Mycopathologia (2014) 178:189–195 DOI 10.1007/s11046-014-9798-5
Killed Saccharomyces cerevisiae Protects Against Lethal Challenge of Cryptococcus grubii Tanya Majumder • Min Liu • Vicky Chen Marife Martinez • Danielle Alvarado • Karl V. Clemons • David A. Stevens
•
Received: 15 April 2014 / Accepted: 31 July 2014 / Published online: 15 August 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Heat-killed Saccharomyces cerevisiae (HKY) vaccination protects mice against aspergillosis, coccidioidomycosis, mucormycosis, or candidiasis. We studied HKY protection against murine cryptococcosis. Once weekly subcutaneous HKY doses (S, 6 9 107; 2S, 1.2 9 108; 3S, 2.4 9 108) began 28 (93), 35 (94), or 42 (96) days prior to intravenous Cryptococcus grubii infection. Survival through 28 days, and CFU in the organs of survivors, were compared to saline-vaccinated controls. In the initial experiment, S, S94, or 2S reduced brain CFU; liver or spleen CFU was reduced by S94 or 2S. In a more lethal second experiment, 2S96, 2S, or 3S94 improved survival, and HKY regimens reduced CFU in the brain, liver, or spleen, with 2S96, 2S, or 3S94 most efficacious. Dose size appears more important than the number of doses: Regimens[S were superior,
T. Majumder M. Liu V. Chen M. Martinez D. Alvarado K. V. Clemons (&) D. A. Stevens California Institute for Medical Research, 2260 Clove Dr., San Jose, CA 95128, USA e-mail: [email protected] K. V. Clemons D. A. Stevens Division of Infectious Diseases, Department of Medicine, Santa Clara Valley Medical Center, San Jose, CA 95128, USA K. V. Clemons D. A. Stevens Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University, Stanford, CA 94305, USA
and 2S and 2S96 were equivalent. 2S and 3S were equivalent, suggesting doses [2S do not provide additional protection. HKY protects against Cryptococcus, supporting components of HKY as a basis for the development of a panfungal vaccine. Keywords Cryptococcus grubii Heat-killed yeast Vaccine Murine model Systemic infection Meningitis
Introduction Cryptococcosis is a fungal infection that often causes mortality in immunocompromised patients, largely owing to meningeal disease [1]. Although the frequency of this disease has declined in the USA and in Europe with the advent of antiretroviral therapies, it remains a frequent cause of mortality in AIDS patients in developing countries [1]. Antifungal therapy is effective, but failures do occur, therapy is prolonged, and resistance to most antifungals occurs [1]. Thus, prevention of cryptococcosis is a desirable alternative. Currently, there is no vaccine commercially available that prevents cryptococcosis. Numerous investigators have worked toward the development of a vaccine. These studies have included killed whole cell preparations, attenuated cryptococci, capsule components, and specific proteins, as well as capsule material conjugated with a protein carrier such
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as tetanus toxoid [2–11], against challenge by various routes. To date, these vaccine studies have either produced nonprotective antibodies against C. neoformans or required an adjuvant for efficacy [3]. Heat-killed Saccharomyces cerevisiae (HKY) has been shown previously to provide protection against other fungal infections such as experimental aspergillosis, coccidioidomycosis, candidiasis, and zygomycosis [12–15]. Our aim in the current study was to examine the potential efficacy of S. cerevisiae as a vaccine against C. grubii and further delineate the spectrum of the protective efficacy induced by heatkilled yeast against fungal infections. These studies used a murine model of systemic cryptococcosis, including meningeal infection. The protective capacity of HKY was assessed by survival and fungal burden in the brain, spleen, and liver.
Materials and Methods Saccharomyces Vaccines Saccharomyces cerevisiae strain 96–108 was revived from long-term storage at -80 °C and cultured on yeast-extract peptone dextrose (YPD) agar for 72 h at 25 °C. It was then cultured in YPD broth for 24 h at 37 °C on a gyratory shaker at 170 rpm. Cultured cells were washed three times by centrifugation in phosphate-buffered saline (PBS). The pelleted yeast were suspended in PBS and placed in a 70 °C water bath for 3 h to prepare the heat-killed yeast (HKY) [12]. Cells were counted using a hemacytometer and diluted with PBS to 4 9 108 cells/mL. The nonviability of the HKY was confirmed by plating on Sabouraud Dextrose Agar (SDA) with chloramphenicol. No growth was found after 3 days of incubation. The HKY was stored at 4 °C for further use.
