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Title:
PROBIOTIC BACTERIA-DIRECTED PREVENTION OR TREATMENT OF FUNGAL INFECTION
Document Type and Number:
WIPO Patent Application WO/2018/026719
Kind Code:
A1
Abstract:
The invention provides a method and bacterial compositions for reducing or preventing an infection of a bodily tissue by a fungal microorganism by contacting the tissue with a composition comprising a purified Lactobacillus paracasei bacterium.

Inventors:
JUNQUEIRA, Juliana, Campos (159 Ana Rosa De Almeida Carvalho, Sao Jose Dos Campos, -514 Sao Jose Dos Campos, Campos, BR)
FUCHS, Helen, Burgwyn (876 Furnace Brook Pkwy, Quincy, MA, 02169, US)
MYLONAKIS, Eleftherios (255 Promenade, Unit 427Providence, RI, 02906, US)
Application Number:
US2017/044703
Publication Date:
February 08, 2018
Filing Date:
July 31, 2017
Export Citation:
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Assignee:
RHODE ISLAND HOSPITAL (593 Eddy Street, Providence, RI, 02903, US)
UNIVERSIDADE ESTADUAL PAULISTA/UNESP (Av. Eng. Francisco José Longo, -000 Seo José Dos Campos, Campos, BR)
International Classes:
A61K35/747; A23L33/135; A61P3/00; A61P31/10; C12N1/20; C12R1/225
Foreign References:
US20160101136A12016-04-14
Other References:
ANWAR, KP ET AL.: "Profile of candidiasis in HIV infected patients", IRANIAN JOURNAL OF MICROBIOLOGY, vol. 4, no. 4, 4 December 2012 (2012-12-04), pages 204 - 209, XP055459806
COMAN, MM ET AL.: "In vitro evaluation of antimicrobial activity of Lactobacillus rhamnosus IMC 501, Lactobacillus paracasei IMC 502 and SYNBIO against pathogens", JOURNAL OF APPLIED MICROBIOLOGY, vol. 117, no. 2, August 2014 (2014-08-01), pages 518 - 527, XP055455565
HASSLOF, P ET AL.: "Growth inhibition of oral mutans streptococci and candida by commercial probiotic lactobacilli - an in vitro study", BMC ORAL HEALTH, vol. 10, no. 1, 2 July 2010 (2010-07-02), pages 1 - 6, XP021076575
ROSSONI, RD ET AL.: "Lactobacillus paracasei modulates the immune system of Galleria mellonella and protects against Candida albicans infection", PLOS ONE, vol. 12, no. 3, 7 March 2017 (2017-03-07), pages 1 - 17, XP055459816
Attorney, Agent or Firm:
BEATTIE, Ingrid, A. et al. (Mintz Levin Cohn Ferris Glovsky And Popeo, P.C.One Financial Cente, Boston MA, 02111, US)
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Claims:
CLAIMS

1. A composition for or method of reducing or preventing an infection of a bodily tissue by a fungal microorganism, comprising contacting said tissue with said composition comprising a purified Lactobacillus paracasei bacterium, wherein said bacterium is not substantially disseminated to lower gastrointestinal tissue.

2. The method of claim 1, wherein said tissue comprises tissues of the oral cavity.

3. The method of claim 1, wherein said tissue comprises mouth, lips, tongue, throat, or esophageal tissue.

4. The method of claim 1, wherein said tissue does not substantially comprise stomach or intestinal tissue.

5. The method of claim 1, wherein said Lactobacillus paracasei comprises strain 28.4.

6. The method of claim 1, wherein said composition is in the form of a cream, liquid, ointment, or chewing gum.

7. A composition comprising a purified Lactobacillus paracasei bacterium and a gellan.

8. The composition of claim 7, wherein said composition is in the form of a cream, liquid, ointment, or chewing gum.

9. The method of claim 1, wherein said fungal microorganism comprises Candida albicans.

10. The method of claim 1, wherein said subject had or has an HIV infection.

11. The method of claim 1, wherein said fungal microorganism is resistant to fluconazole.

Description:
PROBIOTIC BACTERIA-DIRECTED PREVENTION OR TREATMENT OF

FUNGAL INFECTION

RELATED APPLICATIONS

This application claims the priority and the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No: 62/369,735, filed August 1, 2016, which is incorporated herein by reference in its entirety.

INCORPORATION BY REFERENCE TO THE SEQUENCE LISTING

The content of the text file named 21486-631001WO_ST25" which was created on July 28, 2017 and is 1.45 kilobytes in size is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to immune modulation.

BACKGROUND OF THE INVENTION

Among patients with Human Immunodeficiency Virus (HIV), Candida albicans infections continue to be a problem. Patients are plagued by persistent infections, with the oral cavity being a primary infection site. The fungi C. albicans, a natural colonizer of the human intestine, mouth, and skin, can cause conditions such as thrush: hallmarked by raised, white lesions in the mouth that cause pain. Providing patients with prophylactic antifungal chemotherapy can be suboptimal in the case of a chronic infection such as HIV. With continued prevalence of an antifungal such as fluconazole in the system, C. albicans can develop resistance, whereby the treatment can become further challenging. Among susceptible patients, persistent infections can lead to drug resistance due to the need for recurrent therapy.

SUMMARY OF THE INVENTION

The methods and compositions of the invention provide a solution to drawbacks of previous approaches to treat and prevent fungal infections. For example, the invention features a method of reducing or preventing an infection of a bodily tissue by a fungal microorganism by contacting the tissue with a composition comprising a purified Lactobacillus paracasei bacterium. The fungal microorganism can include Candida albicans. The bacterium or bacterial composition is preferably not substantially disseminated to lower gastrointestinal tissue. For example, the L. paracasei need not be ingested or is not ingested. For example, the L. paracasei is ingested less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1% or less by weight). The composition is administered to or is applied to tissues of the oral cavity or adjacent tissues. For example, the tissues include mouth, lips, tongue, throat, or esophageal tissue. The tissue does not substantially comprise stomach or intestinal tissue.

