), and lesions of the ramos of the man-dible and the eyelid of one of the disseminationcontrols, resulted from direct transmission fromthe arm lesion at the site of vaccination.

Further studies are in progress to evaluatethe safety of a ten-spore viable vaccine, to es-

tablish statistically the nondissemination fromthe subcutaneous dose of this magnitude, andto examine the tissue response to injection ofvarious strains of the organism. Preliminaryevidence (Converse et al., 1962b) has indicatedstrain differences, dose level differences, andinoculation site differences in the monkeys' re-sponse to experimental primary cutaneous in-fections with C. immitis.

ACKNOWLEDGMENTS

The authors wish to thank John G. Ray andLouis F. Maire, III, for performing the serologicaltests and Robert K. Castle and Eugene F. Bishopfor making the photographs.A portion of the information contained in this

manuscript was presented at the InternationalConference on Mycobacterial and Fungal Anti-gens, sponsored by the George Washington Uni-versity Medical School, the American Thoracic

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Society, and Chas. Pfizer & Co., Inc. Some of theinformation presented will be published in theProceedings of the Conference.

LITERATURE CITED

BLUNDELL, G. P., M. W. CASTLEBERRY, E. P.LOWE, AND J. L. CONVERSE. 1961. The pathol-ogy of Coccidioides immitis in the Macacamulatta. Am. J. Pathol. 39:613430.

CHIPMAN, E. D., AND H. J. TEMPLETON. 1930.Coccidioidal granuloma. A.M.A. Arch. Der-matol. Syphilol. 21:259-278.

CONVERSE, J. L., M. W. CASTLEBERRY, A. R.BESEMER, AND E. M. SNYDER. 1962a. Im-munization of mice against coccidioidomyco-sis. J. Bacteriol. 84:46-52.

CONVERSE, J. L., S. P. PAKES, E. M. SNYDER,AND M. W. CASTLEBERRY. 1962b. Experi-mental primary cutaneous coccidioidomycosisin the monkey. Trans. 7th Ann. VeteransAdministration-Armed Forces Coccidioido-mycosis Study Group, Los Angeles, Calif.

DAVIS, D. J. 1924. Coccidioidal granuloma withcertain serologic and experimental observa-tions. A.M.A. Arch. Dermatol. Syphilol.9:577-588.

FRIEDMAN, L., C. E. SMITH, W. G. ROESSLER,AND R. J. BERMAN. 1956. The virulence andinfectivity of twenty-seven strains of Coc-cidioides immitis. Am. J. Hyg. 64:198-210.

Guy, W. H., AND F. M. JACOBS. 1926. Granulomacoccidiodes. A.M.A. Arch. Dermatol. Syphi-lol. 14:596.

Guy, W. H., AND F. M. JACOBS. 1927. Granulomacoccidioides. A.M.A. Arch. Dermatol. Syphi-lol. 16:308-311.

MEIS, P. R. 1961. Discussion from the floor.Trans. 6th Ann. Meeting Veterans Adminis-

tration-Armed Forces CoccidioidomycosisCoop Study, p. 29.

OPHULS, W. 1905. Further observations on apathogenic mould formerly described as aprotozoon (Coccidioides immitis, Coccidioidespyogenes). J. Exptl. Med. 6:443-486.

PAPPAGIANIS, D., R. L. MILLER, C. E. SMITH,AND G. S. KOBAYASHI. 1960. Response ofmonkeys to respiratory challenge followingsubcutaneous inoculation with Coccidioidesimmitis. Am. Rev. Respirat. Diseases 82:244-250.

PAPPAGIANIS, D., C. E. SMITH, R. J. BERMAN,AND G. S. KOBAYASHI. 1959. Experimentalsubcutaneous coccidioidal infection in themouse. J. Invest. Dermatol. 32:589-598.

RIXFORD, E. 1894a. Case for diagnosis presentedbefore the San Francisco Medico-ChirurgicalSociety, 5 March 1894. Occident Med. Times8:326.

RIXFORD, E. 1894b. A case of protozoic dermatitis.Occident Med. Times 8:704-707.

ROESSLER, W. G., E. J. HERBST, W. G. MCCUL-LOUGH, R. C. MILLS, AND C. R. BREWER.1946. Studies with Coccidioides immitis. I.Submerged growth in liquid mediums. J.Infect. Diseases 79:12-22.

SMITH, C. E. 1957. The public health significanceof coccidioidomycosis. Proc. Symp. Coc-cidioidomycosis, U.S. Public Health Service.

WILSON, J. W., C. E. SMITH, AND 0. A. PLUNKETT.1953. Primary cutaneous coccidioidomycosis.The criteria for diagnosis and report of acase. Calif. Med. 79:233-239.

WINN, W. A. 1961. Discussion from the floor.Trans. 6th Ann. Meeting Veterans Admin-istration-Armed Forces CoccidioidomycosisCoop Study, p. 29.

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