Mycopathologia (2014) 178:189–195 Table 1 Vaccination regimens and doses for low inoculum challenge experiment Groupa
Vaccine dose (HKY/mouse)
Days of vaccination prior to infection
PBS
PBS
-28, -21, -14
HKY93
6 9 107
-28, -21, -14
HKY94
6 9 107
29 HKY93
-35, -28, -21, -14 7
1.2 9 10
-28, -21, -14
a
The group names (column 1) indicate the regimens given (columns 2 and 3). The prefix (e.g., 29) in column 1 indicates the double dose of vaccine (1.2 9 107 cells, as in column 2), and the suffix (93, 94) indicates the number of doses (days given are shown in column 3) Table 2 Vaccination regimens and doses for high inoculum challenge experiment Groupa
Vaccine dose (HKY/mouse)
Days of vaccination prior to infection
PBS
PBS
-28, -21, -14 7
-28, -21, -14
HKY93
6 9 10
HKY94
6 9 107
-35, -28, -21, -14
29 HKY93
1.2 9 107
-28, -21, -14
29 HKY96
1.2 9 107
-48, -42, -35, -28, -21, -14
39 HKY94
1.8 9 107
-35, -28, -21, -14
a
Group names as explained in Table 1
Vaccinations All mice were vaccinated by subcutaneous injection. Mice received 6 9 107 HKY cells/mouse given in 150 lL (dose split between two sites) or PBS. Mice receiving twice the standard dose were given 300 lL of HKY (dose split between two sites), and mice receiving thrice the standard dose were given 450 lL of HKY (dose split between two sites). Prior to infection, vaccinations were given once a week on the vaccination schedules shown in Tables 1 and 2.
Mice Infection Five-week-old female CD-1 mice were used in all experiments. Mice were housed 5 per cage under standard conditions. Mice were provided irradiated food and acidified water ad libitum. All animal experimentation was done under a protocol approved by the Institutional Animal Care and Use Committee at the California Institute for Medical Research.
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Cryptococcus grubii 9-759 [16, 17] was previously animal-passed and stored frozen at -80 °C in 40 % glycerol. This organism was revived by plating onto SDA with chloramphenicol and grown for 5 days at 35 °C. Isolated colonies were subcultured in defined medium (SAAMF broth) [18] for 72 h at 35 °C on a gyratory shaker at 140 rpm. A second subculture was
Mycopathologia (2014) 178:189–195
done in SAAMF to prepare the infectious inoculum. The cells were washed three times in saline by centrifugation (1,0009g for 10 min) and counted using a hemacytometer to adjust the number to 107 cells/mL [17]. Mice were infected via intravenous injection of 0.25 mL (2.5 9 106 cells/mouse). The viable number, as determined by verification plating on SDA with chloramphenicol, was found to be 9.125 9 105 cells/mouse (first experiment) and 1.7 9 106 cells/mouse (second experiment). Fungal Burdens Survival was tallied for 28 days. Surviving mice were euthanatized with CO2 anoxia, and the brain, spleen, and liver were removed aseptically and homogenized as previously described [17]. Dilutions were plated in duplicate on SDA with chloramphenicol to determine the CFU remaining in each organ. Statistical Analysis Survival was analyzed using the log-rank test. Fungal burdens were analyzed using the Mann–Whitney U test using GraphPad Prism for Windows. A log10 value of 8.50 was chosen as an arbitrarily high value that assigns dead mice a worse outcome than survival with any infectious burden [19, 20]. Prior experiments [16, 17] also determined that mice dying after challenge have higher infectious burdens than survivors.
Results Low Inoculum Challenge The aim of the initial study was that of assessing whether vaccination with HKY given in a regimen that has been shown to protect against experimental aspergillosis, coccidioidomycosis, and candidosis would be protective against cryptococcosis. Three HKY regimens were tested and corresponded to regimens previously shown effective against the other fungal infections mentioned above. Survival The cryptococcal infection established in this study was of low lethality, with only 5 of the 40 mice
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succumbing to infection after 28 days. Thirty percent of the PBS controls died, whereas only 10 % or none of the HKY-vaccinated mice died. Statistical comparison indicated that there were no significant differences between the different regimens, though the most intense vaccine regimen (29 HKY 93) showed a trend toward being superior to PBS (P = 0.07). Fungal Burdens Fungal burdens of C. grubii remaining in the organs were determined and the results presented in Fig. 1. These data showed that PBS controls had a significantly higher burden of C grubii than did the groups given HKY94 and 29 HKY 93 in all organs (P \ 0.002), and the HKY93 group in the brain (P = 0.03). The HKY94 group also had significantly lower burdens in all organs than did the HKY93 group (P \ 0.03). Mice given 29 HKY93 had a significantly lower burden in the liver than did those given HKYx3 (P = 0.007). Two mice from the HKY 94 group completely cleared the infection in the liver, and two mice from the HKY x4 group and two mice from the 29 HKY 93 completely cleared the infection in the spleen. High Inoculum Challenge The results of the initial study indicated that HKY vaccination appeared to be protective against systemic cryptococcosis, but primarily with more intense regimens. To further examine this, we chose to replicate the regimens used in the first study and also include vaccine regimens using higher doses and more frequent dosing. Survival A higher inoculum of C. grubii was used to establish infection in this study and resulted in a somewhat more lethal model than that of the initial study (Fig. 2). Forty percent of the PBS-vaccinated mice succumbed to infection by day 28, compared with 10 % or none given one of the HKY regimens of vaccination. The survival of mice given PBS was significantly lower than mice given the HKY vaccine regimens of 29 HKY 93, 29 HKY 96, or 39 HKY94 (P \ 0.04). No other comparisons were significantly different.