In some examples, the fungal microorganism, e.g., pathogen, comprises Candida albicans. For example, the fungal microorganism is resistant to fluconazole. A subject to be treated may be immunocompromised, e.g., the subject had or has an HIV infection.

The Lactobacillus paracasei is purified. By "purified" or "substantially purified" is meant a L. paracasei bacterium that is substantially free of contaminating microorganisms or other macromolecules, e.g. , polysaccharides, nucleic acids, or proteins. For example, the L. paracasei comprises strain 28.4. The bacterium or bacterial composition is in a form suitable for administration to oral cavity tissues, e.g., composition is in the form of a cream, liquid, or ointment for spreading on oral cavity tissues or in the form of a chewing gum, whereby oral cavity tissues are contacted with the bacteria as it is liberated from the gum or dissolvable or chewable tablet as the subject sucks on or chews the composition. Other formulations include a liquid mouth wash/rinse or spray to be applied to oral cavity tissues and associated tissues.

Also within the invention is a composition comprising a purified Lactobacillus paracasei bacterium and a gellan. For example, the composition is in the form of a cream ointment, or chewing gum or chewable tablet.

The terms "subject," "individual," and "patient" are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. The mammal can be e.g., a human or appropriate non-human mammal, such as primate, mouse, rat, dog, cat, cow, horse, goat, camel, sheep or a pig. The subject can also be a bird or fowl. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed. As used herein, a "subject in need thereof or "a patient" may be a subject having a HIV or a subject who had HIV.

Alternatively, a subject may be a subject comprises a fungal pathogen that is resistant to an antifungal agent (other than the composition described herein), such as fluconazole.

As used herein, "treatment" or "treating," or "palliating" or "ameliorating" or "reducing" are used interchangeably herein. These terms refer to an approach for obtaining beneficial or desired results including but not limited to therapeutic benefit and/or a prophylactic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient may still be afflicted with the underlying disorder. For prophylactic benefit, the compositions may be administered to a patient at risk of developing a particular disease, or to a patient reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made. Treatment includes preventing the disease, that is, causing the clinical symptoms of the disease not to develop by administration of a protective composition prior to the induction of the disease; suppressing the disease, that is, causing the clinical symptoms of the disease not to develop by administration of a protective composition after the inductive event but prior to the clinical appearance or reappearance of the disease; inhibiting the disease, that is, arresting the development of clinical symptoms by administration of a protective composition after their initial appearance; preventing re-occurring of the disease and/or relieving the disease, that is, causing the regression of clinical symptoms by administration of a protective composition after their initial appearance.

The term "lower gastrointestinal tissue" includes the lower gastrointestinal tract such as most of the small intestine and all of the large intestine. The

gastrointestinal (GI) tract is conventionally divided into upper (mouth to ileum) and lower (cecum to anus). The lower digestive tract, also known as the bowel, is approximately 25 feet long and encompasses or consists of the small intestine and large intestine. Food from the stomach passes through the pyloric valve into the small intestine, a 20-foot tube with three sections: the duodenum, the jejunum, and the ileum. In some clinical contexts, e.g., gastrointestinal bleeding, the demarcation between the upper and lower GI tract is the duodenojejunal (DJ) junction (ligament of Treitz

All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a series of line graphs showing susceptibility of G. mellonella to infection with Lactobacillus spp. Serial concentrations of Lactobacillus/lsLrvae, were delivered to observe the effects to larvae longevity.

Figure 2A is a line graph showing the survival rate for different concentrations of L. paracasei: significant differences were observed for the groups 10 5 cells of 28.4 + Candida (p=0.0166), 10 6 cells of 28.4 + Candida (p=0.0003) and 10 7 cells of 28.4 + Candida (/?=0.0001) in relation to the control group (PBS + 10 6 cells of Candida).

Figure 2B is a line graph showing the survival rate for Heat- Killed (HK) L. paracasei: no significant statistically differences were observed for the groups 10 5 cells of HK28.4 + Candida (p=1.000), 10 6 cells of HK28.4 + Candida (p=1.000) and 10 7 cells of HK28.4 + Candida (/?=0.0733) when compared to the control group PBS + Candida. Kaplan-Meier test, p< 0.05. Figure 2. L. paracasei strain 28.4 prolonged survival of G. mellonella larvae infected with C. albicans.

Figure 3 is a bar graph showing G. mellonella hemocyte density increased with the injection of L. paracasei strain 28.4. Hemocyte density of L. paracasei group (28.4 + 28.4) increased 2.38 x 10 6 more cells/mL compared to a PBS control (PBS + PBS). The group L. paracasei + C. albicans (28.4 + C. albicans) also increased the hemocyte density 1.24 x 10 6 more cells/mL compared to C. albicans group (PBS + C. albicans). Student t test, p < 0.05 value was considered significant. **p=0.0001, */?=0.0267.

Figures 4A and B are bar graphs showing that. L. paracasei strain 28.4 increased the expression of antifungal peptides of G. mellonella. Relative

quantification (log) of Galiomicin (Figure 4 A) and Gallerymicin (Figure 4B) compared from larvae treated with only PBS (Control) to larvae pre-treated with PBS and infected with C. albicans, larvae only treated with L. paracasei, and larvae pre- treated with L. paracasei and infected with C. albicans. Values are expressed as the means and standard deviation. ANOVA and Tukey Tests (p < 0.05). ***p < 0.001.