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Mycopathologia (2014) 178:189–195
Fig. 2 Survival curve of mice vaccinated with HKY according to various regimens and infected with C. grubii as indicated in the figure. The 29 and 39 regimens resulted in significantly (P \ 0.04) improved survival compared to controls
Fungal Burdens All mice given one of the HKY vaccine regimens had significantly lower fungal burden than did PBSvaccinated mice in all organs (P \ 0.02), except in the liver for the group given HKY93 (Fig. 3). Mice given HKYx3 had significantly higher burdens of C. grubii in all organs than did mice given 29 HKY93, 29 HKY96, or 39 HKY94 (P \ 0.04). Mice vaccinated with HKY94 had significantly higher burdens of C. grubii than did those vaccinated with 2X HKYx6 in all organs, 3X HKYx4 in the spleen and liver, and 29 HKY93 in the liver (P \ 0.02). Two mice from the 29 HKY 93 group cleared infection in the spleen and the liver. One mouse in the 29 HKY 96 group cleared infection in the spleen, and two mice in the 29 HKY 96 group cleared infection in the brain. Two mice from the 3X HKY x4 group cleared infection in the spleen, and one mouse from this group cleared infection in the liver.
Discussion
Fig. 1 Scattergram and median (horizontal bar) of CFU recovered from the brain, liver, and spleen of mice vaccinated with the indicated regimen that survived through 27 days of infection with C. grubii. Comparisons of vaccine results versus the PBS control, and between vaccine regimens, are given in the text
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In our current study, we assessed whether vaccination with HKY could provide significant protection against systemic cryptococcal infection similar to what we have demonstrated for aspergillosis, coccidioidomycosis, candidiasis, and zygomycosis. Our results clearly demonstrate that HKY provided significant protection against lethal infection, as well as a significant reduction of the fungal burden in the organs, including the brain. These results extend the
Mycopathologia (2014) 178:189–195
Fig. 3 Scattergram and median (horizontal bar) of CFU recovered from the brain, liver, and spleen of mice vaccinated with the indicated regimen that survived through 27 days of infection with C. grubii. All vaccine regimens resulted in lower CFU in all organs (except liver, HKY93 regimen) than control. The 29 and 39 regimens resulted in lower burdens in all organs than HKY93, and in several organs, lower burdens than HKY94 (see text)
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range of the cross-protective effects of using HKY as a vaccine to now include the basidiomycetous yeast, Cryptococcus, in addition to the ascomycetous organisms, and Rhizopus, studied previously. Induction of protection by HKY against cryptococcal disease improved with doses 2 or 3 times higher than those we have found effective against aspergillosis and coccidioidomycosis. In those studies, we found that a standard dose of 6 9 107 HKY given 3 times provided excellent protection [12–14], whereas against cryptococcal disease doses of HKY at 1.2 9 108 given 3 times were superior to the standard 6 9 107 HKY dose. This improved efficacy with increased dose did, however, plateau, since comparison of the 29 HKY with the 39 HKY doses showed no improvement with the higher dose. Furthermore, it appears that the number of HKY present in the dosage is more important than the number of doses administered, as there was no significant improvement using 4 or 6 doses compared to using 3 doses given 1 week apart. In the development of a vaccine, the ideal candidate would result in the complete clearance of the infectious fungal challenge from the tissues. However, the severity of the challenges used in experimental studies must be taken into account and is likely more severe than that seen in a clinical setting. Although the infection was rapidly lethal to a sizable proportion of the unvaccinated controls, our studies with HKY have shown prolongation of survival and reduction of fungal burden from the tissues, with some cured. Which component or components of HKY are responsible for protection against C. grubii infection are unknown at this time. In other studies, we have demonstrated that HKY vaccination induced both an antibody response and a specific cell-mediated immune response with the induction of Th1 cells and cytokine response to re-stimulation with HKY [21]. Th1-related immunity appears more important than Th17-related immunity in protecting against cryptococcosis [10]. It has been suggested that stimulating innate immunity by immunization with another microbe or antigen can prime the immune response to increased activity against Cryptococcus [22]. It is possible that protection is related to antibodies produced, cross-reactive against mannans or b-glucans in the cell wall of the cryptococci. Mannoproteins also appear responsible for cellular immunity against cryptococci [10], and mannoprotein glycosylation
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Mycopathologia (2014) 178:189–195
appears important for the T cell response [23]. Antibodies against b-glucan inhibit cryptococcal growth in vitro and are effective therapy against cryptococcosis in vivo [24]. However, antibodies do not appear responsible for protection after HKY immunization, at least against aspergillosis [25]. We believe it more desirable to achieve maximum protection with purified components of the HKY vaccine. We have demonstrated protection against aspergillosis and coccidioidomycosis with purified preparations of mannan or glucan, or conjugates of them to bovine serum albumin, as vaccines [26–28]. We have demonstrated some protein homologies between Cryptococcus, Aspergillus, Coccidioides, and Saccharomyces, as well as other fungi [29]. Protein components of HKY homologous with proteins of C. grubii, and cross-reacting immunity against these could explain protection. Thus, several possibilities exist that could contribute to the induction of host resistance by HKY that is protective against an unrelated organism like C. grubii. We noted that the durability of protection arising from HKY vaccination is at least 28 days in two previous studies [12, 13], which is suggestive that protection may be due to a memory response. However, additional studies on this aspect are necessary. Overall, our results provide further evidence that a panfungal vaccine based on components of HKY may be attainable. Studies in human cancer patients have shown the safety of administering heat-killed Saccharomyces cerevisiae and yeast-based vaccines [30–32]. This, coupled with our results, is suggestive that a panfungal yeast-based vaccine is possible. To that end, we have suggested potential recipient groups previously, and recognized the difficulty of immunizing and inducing protection in immunosuppressed individuals [28, 33]. Conflict of interest
None.