Figures 5A-5D are photographs showing G. mellonella larvae. Analyses of melanization process after 24h of prophylactic treatment with L. paracasei and infection with C. albicans: control group treated with PBS (Figure 5A), group treated with 10 5 cells/larva of L. paracasei (Figure 5B), group treated with 10 6 cells/larva of L. paracasei (Figure 5C), and group treated with 10 7 cells/larva of L. paracasei (Figure 5D).

Figure 6 is a series of line graphs showing ictobacillus spp. prolongs the survival of G. mellonella larvae infected with C. albicans. There was a significant difference between the Lactobacillus strain 4- C. albicans group" and "PBS + C. albicans control group": A. p = 0.0097; B. p = 0.0013; C. p = 0.0044; D. p = 0.0001 ; E. p = 0.0245; F. p = 0.0001; G. p = 0.0075; H, p = 0.0003 and h p = 0.0733. Kaplan- Meier test, p< 0.05.

Figure 7 is a bar graph showing G. mellonella hemocyte number increased with the injection of L. paracasei strain 28.4. The group of L. paracasei 28.4 + PBS increased the hemocyte number compared to a PBS control (PBS - PBS) at all different time points studied. The group L. paracasei + C. albicans also increased the hemocyte quantity compared to C. albicans group (PBS + C. albicans). PBS + C. albicans group showed a reduction of hemocyte quantity in relation to the PBS control group, but when the larvae were pretreated with Lactobacillus (L. paracasei + C. albicans group) the hemocyte quantity was very similar to the values found in the PBS control group. The four groups were compared in each time point studied by ANOV A test (4h: p = 0.0001, 8h: p = 0.0003, 24h: p = 0.0006). The results of Tukey test are indicated by letters: different letters (A, B, and C) represent statistically significant differences among the groups for each time point studied. A p < 0.05 value was considered significant.

Figure 8 is a series of bar graphs showing L. paracasei strain 28.4 increased the expression of antifungal peptides of G. mellonella. Relative quantification (log) of Galiomicin (A) and Gallerymicin (B) for the groups treated with only PBS

(Control), pre- treated with PBS and infected with C. albicans, only treated with L. paracasei, and pre-treated with L. paracasei and infected with C. albicans. The units in the Y-axis were calculated based on the 2 ~ A T method, and they are expressed as the means and standard deviation. Each gene was normalized and compared with the expression of insects exposed to the control (PBS) using the reference gene β-aetin. Different letters (A, B, and C) represent statistically significant differences among the groups. ANOVA and Tukey Tests (p<0.05). ***p < 0.001.

Figure 9 is a series of bar graphs showing L. paracasei strain 28.4 decreased the number of fungal cells in G. melioneila hemolvmph. Mean and standard deviation of C. albicans counts (CFU/larvae) in the hemolymph of Galleria melioneila after 4, 8 and 24 h of experimental infection. The following groups were compared at each time of infection: PBS + C. albicans (control) and L. paracasei + C. albicans. A significant difference between groups was only observed after 24 h of infection, with a larger number of CFU/larvae in the control group compared to the L. paracasei + C. albicans ( p - 0.0199). Student t test, p < 0.05.

DETAILED DESCRIPTION

Recurrent fungal infections are a problem in immunocompromised subjects, e.g., those infected with HIV, those suffering from autoimmune diseases including diabetes, those with immune systems weakened from the disease and/or from treatments for disease such as chemotherapy and radiation used for treatment of cancer, and the elderly whose immune system may be weakened from age.

Oral thrush, also called oral candidiasis is a condition in which the fungus, Candida albicans, accumulates on the lining of the mouth. Candida is a normal organism in the mouth, but sometimes it can overgrow and cause clinical symptoms. Oral thrush causes creamy white lesions, usually on the tongue or inner cheeks.

Sometimes oral thrush may spread to the roof of the mouth, gums or tonsils, or the back of the throat. Although oral thrush can affect anyone, it's more likely to occur in babies, the elderly, and in people with suppressed immune systems or or those who take certain medications as described above.

We sought to address the problem of recurrent infection by looking for a non- drug method of treatment. The use of probiotics has been successfully used to influence gut colonization. Thus, we endeavored to determine if bacteria could affect C. albicans. We previously found that prokary otic -eukary otic interactions can inhibit C. albicans, in particular the ability to form hyphae (Peleg et al. PNAS. 2008;

105(38): 14585-14590; Vilela et al., Virulence. 2015; 6(l):29-39.). Knowing that bacteria can directly influence C. albicans we searched for a strain of Lactobacillus, a bacterium that can be used as a probiotic, that is native to the oral cavity and can inhibit a C. albicans infection. To this end, we generated a collection of

Lactobacillus strains derived from the oral cavity of HIV patients in Brazil. We then determined if any of the collected species could inhibit C. albicans.

The data described herein identified a species that is superior to other

Lactobacillus in inhibiting C. albicans infections: Lactobacillus paracasei strain 28.4, providing prophylactic protection. Using an invertebrate infection model, the greater wax moth, Galleria mellonella exhibited prolonged survival when L. paracasei 28.4 was provided prior to a lethal dose of C. albicans. Our interrogation of how

Lactobacillus affects the host found it is able to modulate the immune response of G. mellonella, providing protection against C. albicans infection. This result is mediated by recruitment of circulating hemocytes and the production of elevated levels of antimicrobial peptides (AMPs), which protect the insect exposure to the pathogen. Identification of Lactobacillus strains with immunomodulatory activity against fungal pathogens

The human mouth is naturally colonized by a multitude of microbes. When individuals become immunocompromised, they can become susceptible to this reservoir of microbes. We collected bacterial samples from the oral cavity of HIV patients in an effort to identify Lactobacillus strains that could provide protection against infectious fungal microbes, serving as a probiotic. Others have used the idea of providing probiotics to prevent dental cavities using Lactobacillus acidophilus to inhibit the bacteria Streptococcus mutans. By contract, we are using a different Lactobacillus species to inhibit C. albicans.