References 1. Heitman J, Kozel TR, Kwon-Chung KJ, Perfect JR, Casadevall A. Cryptococcus: from human pathogen to model yeast. Washington: ASM Press; 2011. 2. Casadevall A, Mukherjee J, Devi SJ, Schneerson R, Robbins JB, Scharff MD. Antibodies elicited by a Cryptococcus neoformans-tetanus toxoid conjugate vaccine have the same specificity as those elicited in infection. J Infect Dis. 1992;165(6):1086–93.
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3. Casadevall A, Pirofski LA. Feasibility and prospects for a vaccine to prevent cryptococcosis. Med Mycol. 2005;43(8):667–80. 4. Anderson DM, Dykstra MA. Resistance to challenge and macrophage activity in mice previously vaccinated with formalin-killed Cryptococcus neoformans. Mycopathologia. 1984;86(3):169–77. 5. Baronetti JL, Chiapello LS, Aoki MP, Gea S, Masih DT. Heat killed cells of Cryptococcus neoformans var. grubii induces protective immunity in rats: immunological and histopathological parameters. Med Mycol. 2006;44(6):493–504. doi:10.1080/13693780600750022. 6. Chow SK, Casadevall A. Evaluation of Cryptococcus neoformans galactoxylomannan-protein conjugate as vaccine candidate against murine cryptococcosis. Vaccine. 2011;29(10):1891–8. doi:10.1016/j.vaccine.2010.12.134. 7. Datta K, Lees A, Pirofski LA. Therapeutic efficacy of a conjugate vaccine containing a peptide mimotope of cryptococcal capsular polysaccharide glucuronoxylomannan. Clin Vaccine Immunol. 2008;15(8):1176–87. doi:10.1128/ CVI.00130-08. 8. Fleuridor R, Lees A, Pirofski L. A cryptococcal capsular polysaccharide mimotope prolongs the survival of mice with Cryptococcus neoformans infection. J Immunol. 2001;166(2):1087–96. 9. Wormley FL Jr, Cox GM, Perfect JR. Evaluation of host immune responses to pulmonary cryptococcosis using a temperature-sensitive C. neoformans calcineurin A mutant strain. Microb Path. 2005;38(2–3):113–23. doi:10.1016/j. micpath.2004.12.007. 10. Hole CR, Wormley FL Jr. Vaccine and immunotherapeutic approaches for the prevention of cryptococcosis: lessons learned from animal models. Frontiers Microbiol. 2012;3:291. doi:10.3389/fmicb.2012.00291. 11. Mansour MK, Yauch LE, Rottman JB, Levitz SM. Protective efficacy of antigenic fractions in mouse models of cryptococcosis. Infect Immun. 2004;72(3):1746–54. 12. Capilla J, Clemons KV, Liu M, Levine HB, Stevens DA. Saccharomyces cerevisiae as a vaccine against coccidioidomycosis. Vaccine. 2009;27:3662–8. 13. Liu M, Capilla J, Johansen ME, Alvarado D, Martinez M, Chen V, et al. Saccharomyces as a vaccine against systemic aspergillosis: ‘the friend of man’ a friend again? J Med Microbiol. 2011;60(Pt 10):1423–32. doi:10.1099/jmm.0. 033290-0. 14. Liu M, Clemons KV, Johansen ME, Martinez M, Chen V, Stevens DA. Saccharomyces as a vaccine against systemic candidiasis. Immunol Invest. 2012;41(8):847–55. doi:10. 3109/08820139.2012.692418. 15. Luo G, Gebremariam T, Clemons KV, Stevens DA, Ibrahim AS. Heat killed yeast protects diabetic ketoacidotic/steroidtreated mice from pulmonary mucormycosis. Vaccine. 2014;32(29):3573–6. 16. Capilla J, Maffei CM, Clemons KV, Sobel RA, Stevens DA. Experimental systemic infection with Cryptococcus neoformans var. grubii and Cryptococcus gattii in normal and immunodeficient mice. Med Mycol. 2006;44(7):601–10. 17. Hostetler JS, Clemons KV, Hanson LH, Stevens DA. Efficacy and safety of amphotericin B colloidal dispersion compared with those of amphotericin B deoxycholate suspension for treatment of disseminated murine