In order to evaluate the hypothesis that bacteria of the genus Lactobacillus have the ability to be immunomodulatory, we first evaluated whether some lactobacilli from our collection can prolong survival after infection with C. albicans. Since there are no studies in the literature inoculating L. paracasei, L. fermentum and L. rhamnosus in G. mellonella, we first evaluated the susceptibility of G. mellonella to infection with Lactobacillus ssp. In this series of experiments, we tested concentrations between 10 5 -10 9 cells/larva, and only in the 2 highest concentrations (10 8 and 10 9 cells/larvae) did we observe larval death.

Lactobacillus paracasei

L. paracasei is a gram-positive, facultatively heterofermentative, non-spore forming microorganism in the family of lactic acid bacteria. The cells of L. paracasei are typically rod shaped, with a size range of 2.0μηι to 4.0μιη in width, and 0.8 to Ι.Ομηι in length. The organism is nonmotile. L. paracasei cells often have square ends, and may exist either in single form or in chains. A number of different strains of L. paracasei have been isolated from a variety of environments including dairy, plants, and human and animal gastrointestinal tracts. L. paracasei is genotypically and phenotypically indistinguishable from other members of its genus such as Lactobacillus casei and Lactobacillus rhamnosus. However, they are easily differentiated from each other by their fermentation profiles. L. paracasei grows optimally in a temperature range between 10 and 37 °C.

Example 1 : Lactobacillus paracasei modulates the immune system of Galleria mellonella and protects against Candida albicans infection

Probiotics can control opportunistic infectious due to their ability to stimulate the immune system. Using the non-vertebrate model host Galleria mellonella, we evaluated whether Lactobacillus spp. are able to modulate the immune system and provide protection against Candida albicans infection. Among different strains of Lactobacillus paracasei, Lactobacillus rhamnosus and Lactobacillus fermentum, we verified that L. paracasei 28.4 strain had the greatest ability to prolong the survival of larvae infected with a lethal dose of C. albicans. We found that the injection of 10 7 cells/larvae of L. paracasei into G. mellonella larvae infected by C. albicans increased the survival of these animals in 50% compared to the control group.

The immune mechanisms involved in the protection against C. albicans infection were investigated, evaluating the number of hemocytes and the gene expression of antifungal peptides. We found that L. paracasei increased the hemocyte density (2.38 x 10 6 cells/mL) in relation to the control group (1.29 x 10 6 cells/mL), indicating that this strain is capable to recruit hemocytes into the G. mellonella hemolymph. Further, we found that the L. paracasei upregulated the genes that encode the antifungal peptides galiomicin and gallerymicin. In relation to the control group, L. paracasei induced an increase of gene expression of 6.67-fold for galiomicin and of 17.29-fold for gallerymicin. These data indicate that L. paracasei modulates the immune system of G. mellonella in an insect model of C. albicans and therefore indicates that this probiotic strain is useful to reduce the severity of and protect against candidiasis in mammalian subjects such as humans.

Probiotics to reduce infection by fungal pathogens

C. albicans is a commensal yeast that colonizes the gastrointestinal track in approximately 53% of healthy individuals (Coronado-Castellote L, Jimenez-Soriano Y. 2013. J Clin Exp Dent 5:e279-286). This yeast is an opportunistic pathogen that can cause severe and recurrent infections in the mucosa, as well as life-threatening systemic infections (Mayer FL, Wilson D, Hube B. 2013. Virulence 4: 119-128). The development of mucosa or systemic candidiasis occurred frequently in

immunosuppressed patients (Martinez- Alvarez JA, Perez-Garcia LA, Flores-Carreon A, Mora-Montes HM. 2014. Rev Iberoam Micol 31:62-66) and is associated with a complex interplay between the fungal virulence factors and the host immune system (Hofs S, Mogavero S, Hube B. 2016. J Microbiol 54: 149-169). The host response to C. albicans is characterized by a rapid activation of the innate immune system upon first contact with the fungal cell. During the innate immune response, macrophages, neutrophils and dendritic cells provide primary protective effect via direct antifungal activities, including phagocytosis and release of antimicrobial peptides. The activation of the innate immune system is the basis for a more specific immune response mediated by T or B lymphocytes (adaptative immune system) (4, 5).

Probiotics bacteria have been studied as a potential method for prevent opportunistic infectious diseases due to their ability to stimulate the immune system (Wickens K, Black PN, Stanley TV, Mitchell E, Fitzharris P, Tannock GW, Purdie G, Crane J, Probiotic Study G. 2008. J Allergy Clin Immunol 122:788-794; Ryan KA, O'Hara AM, van Pijkeren JP, Douillard FP, O'Toole PW. 2009. Lactobacillus. J Med Microbiol 58:996-1005; Jorjao AL, de Oliveira FE, Leao MV, Carvalho CA, Jorge AO, de Oliveira LD. 2015.. ScientificWorldJournal 2015:716749). According to World Health Organization probiotics, are live microorganisms that confer health benefits on the host when administered in adequate amounts (Guarner F, Khan AG, Garisch J, Eliakim R, Gangl A, Thomson A, Krabshuis J, Lemair T, Kaufmann P, de Paula JA, Fedorak R, Shanahan F, Sanders ME, Szajewska H, Ramakrishna BS, Karakan T, Kim N, World Gastroenterology O. 2012. World Gastroenterology Organisation Global Guidelines: probiotics and prebiotics October 2011. J Clin Gastroenterol 46:468-481). In this context, several Lactobacillus strains have been investigated as a probiotic bacterium to inhibit the colonization of pathogens and to stimulate the immune system (Aoudia N, Rieu A, Briandet R, Deschamps J, Chluba J, Jego G, Garrido C, Guzzo J. 2016. Food Microbiol 53:51-59). Different lactobacilli strains can modulate innate and adaptive immune system preventing the initiation and progression of cancer cells (Abedin-Do A, Taherian-Esfahani Z, Ghafouri-Fard S, Ghafouri-Fard S, Motevaseli E. 2015. Immunotherapy 7: 1307-1329). Moreover, other previous studies showed that certain strains of lactobacilli were capable to modulate the expression of several genes involved in the regulation of the immune system (Chon H, Choi B, Jeong G, Lee E, Lee S. 2010. Comp Immunol Microbiol Infect Dis 33:e41-49; Chon H, Choi B, Lee E, Lee S, Jeong G. 2009. J Appl