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cryptococcosis. Antimicrob Agents Chemother. 1992;36(12):2656–60. Hoeprich P, Finn P. Obfuscation of the activity of antifungal antimicrobics by culture media. J Infect Dis. 1972;126(4):353–61. Lachin JM. Worst-rank score analysis with informatively missing observations in clinical trials. Control Clin Trials. 1999;20:408–22. Shih W. Problems in dealing with missing data and informative censoring in clinical trials. Curr Control Trials Cardiovasc Med. 2002;3(1):4. Liu M, Clemons KV, Bigos M, Medovarska I, Brummer E, Stevens DA. Immune responses induced by heat killed Saccharomyces cerevisiae: a vaccine against fungal infection. Vaccine. 2011;29(9):1745–53. doi:10.1016/j.vaccine. 2010.12.119. Casadevall A, Dadachova E, Pirofski L-A. Vaccines and antibody therapies from Cryptococcus neoformans to melanoma. In: Heitman J, Kozel TR, Kwon-Chung KJ, Perfect JR, Casadevall A, editors. Cryptococcus: from human pathogen to model yeast. Washington: American Society for Microbiology; 2011. p. 537–46. Mansour MK, Schlesinger LS, Levitz SM. Optimal T cell responses to Cryptococcus neoformans mannoprotein are dependent on recognition of conjugated carbohydrates by mannose receptors. J Immunol. 2002;168(6):2872–9. Rachini A, Pietrella D, Lupo P, Torosantucci A, Chiani P, Bromuro C, et al. An anti-b-glucan monoclonal antibody inhibits growth and capsule formation of Cryptococcus neoformans in vitro and exerts therapeutic, anticryptococcal activity in vivo. Infect Immun. 2007;75(11):5085–94. doi:10.1128/IAI.00278-07. Clemons KV, Martinez M, Chen V, Liu M, Yoon HJ, Stevens DA. Protection against experimental aspergillosis by heat-killed yeast is not antibody dependent. Med Mycol. 2014;52(4):422–6. doi:10.1093/mmy/myt015.
195 26. Clemons KV, Danielson M, Michel KS, Liu M, Martinez M, Chen V, et al. Whole glucan particles (WGP) as a vaccine against murine aspergillosis. 53rd Intersci. Conf. Antimicrob. Agents Chemother.; Denver: Amer. Soc. Microbiol.; 2013. p. Abst. M790. 27. Liu M, Machova E, Nescakova Z, Medovarska I, Clemons KV, Martinez M, et al. Vaccination with mannan protects mice against systemic aspergillosis. Med Mycol. 2012;50(8):818–28. doi:10.3109/13693786.2012.683539. 28. Stevens DA, Clemons KV, Liu M. Developing a vaccine against aspergillosis. Med Mycol. 2010;49(Suppl 1):S170–6. 29. Champer J, Diaz-Arevalo D, Champer M, Hong TB, Wong M, Shannahoff M, et al. Protein targets for broad-spectrum mycosis vaccines: quantitative proteomic analysis of Aspergillus and Coccidioides and comparisons with other fungal pathogens. Ann N Y Acad Sci. 2012;1273(1):44–51. doi:10.1111/j.1749-6632.2012.06761.x. 30. DiMiceli L, Pool V, Kelso JM, Shadomy SV, Iskander J. Vaccination of yeast sensitive individuals: review of safety data in the US vaccine adverse event reporting system (VAERS). Vaccine. 2006;24(6):703–7. doi:10.1016/j. vaccine.2005.07.069. 31. Franzusoff A, Duke RC, King TH, Lu Y, Rodell TC. Yeasts encoding tumour antigens in cancer immunotherapy. Expert Opin Biol Ther. 2005;5(4):565–75. 32. Munson S, Parker J, King TH, Lu Y, Kelley V, Guo Z, et al. Coupling innate and adaptive immunity with yeast-based cancer immunotherapy. In: Orentas RJ, Hodge JW, Johnson BD, editors. Cancer vaccines and tumor immunity. Hoboken: Wiley; 2008. p. 131–49. 33. Stevens DA. Vaccinate against aspergillosis! A call to arms of the immune system. Clin Infect Dis. 2004;38(8):1131–6.