Microbiol 107: 1588-1597; Galdeano CM, Perdigon G. 2006. Clin Vaccine Immunol 13:219-226; Kim YG, Ohta T, Takahashi T, Kushiro A, Nomoto K, Yokokura T, Okada N, Danbara H. 2006. Microbes Infect 8:994-1005; Wagner RD, Johnson SJ. 2012. J Biomed Sci 19:58).

The study of immune response against C. albicans was performed in a Galleria mellonella model that is an art-recongnized tool to the study of innate immune response. The immune system of G. mellonella demonstrates a number of structural and functional similarities to the innate immune system of mammals (Vilmos P, Kurucz E. 1998. Immunol Lett 62:59-66) and is composed by cellular and humoral components (Fallon JP, Troy N, Kavanagh K. 2011. Virulence 2:413-421). Their cellular immune response consists of the synthesis and mobilization of immune cells called hemocytes, which can surround and engulf invading pathogens (Mowlds P, Kavanagh K. 2008. Mycopathologia 165:5-12). The humoral element of these larvae consists in the production of a wide range of antimicrobial peptides (AMP) (Ganz T, Lehrer RI. 1995. Pharmacol Ther 66: 191-205; Zdybicka-Barabas A, Mak P, Jakubowicz T, Cytrynska M. 2014. Arch Insect Biochem Physiol 87: 1-12). In addition, G. mellonella is a facile infection model that have been used as a screening tool for studies using vertebrate models (Barbosa JO, Rossoni RD, Vilela SF, de Alvarenga JA, Velloso Mdos S, Prata MC, Jorge AO, Junqueira JC. 2016. PLoS One l l:e0150457). Prophylactic or therapeutic inoculation of L. acidophilus ATCC 4356 into G. mellonella infected by C. albicans reduced the number of yeast cells in the larval hemolymph and increased the survival of these animals (Barbosa JO, Rossoni RD, Vilela SF, de Alvarenga JA, Velloso Mdos S, Prata MC, Jorge AO, Junqueira JC. 2016. PLoS One l l :e0150457).

Due to the high recurrence of Candida spp. lesions in immunosuppressed patients, the continuous prophylactic use of probiotics to prevent Candida spp.

infections represents a potential strategy in thwarting recurrent infections. Using G. mellonella as a model for the study of immunomodulatory effects of probiotics, we screened different clinical strains of Lactobacillus in order to identify new probiotics strains capable to prevent candidiasis. Since L. paracasei strain 28.4 showed the greatest ability to reduce Candida infections, we identified a number of insect immune responses to evaluate its probiotic activity. Based on these investigations, we were able to describe the specific responses stimulated by L. paracasei 28.4 that protected G. mellonella against C. albicans infections.

The following materials and methods were used to generate the data described herein.

Organisms and strains. In this study we used 9 clinical strains of Lactobacillus spp. recovered from the oral cavity and 1 reference strain of C. albicans from the American Type Culture Collection (ATCC 18804). The oral Lactobacillus spp. strains were isolated from the saliva of 41 healthy patients and included: L. paracasei (n=5), Lactobacillus rhamnosus (n=3) and Lactobacillus fermentum (n=l) (Table 1). All the strains were stored as frozen stocks with 25% glycerol at -80°C until used.

TABLE 1. Clinical strains of Lactobacillus used in this study

Species Strain designation

L. fermentum 20.4

L. paracasei 20.3

L. paracasei 26.1

L. paracasei 27.1

L. paracasei 28.4

L. paracasei 30.1

L. rhamnosus 5.2

L. rhamnosus 19.3

L. rhamnosus 19.9

Microbial inoculum preparation. C. albicans cells were grown in YPD medium (1% yeast extract, 2% bacto-peptone, 2% dextrose) overnight at 30°C with agitation. Cells were collected by centrifugation and washed 3 times with PBS. Yeast cells were counted using a hemocytometer. The cell number was confirmed by determining CFU/mL on YPD plates. Lactobacillus spp. were grown in Lactobacillus MRS Broth (Difco, Detroit, USA) for 24 h at 37°C in a bacteriological incubator under microaerophilic conditions. Cells were collected by centrifugation and washed 3 times with PBS, after this the number of cells in suspension was determined with a spectrophotometer (Eppendorf Biophotometer Plus, Eppendorf, Hamburg, Germany). For the assay with heat-killed (HK) Lactobacillus spp., we incubated bacteria at 80°C for 20 min and subsequently plated the cells on MRS agar to ensure that no viable cells remained.

G. mellonella survival. For this study, the standard methodologies described by Mylonakis et al. (Mylonakis E, Moreno R, El Khoury JB, Idnurm A, Heitman J, Calderwood SB, Ausubel FM, Diener A. 2005. Infect Immun 73:3842-3850) and Vilela et al. (Barbosa JO, Rossoni RD, Vilela SF, de Alvarenga JA, Velloso Mdos S, Prata MC, Jorge AO, Junqueira JC. 2016. PLoS One l l :e0150457) were used. G. mellonella (Vanderhorst Wholesale, St. Marys, OH) in their final larval stage were stored in the dark and used within 7 days from shipment. Sixteen randomly chosen G. mellonella larvae with similar weight and size (250-350 mg) were used per group in all assays. Two control groups were included in the assays that form part of this study: one group was inoculated with PBS, and the other received no injection as a control for general viability.

We initially determined the sub-lethal inoculum concentration of

Lactobacillus by injecting larvae with serial dilutions of the bacteria. For this purpose, different concentrations of each Lactobacillus strain (10 5 to 10 9 cells/larvae) were inoculated into G. mellonella through the last left proleg. The larvae were kept on Petri dishes at 37°C and monitored daily for survival.

To evaluate the effects of probiotics on C. albicans infections, the larvae were pre-infected with Lactobacillus by injecting the bacteria (concentration previously determined) through the last left proleg. After 1 h, larvae were infected with 10 6 cells/larvae of C. albicans suspended in PBS at the last right proleg. Larvae were incubated at 37°C and monitored daily for survival. The experimental groups used in this study are presented in Table 2. Among all the strains tested, L. paracasei 28.4 reached the highest survival rate and it was selected for the following experiments. TABLE 2. Experimental groups used to evaluate the effects of Lactobacillus strains on Candida infections

Groups 1 st Injection 2 nd Injection

(Last left proleg) (Last right proleg) PBS PBS PBS

C. albicans PBS C. albicans

Lactobacillus Lactobacillus PBS

Lactobacillus + C. albicans Lactobacillus C. albicans

No Injection

Evaluating G. mellonella hemocyte density. Larvae were pre-infected with L paracasei strain 28.4 by injecting the bacteria at the last left proleg. After lh, larvae were infected with C. albicans at the last right proleg. Hemocytes were collected from the hemocoel at 4 h post-injection with C. albicans. Larvae were bled into tubes containing cold, sterile insect physiologic saline (IPS) (150 mM sodium chloride; 5 mM potassium chloride; 100 mM Tris-hydrochloride, pH 6.9 with 10 mM EDTA, and 30 mM sodium citrate). The hemocytes were enumerated using a hemocytometer and the results were averaged from four replicates.

Analysis of peptide expression. Larval RNA was extracted using a TRIzol (Ambion, Inc., Carlsbad, CA, USA) as recommended by the manufacturer at 4, 8, and 24 h post-injection of L. paracasei strain 28.4. In brief, a 2 mL volume of TRIzol was added to a 15 mL tube containing the homogenized frozen tissue of larvae and incubated at room temperature (RT) for 10 min. Subsequently, 400 iL of chloroform (Sigma- Aldrich, St. Louis, MO, USA) was added and the tubes were centrifuged at 12,000 x g for 15 min at 4°C. The supernatant was then transferred to a new tube, and 1 mL of isopropanol (Sigma- Aldrich, St. Louis, MO, USA) was added. After centrifugation, the obtained pellet was washed with 70% ethanol (Sigma- Aldrich, St. Louis, MO, USA), centrifuged again, and suspended in 50 of nuclease-free water (Ambion Inc., Carlsbad, CA, USA). The concentration, purity and quality of the RNA were verified using a NanoVue Plus spectrophotometer (GE Healthcare Bio-Sciences, Pittsburgh, USA).

The extracted total RNA (1 μg) was transcribed into complementary DNA (cDNA) using the Verso cDNA Synthesis Kit (Thermo Fisher Scientific Inc, Waltham, MA, USA), according to the protocols recommended by the manufacturer. The primers for the gene encoding gallerymicin were described and used as indicated by Bergin et al. (25) (Table 3). The transcribed cDNAs were amplified for relative quantification of the expression of the genes encoding galiomicin and gallerymicin in relation to the concentration of the reference gene (β-actin).

TABLE 3. PCR primer pairs used to amplify regions of the genes involved in the immune system of G. mellonella and a reference gene

aF indicates a forward primer b R indicates a reverse primer

''Base pair (Fragment size)

The qPCR method was applied to evaluate the amount of the cDNA products in the exponential phase of the amplification reaction. As a detection system, the iTaq™ Universal SYBR® Green Supermix (Bio-Rad Laboratories, Inc, Hercules, CA, USA) was used in the following reaction mixture: 5 of iTaq Universal SYBR Green (2x), 300 nM of the forward primer, 300 nM of the reverse primer, 2 μΐ ^ of cDNA solution (diluted 1: 10) and 2 μΐ ^ of nuclease-free water (Ambion Inc., Carlsbad, CA, USA), to obtain a final volume of 10 in each well of a 96-well plate (Bio-Rad Laboratories, Inc, Hercules, CA, USA). As a negative control for the reaction, all the reagents were added to the last wells of the plates except for cDNA, and the wells were sealed with optical adhesive (Bio-Rad Laboratories, Inc, Hercules, CA, USA). Subsequently, the plate was placed in a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad Laboratories, Inc, Hercules, CA, USA) device. The following cycling parameters were used: 95 °C for 2 min for an initial denaturation followed by 40 cycles of 95°C for 15 s and 60°C for 30 s. After the end of the last cycle, the samples were subjected to dissociation (melting) curve analysis, and the absence of any bimodal curve or abnormal amplification signal was observed and analyzed every 0.1 °C. The 2 "AACT method was used to analyze the relative changes in gene expression from the quantitative RT-PCR experiment (Livak KJ, Schmittgen TD. 2001. Methods 25:402-408).

Quantification of Candida CFU/larvae in G. mellonella hemolymph. The methodology described by Vilela et al. was used with some modifications. Larvae were infected with the same method used for the Galleria mellonella survival assay. For quantification of the presence of C. albicans in infected G. mellonella, the larvae were euthanized 4, 8 and 24 h after infection in the following groups: PBS + C. albicans and 28.4 + C. albicans. A pool of 4 larvae was used per group and time. The experiment was carried out in triplicate using 16 larvae per group, for a total of 96 infected larvae. A control group was included for each time point, which was injected with 10 | L PBS into the last left proleg.