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Killed Saccharomyces cerevisiae Protects Against Lethal Challenge of Cryptococcus grubii Tanya Majumder • Min Liu • Vicky Chen Marife Martinez • Danielle Alvarado • Karl V. Clemons • David A. Stevens
•
Received: 15 April 2014 / Accepted: 31 July 2014 / Published online: 15 August 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Heat-killed Saccharomyces cerevisiae (HKY) vaccination protects mice against aspergillosis, coccidioidomycosis, mucormycosis, or candidiasis. We studied HKY protection against murine cryptococcosis. Once weekly subcutaneous HKY doses (S, 6 9 107; 2S, 1.2 9 108; 3S, 2.4 9 108) began 28 (93), 35 (94), or 42 (96) days prior to intravenous Cryptococcus grubii infection. Survival through 28 days, and CFU in the organs of survivors, were compared to saline-vaccinated controls. In the initial experiment, S, S94, or 2S reduced brain CFU; liver or spleen CFU was reduced by S94 or 2S. In a more lethal second experiment, 2S96, 2S, or 3S94 improved survival, and HKY regimens reduced CFU in the brain, liver, or spleen, with 2S96, 2S, or 3S94 most efficacious. Dose size appears more important than the number of doses: Regimens[S were superior,
T. Majumder M. Liu V. Chen M. Martinez D. Alvarado K. V. Clemons (&) D. A. Stevens California Institute for Medical Research, 2260 Clove Dr., San Jose, CA 95128, USA e-mail: [email protected] K. V. Clemons D. A. Stevens Division of Infectious Diseases, Department of Medicine, Santa Clara Valley Medical Center, San Jose, CA 95128, USA K. V. Clemons D. A. Stevens Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University, Stanford, CA 94305, USA
and 2S and 2S96 were equivalent. 2S and 3S were equivalent, suggesting doses [2S do not provide additional protection. HKY protects against Cryptococcus, supporting components of HKY as a basis for the development of a panfungal vaccine. Keywords Cryptococcus grubii Heat-killed yeast Vaccine Murine model Systemic infection Meningitis
Introduction Cryptococcosis is a fungal infection that often causes mortality in immunocompromised patients, largely owing to meningeal disease [1]. Although the frequency of this disease has declined in the USA and in Europe with the advent of antiretroviral therapies, it remains a frequent cause of mortality in AIDS patients in developing countries [1]. Antifungal therapy is effective, but failures do occur, therapy is prolonged, and resistance to most antifungals occurs [1]. Thus, prevention of cryptococcosis is a desirable alternative. Currently, there is no vaccine commercially available that prevents cryptococcosis. Numerous investigators have worked toward the development of a vaccine. These studies have included killed whole cell preparations, attenuated cryptococci, capsule components, and specific proteins, as well as capsule material conjugated with a protein carrier such
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as tetanus toxoid [2–11], against challenge by various routes. To date, these vaccine studies have either produced nonprotective antibodies against C. neoformans or required an adjuvant for efficacy [3]. Heat-killed Saccharomyces cerevisiae (HKY) has been shown previously to provide protection against other fungal infections such as experimental aspergillosis, coccidioidomycosis, candidiasis, and zygomycosis [12–15]. Our aim in the current study was to examine the potential efficacy of S. cerevisiae as a vaccine against C. grubii and further delineate the spectrum of the protective efficacy induced by heatkilled yeast against fungal infections. These studies used a murine model of systemic cryptococcosis, including meningeal infection. The protective capacity of HKY was assessed by survival and fungal burden in the brain, spleen, and liver.
Materials and Methods Saccharomyces Vaccines Saccharomyces cerevisiae strain 96–108 was revived from long-term storage at -80 °C and cultured on yeast-extract peptone dextrose (YPD) agar for 72 h at 25 °C. It was then cultured in YPD broth for 24 h at 37 °C on a gyratory shaker at 170 rpm. Cultured cells were washed three times by centrifugation in phosphate-buffered saline (PBS). The pelleted yeast were suspended in PBS and placed in a 70 °C water bath for 3 h to prepare the heat-killed yeast (HKY) [12]. Cells were counted using a hemacytometer and diluted with PBS to 4 9 108 cells/mL. The nonviability of the HKY was confirmed by plating on Sabouraud Dextrose Agar (SDA) with chloramphenicol. No growth was found after 3 days of incubation. The HKY was stored at 4 °C for further use.
Mycopathologia (2014) 178:189–195 Table 1 Vaccination regimens and doses for low inoculum challenge experiment Groupa
Vaccine dose (HKY/mouse)
Days of vaccination prior to infection
PBS
PBS
-28, -21, -14
HKY93
6 9 107
-28, -21, -14
HKY94
6 9 107
29 HKY93
-35, -28, -21, -14 7
1.2 9 10
-28, -21, -14
a
The group names (column 1) indicate the regimens given (columns 2 and 3). The prefix (e.g., 29) in column 1 indicates the double dose of vaccine (1.2 9 107 cells, as in column 2), and the suffix (93, 94) indicates the number of doses (days given are shown in column 3) Table 2 Vaccination regimens and doses for high inoculum challenge experiment Groupa
Vaccine dose (HKY/mouse)
Days of vaccination prior to infection
PBS
PBS
-28, -21, -14 7
-28, -21, -14
HKY93
6 9 10
HKY94
6 9 107
-35, -28, -21, -14
29 HKY93
1.2 9 107
-28, -21, -14
29 HKY96
1.2 9 107
-48, -42, -35, -28, -21, -14
39 HKY94
1.8 9 107
-35, -28, -21, -14
a
Group names as explained in Table 1
Vaccinations All mice were vaccinated by subcutaneous injection. Mice received 6 9 107 HKY cells/mouse given in 150 lL (dose split between two sites) or PBS. Mice receiving twice the standard dose were given 300 lL of HKY (dose split between two sites), and mice receiving thrice the standard dose were given 450 lL of HKY (dose split between two sites). Prior to infection, vaccinations were given once a week on the vaccination schedules shown in Tables 1 and 2.