At each time point, the larvae were cut in the cephalocaudal direction with a scalpel blade and squeezed to remove the hemolymph, which was transferred to an Eppendorf tube. Serial dilutions were prepared from the hemolymph pool, seeded onto Petri dishes containing Sabouraud dextrose agar (Difco, Detroit, USA) supplemented with chloramphenicol (100 pg/mL), and incubated for 48 h at 37°C. After this period, the colonies were counted for the calculation of CFU/larvae.

Statistical analysis. Percent survival and killing curves of G. mellonella were plotted and statistical analysis was performed by the Kaplan-Meier test using GraphPad Prism statistical software (GraphPad Software, Inc., California, CA, USA). Analysis of variance (ANOVA) was used to compare the results obtained in the survival assays. Student's t-test was used to compare hemocyte densities. A P value < 0.05 was considered significant.

Effects of Lactobacillus spp. on experimental candidiasis In order to evaluate whether bacteria of the genus Lactobacillus have immunomodulatory effects and to identify potential probiotic strains for the prevention of Candida infections, we analyzed different Lactobacillus clinical strains from our collection including some strains of L. fermentum, L. paracasei and L. rhamnosus.

Firstly, we evaluated the susceptibility of G. mellonella to Lactobacillus strains using larvae no infected by C. albicans for determining the sub-lethal inoculum concentration. We tested concentrations ranging 10 5 to 10 9 cells/larva and death of larvae was observed only with the two highest concentrations (10 8 and 10 9 cells/larvae) (Figure 1).

Based on these results, a sub-lethal concentration of 10 6 cells/larva was adopted for the study of the effects of Lactobacillus strains on experimental candidiasis. We screened 9 clinical strains of lactobacilli to determine which strain(s) prolonged survival after infection with C. albicans. In the control group, the infection with C. albicans without previous injection of lactobacilli caused death in 100% of the larvae within 24 h. When the larvae were pretreated with Lactobacillus spp. prior to C. albicans infection, the survival rate of G. mellonela larvae was dependent on the Lactobacillus strain injected. Among the 9 strains analyzed, 6 were capable to prolong the survival of larvae infected with C. albicans by up to 120 h (Table 4). Using an ANOVA test to compare all the Lactobacillus strains, we found that L. paracasei strain 28.4 reached the greatest survival rate with statistically significant difference compared to other strains. Then, this strain was selected for all the subsequent assays. TABLE 4. Effects of Lactobacillus spp. on experimental candidiasis based on the analysis of survival curves of G. mellonella larvae

Strain Survival after 120 h (%)

20.4 - L. fermentum 0

20.3 - L. paracasei 12 26.1 - L. paracasei 19 27.1 - L. paracasei 6

28.4 - L. paracasei 27 30.1 - L. paracasei 0 5.2 - L. rhamnosus 14

19.3 - L. rhamnosus 0

19.9 - L. rhamnosus 14

In order to determine whether different concentrations of L. paracasei strain 28.4 could influence the survival rate of larvae infected with C. albicans, the larvae were pretreated with L. paracasei at concentrations of 10 5 -10 7 cells/larva. We observed a dose dependent survival rate of the larvae, whereby an inoculum of 10 7 cells/larva of L. paracasei reached higher survival rate in relation to the other concentrations (10 5 and 10 6 cells/larvae) (Figure. 2A). The increase of the L.

paracasei concentration was correlated with a decrease of the melanization of G. mellonella, that is part of the infection process with C. albicans (Fig. 5-1 to 5-4). In addition, we evaluated if the survival rate of G. mellonella could be influenced by the viability of L. paracasei strain 28.4. We used the same groups described above, but live L. paracasei was replaced by heat-killed L paracasei. Heat-killed L. paracasei did not provide prophylactic protection, and thus did not increase the survival G. mellonella infected with C. albicans. These data indicate that the probiotic action was a consequence of the living and not dead bacteria (Figure 2B).

Effects of L. paracasei strain 28.4 on G. mellonella hemocyte density

To investigate the immune mechanisms associated with the preventive effects of L. paracasei against Candida infection, we determined the number of available hemocytes in the hemolymph of larvae after 4h of Lactobacillus injection. As the higher survival rate of G. mellonella was achieved with a concentration of 10 7 cells/larvae of L. paracasei, we used this concentration to the hemocyte density assay. Firstly, we analyzed only the larvae not infected by C. albicans. The hemocyte density in the L. paracasei group increased the number of cells compared to the PBS control group (2-fold increase). In the larvae infected with C. albicans, the group pretreated with L. paracasei also increased the hemocyte density compared to C. albicans control group (1.96-fold increase) (Figure 3). These results indicate that L. paracasei strain 28.4 recruits hemocytes into the hemolymph and protect G.

mellonella against Candida infections. Effects of L. paracasei strain 28.4 on the expression of the gene encoding gallerymicin and galiomicin

The presence of an increased hemocyte density indicates that L. paracasei strain 28.4 modulates the immune response of G. mellonella larvae. Thus, we further explored alterations in the immune response examining the expression of antifungal peptides. Using RT-PCR, we evaluated the change in expression of the gene encoding galiomicin, a defensin identified in G. mellonella, and gallerymicin, a cysteine-rich antifungal peptide.

We found that L. paracasei strain 28.4 increased the expression of both antifungal peptides analyzed. For the gene encoding galiomicin, the group pretreated with L. paracasei and then infected with C. albicans had a statistically significant increase (/?=0.0001) in relation to the control group (PBS + C. albicans) only for the observation time of 4h. L. paracasei leaded an increase of gene expression of 6.67 and 1.68-fold compared, respectively, to the control group formed by PBS + PBS and the control group composed by PBS + C. albicans (Figure 4A).

For the gene encoding gallerymicin, the group pretreated with L. paracasei and then infected with C. albicans had a greater increase in gene expression compared to the other groups for all the times evaluated: 4h (p=0.0001), 8h (p=0.0001) and 24h (p=0.0001). L. paracasei increased the expression of the gene encoding gallerymicin according to observation time, achieving 17.29-fold of increase compared to control group formed by PBS + PBS and 1.87 -fold compared to control group formed by PBS + C. albicans after 24h (Figure 4B). These data shows that the pre-treatment with L. paracasei strain 28.4 to stimulates the antimicrobial peptide against C. albicans in G. mellonella model.

Lactobacillus paracasei probiotic strains reduce the severity of fungal infections

Lactobacilli spp. are a probiotic bacteria that have excellent health benefits, such as the ability to inhibit the colonization of pathogenic microorganisms, to produce biosurfactants and hydrogen peroxide and to modulate the host immune response (10, 27-29). In this study, we screened new potential probiotic strains of Lactobacillus spp. capable to prevent and treat Candida infections in G. mellonella host model. We also sought to identify immune responses in G. mellonella that can be used as substitute markers to predict prophylactic efficacy of a probiotic. We found that L. paracasei 28.4 strain improved the survival of G. mellonella infected with a lethal inoculum of C. albicans. Our results demonstrated that the immune response of G. mellonella can be stimulated with a prophylactic provision of probiotic bacteria, making them more resistant to virulent pathogens. These effects were associated with recruitment of hemocytes into the hemolymph and by stimulating antimicrobial peptide response.

Prior to the study of the effects of lactobacilli on the development of candidiasis, we evaluated the susceptibility of G. mellonella to Lactobacillus strains in larvae no infected by C. albicans. We observed that the strains did not cause death of the animals in concentrations up to 10 7 cells/larvae, demonstrating low

pathogenicity for G. mellonella model. There are few studies that used G. mellonella model to the study of probiotics bacteria, in which the strains of Lactococcus lactis and Lactobacillus acidophilus were not also virulent for this host model (23, 30).

By taking sampling of clinical Lactobacillus strains from the oral cavity of healthy patients, we found that some strains provided better protection against C. albicans than others. We found that L. paracasei 28.4 was the best strain to prevent candidiasis in G. mellonella model. Based on these results, we investigated the capacity of this strain to stimulate the immune system of G. mellonella. In contrast to the vertebrate animals, the immune system of insects is not composed by

immunoglobulin and immune cells with long-term memory. More specifically, the cellular immune response of G. mellonella is mediated by hemocytes that represents the main antimicrobial process characterized by phagocytosis (Arvanitis M, Glavis- Bloom J, Mylonakis E. 2013. Biochim Biophys Acta 1832: 1378-1383). The humoral immune response involves the production of a various antimicrobial peptides (AMP) that arrest and kill pathogens that evade the cellular immune response (Fallon JP, Troy N, Kavanagh K. 2011. Virulence 2:413-421).

Provision of L. paracasei 28.4 strain led an increasing of the survival rate of G. mellonella larvae that was accompanied by an increase in the number of hemocytes, demonstrating that L. paracasei is capable to stimulate the cellular immune response of the larvae by recruitment of hemocytes. Bergin et al. (Bergin D, Brennan M, Kavanagh K. 2003. Microbes Infect 5: 1389-1395) performed a study to evaluate whether fluctuations in the number of hemocytes and yeast cells in infected larvae could be used to determine the relative pathogenicity of a range of strains. The results indicated that larvae inoculated with virulent Candida strains showed a significant reduction in density of hemocytes, while the larvae inoculated with strains with low pathogenicity demonstrated only a slight variation in the number of hemocytes. Taken in their totality, these results confirmed that hemocytes could be used to determine the pathogenicity of microorganisms and modulations of the immune response.

The results described herein demonstrate that Lactobacilli spp., e.g., L.

paracasei, lead to broad immunomodulation in the G. mellonella model of fungal disease that impacts in the survival of animals during fungal infections.

We also explored alterations in the immune response examining the expression of AMP, including the genes encoding gallerymicin and galiomicin. In G. mellonella, the production of AMP represents the last line of defense. These peptides are released into the hemolymph in order to attack elements of the bacterial or fungal cell wall (19, 33). AMP are synthesized as pre-proproteins at a rate up to 100 times faster than IgM in mammals (Lowenberger C. 2001. Insect Biochem Mol Biol 31:219-229) and their small size, less than 10 kDa, allows diffusion through the hemolymph to counteract invading pathogens. In general, the mode of action of AMP is through binding to the surface of pathogens that result in damage to the microbial membrane and lead the collapse of the transmembrane electrochemical gradients (Shai Y. 1999. Biochim Biophys Acta 1462:55-70; Shai Y. 2002. Biopolymers 66:236-248; Brown SE, Howard A, Kasprzak AB, Gordon KH, East PD. 2009. Insect Biochem Mol Biol 39:792-800).

AMP expression in G. mellonella treated with probiotic bacteria was found to be modulated such that the immune response against fungal pathogens was augmented. The highest expressions of the genes encoding gallerymicin and galiomicin were found at the times of 8 and 24 h.

The clinical strains of Lactobacillus isolated from the oral cavity of healthy patients shows probiotic activity against C. albicans. L. paracasei 28.4 strain represents a probiotic strain that is useful to control Candida infections. In addition, this study indicates that prior exposure to a L. paracasei dose activates the G.

mellonella immune system and allows the larvae to combat a lethal infection by C. albicans. This effect is mediated by recruitment of circulating hemocytes, and the production of elevated levels of AMP. This study also demonstrate that G. mellonella is a suitable model for analyzing specific aspects of broad probiotic

immunomodulation.