Mice Infection Five-week-old female CD-1 mice were used in all experiments. Mice were housed 5 per cage under standard conditions. Mice were provided irradiated food and acidified water ad libitum. All animal experimentation was done under a protocol approved by the Institutional Animal Care and Use Committee at the California Institute for Medical Research.
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Cryptococcus grubii 9-759 [16, 17] was previously animal-passed and stored frozen at -80 °C in 40 % glycerol. This organism was revived by plating onto SDA with chloramphenicol and grown for 5 days at 35 °C. Isolated colonies were subcultured in defined medium (SAAMF broth) [18] for 72 h at 35 °C on a gyratory shaker at 140 rpm. A second subculture was
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done in SAAMF to prepare the infectious inoculum. The cells were washed three times in saline by centrifugation (1,0009g for 10 min) and counted using a hemacytometer to adjust the number to 107 cells/mL [17]. Mice were infected via intravenous injection of 0.25 mL (2.5 9 106 cells/mouse). The viable number, as determined by verification plating on SDA with chloramphenicol, was found to be 9.125 9 105 cells/mouse (first experiment) and 1.7 9 106 cells/mouse (second experiment). Fungal Burdens Survival was tallied for 28 days. Surviving mice were euthanatized with CO2 anoxia, and the brain, spleen, and liver were removed aseptically and homogenized as previously described [17]. Dilutions were plated in duplicate on SDA with chloramphenicol to determine the CFU remaining in each organ. Statistical Analysis Survival was analyzed using the log-rank test. Fungal burdens were analyzed using the Mann–Whitney U test using GraphPad Prism for Windows. A log10 value of 8.50 was chosen as an arbitrarily high value that assigns dead mice a worse outcome than survival with any infectious burden [19, 20]. Prior experiments [16, 17] also determined that mice dying after challenge have higher infectious burdens than survivors.
Results Low Inoculum Challenge The aim of the initial study was that of assessing whether vaccination with HKY given in a regimen that has been shown to protect against experimental aspergillosis, coccidioidomycosis, and candidosis would be protective against cryptococcosis. Three HKY regimens were tested and corresponded to regimens previously shown effective against the other fungal infections mentioned above. Survival The cryptococcal infection established in this study was of low lethality, with only 5 of the 40 mice
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succumbing to infection after 28 days. Thirty percent of the PBS controls died, whereas only 10 % or none of the HKY-vaccinated mice died. Statistical comparison indicated that there were no significant differences between the different regimens, though the most intense vaccine regimen (29 HKY 93) showed a trend toward being superior to PBS (P = 0.07). Fungal Burdens Fungal burdens of C. grubii remaining in the organs were determined and the results presented in Fig. 1. These data showed that PBS controls had a significantly higher burden of C grubii than did the groups given HKY94 and 29 HKY 93 in all organs (P \ 0.002), and the HKY93 group in the brain (P = 0.03). The HKY94 group also had significantly lower burdens in all organs than did the HKY93 group (P \ 0.03). Mice given 29 HKY93 had a significantly lower burden in the liver than did those given HKYx3 (P = 0.007). Two mice from the HKY 94 group completely cleared the infection in the liver, and two mice from the HKY x4 group and two mice from the 29 HKY 93 completely cleared the infection in the spleen. High Inoculum Challenge The results of the initial study indicated that HKY vaccination appeared to be protective against systemic cryptococcosis, but primarily with more intense regimens. To further examine this, we chose to replicate the regimens used in the first study and also include vaccine regimens using higher doses and more frequent dosing. Survival A higher inoculum of C. grubii was used to establish infection in this study and resulted in a somewhat more lethal model than that of the initial study (Fig. 2). Forty percent of the PBS-vaccinated mice succumbed to infection by day 28, compared with 10 % or none given one of the HKY regimens of vaccination. The survival of mice given PBS was significantly lower than mice given the HKY vaccine regimens of 29 HKY 93, 29 HKY 96, or 39 HKY94 (P \ 0.04). No other comparisons were significantly different.
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Fig. 2 Survival curve of mice vaccinated with HKY according to various regimens and infected with C. grubii as indicated in the figure. The 29 and 39 regimens resulted in significantly (P \ 0.04) improved survival compared to controls
Fungal Burdens All mice given one of the HKY vaccine regimens had significantly lower fungal burden than did PBSvaccinated mice in all organs (P \ 0.02), except in the liver for the group given HKY93 (Fig. 3). Mice given HKYx3 had significantly higher burdens of C. grubii in all organs than did mice given 29 HKY93, 29 HKY96, or 39 HKY94 (P \ 0.04). Mice vaccinated with HKY94 had significantly higher burdens of C. grubii than did those vaccinated with 2X HKYx6 in all organs, 3X HKYx4 in the spleen and liver, and 29 HKY93 in the liver (P \ 0.02). Two mice from the 29 HKY 93 group cleared infection in the spleen and the liver. One mouse in the 29 HKY 96 group cleared infection in the spleen, and two mice in the 29 HKY 96 group cleared infection in the brain. Two mice from the 3X HKY x4 group cleared infection in the spleen, and one mouse from this group cleared infection in the liver.
Discussion
Fig. 1 Scattergram and median (horizontal bar) of CFU recovered from the brain, liver, and spleen of mice vaccinated with the indicated regimen that survived through 27 days of infection with C. grubii. Comparisons of vaccine results versus the PBS control, and between vaccine regimens, are given in the text
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In our current study, we assessed whether vaccination with HKY could provide significant protection against systemic cryptococcal infection similar to what we have demonstrated for aspergillosis, coccidioidomycosis, candidiasis, and zygomycosis. Our results clearly demonstrate that HKY provided significant protection against lethal infection, as well as a significant reduction of the fungal burden in the organs, including the brain. These results extend the
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Fig. 3 Scattergram and median (horizontal bar) of CFU recovered from the brain, liver, and spleen of mice vaccinated with the indicated regimen that survived through 27 days of infection with C. grubii. All vaccine regimens resulted in lower CFU in all organs (except liver, HKY93 regimen) than control. The 29 and 39 regimens resulted in lower burdens in all organs than HKY93, and in several organs, lower burdens than HKY94 (see text)
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range of the cross-protective effects of using HKY as a vaccine to now include the basidiomycetous yeast, Cryptococcus, in addition to the ascomycetous organisms, and Rhizopus, studied previously. Induction of protection by HKY against cryptococcal disease improved with doses 2 or 3 times higher than those we have found effective against aspergillosis and coccidioidomycosis. In those studies, we found that a standard dose of 6 9 107 HKY given 3 times provided excellent protection [12–14], whereas against cryptococcal disease doses of HKY at 1.2 9 108 given 3 times were superior to the standard 6 9 107 HKY dose. This improved efficacy with increased dose did, however, plateau, since comparison of the 29 HKY with the 39 HKY doses showed no improvement with the higher dose. Furthermore, it appears that the number of HKY present in the dosage is more important than the number of doses administered, as there was no significant improvement using 4 or 6 doses compared to using 3 doses given 1 week apart. In the development of a vaccine, the ideal candidate would result in the complete clearance of the infectious fungal challenge from the tissues. However, the severity of the challenges used in experimental studies must be taken into account and is likely more severe than that seen in a clinical setting. Although the infection was rapidly lethal to a sizable proportion of the unvaccinated controls, our studies with HKY have shown prolongation of survival and reduction of fungal burden from the tissues, with some cured. Which component or components of HKY are responsible for protection against C. grubii infection are unknown at this time. In other studies, we have demonstrated that HKY vaccination induced both an antibody response and a specific cell-mediated immune response with the induction of Th1 cells and cytokine response to re-stimulation with HKY [21]. Th1-related immunity appears more important than Th17-related immunity in protecting against cryptococcosis [10]. It has been suggested that stimulating innate immunity by immunization with another microbe or antigen can prime the immune response to increased activity against Cryptococcus [22]. It is possible that protection is related to antibodies produced, cross-reactive against mannans or b-glucans in the cell wall of the cryptococci. Mannoproteins also appear responsible for cellular immunity against cryptococci [10], and mannoprotein glycosylation
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appears important for the T cell response [23]. Antibodies against b-glucan inhibit cryptococcal growth in vitro and are effective therapy against cryptococcosis in vivo [24]. However, antibodies do not appear responsible for protection after HKY immunization, at least against aspergillosis [25]. We believe it more desirable to achieve maximum protection with purified components of the HKY vaccine. We have demonstrated protection against aspergillosis and coccidioidomycosis with purified preparations of mannan or glucan, or conjugates of them to bovine serum albumin, as vaccines [26–28]. We have demonstrated some protein homologies between Cryptococcus, Aspergillus, Coccidioides, and Saccharomyces, as well as other fungi [29]. Protein components of HKY homologous with proteins of C. grubii, and cross-reacting immunity against these could explain protection. Thus, several possibilities exist that could contribute to the induction of host resistance by HKY that is protective against an unrelated organism like C. grubii. We noted that the durability of protection arising from HKY vaccination is at least 28 days in two previous studies [12, 13], which is suggestive that protection may be due to a memory response. However, additional studies on this aspect are necessary. Overall, our results provide further evidence that a panfungal vaccine based on components of HKY may be attainable. Studies in human cancer patients have shown the safety of administering heat-killed Saccharomyces cerevisiae and yeast-based vaccines [30–32]. This, coupled with our results, is suggestive that a panfungal yeast-based vaccine is possible. To that end, we have suggested potential recipient groups previously, and recognized the difficulty of immunizing and inducing protection in immunosuppressed individuals [28, 33]. Conflict of interest
None.
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