History and Perspective of Immunotherapy for Pythiosis

The fungus-like microorganism Pythium insidiosum causes pythiosis, a life-threatening infectious disease increasingly reported worldwide. Antimicrobial drugs are ineffective. Radical surgery is an essential treatment. Pythiosis can resume post-surgically. Immunotherapy using P. insidiosum antigens (PIA) has emerged as an alternative treatment. This review aims at providing up-to-date information of the immunotherapeutic PIA, with the focus on its history, preparation, clinical application, outcome, mechanism, and recent advances, in order to promote the proper use and future development of this treatment modality. P. insidiosum crude extract is the primary source of immunotherapeutic antigens. Based on 967 documented human and animal (mainly horses) pythiosis cases, PIA immunotherapy reduced disease morbidity and mortality. Concerning clinical outcomes, 19.4% of PIA-immunized human patients succumbed to vascular pythiosis instead of 41.0% in unimmunized cases. PIA immunotherapy may not provide an advantage in a local P. insidiosum infection of the eye. Both PIA-immunized and unimmunized horses with pythiosis showed a similar survival rate of ~70%; however, demands for surgical intervention were much lesser in the immunized cases (22.8% vs. 75.2%). The proposed PIA action involves switching the non-protective T-helper-2 to protective T-helper-1 mediated immunity. By exploring the available P. insidiosum genome data, synthetic peptides, recombinant proteins, and nucleic acids are potential sources of the immunotherapeutic antigens worth investigating. The PIA therapeutic property needs improvement for a better prognosis of pythiosis patients.

Prompt and effective treatment improves the patient's prognosis [56,[85][86][87][88][89][90][91][92][93][94]. The use of conventional antifungal drugs provides limited efficacy in treating pythiosis because P. insidiosum lacks the drug-target sterol biosynthesis enzymes [95]. A few antibacterial drugs tocol of choice. In brief, the organism is cultured in a liquid medium (i.e., Sabouraud dextrose or nutrient broth), with or without shaking (100-150 rpm), at 37 • C for 5-10 days [119,120,122,123,136]. Growing P. insidiosum hyphae are harvested from the liquid culture by discarding the fluid [119] or filtrating through a membrane [123] to prepare cytoplasmic antigens. The remaining cell-free culture broth is collected for the preparation of exoantigens. The harvested P. insidiosum hyphae are ruptured to release the cytoplasmic antigens by using one of the following tools: cell homogenizer [123], sonicator [119,122,136], or vortex shaker [122]. The hyphal cell lysate (resuspended in water, saline, or phosphate-buffered saline pH 7.2) is centrifuged to collect the supernatant containing soluble cytoplasmic antigens [119,122,136,138]. As a part of exoantigen preparation, an appropriate amount of ether (i.e., an ether-to-broth ratio of 1:1) or acetone (i.e., an acetone-to-broth ratio of 1:1 or 2:1) is added to the cell-free culture broth to precipitate the extracellular proteins of P. insidiosum [120,123,136,138]. The precipitated exoantigens are collected by centrifugation.
Some investigators combined different antigens to formulate a new PIA. For example, Santurio et al. mixed the cytoplasmic antigens prepared by different methods (i.e., vortex and sonication) [122], whereas Mendoza et al. merged the cytoplasmic antigens and exoantigens extracted from the same isolate [136,138]. Inactivation of P. insidiosum, before or after the protein extraction, can be achieved using 0.02% thiomersal or 0.5% phenol [119,122,123,136]. Microbial contamination is checked by spreading 100 µL of the crude extracted proteins onto Sabouraud dextrose and blood agar plates before incubation at 37 • C for one week [119,123,136]. The obtained PIA can be kept at 4 • C for short-term storage (i.e., several weeks to months) or at freezing temperature (i.e., −21 or −80 • C) for a longer period [106,119,120,123,127,131,135,136]. Lyophilization can preserve the extracted antigens at room temperature for at least one year [122].

Clinical Application of Immunotherapy in Human Pythiosis
Primary clinical forms of human pythiosis include vascular pythiosis (an infection of a medium-to-large artery) and ocular pythiosis (an eye infection). A few patients come with cutaneous pythiosis (an infection of the skin) [43,147,148] or disseminated pythiosis (an infection of multiple organs) [12,43,60]. Surgical intervention is the primary treatment of pythiosis [13,61,106,109,127,132,149,150]. The therapeutic goal is to remove all infected tissues to achieve an organism-free surgical margin [13,90,106,109,151,152]. The first use of PIA immunotherapy in a human patient with pythiosis was reported in 1998 [107]. After being treated with PIA immunotherapy, this patient has recovered from the P. insidiosum infection of external and internal carotid arteries, where radical surgery is impossible. After excluding potentially-overlapping or clinical data-lacking cases (i.e., no treatment outcome), the literature search identified 108 patients with vascular pythiosis (Table 1),  35 patients with ocular pythiosis (Table 2), and 2 patients with a periorbital infection that received the PIA immunotherapy as an adjunctive treatment, together with antimicrobial drugs or surgery [2,7,13,43,90,[106][107][108][109]131,132,[149][150][151][152][153][154][155][156][157][158][159][160]. The PIA formulation used in these patients contained both extracellular and intracellular proteins of P. insidiosum [136][137][138]. The final clinical outcomes of these 145 PIA-immunized patients were "cured" in 84.1% of cases (with or without losing an affected organ; n = 122) and "dead" in 15.9% of cases (n = 23).    The P. insidiosum infection usually progresses along an affected artery from the distal to the proximal part of the leg. Pythiosis involving the aorta and iliac artery possessed a remarkably high mortality rate of 88.2% in the patients without PIA immunotherapy and a relatively lower rate of 61.9% in the patients with such treatment ( Table 3). Regardless of PIA immunotherapy, the mortality rate appeared to be lower (up to 15.2%) in the pythiosis patients with an infection of a lower-level artery, such as femoral, popliteal, tibial, peroneal, and dorsalis pedis arteries. Six PIA-immunized patients survived vascular pythiosis without leg amputations [13,106,107,131] (Table 3).
Concerning the disease duration, up to 2 months before treatment, the PIA-immunized and PIA-unimmunized vascular pythiosis patients showed similar mortality rates (28.6% vs. 25.0%; Table 3). However, if the disease duration was longer than 2 months (described as chronic infection by Mendoza et al. [121]), the PIA-immunized patients had~17% lower mortality rate than the PIA-unimmunized cases (33.3% vs. 50.0%; Table 3). Thus, a longer disease duration could lead to a higher mortality rate, and PIA immunotherapy could notably reduce disease mortality in patients with a chronic P. insidiosum infection.
Because the PIA-immunized patients usually obtained a combination treatment (including surgery and antimicrobial agents), the sole immunotherapy efficacy against P. insidiosum cannot be evaluated directly. The PIA-immunized patients with a marked inflammatory reaction (i.e., local swelling, pruritus, and erythema at the injection site and regional lymphadenopathy) showed a more favorable treatment outcome than patients with minimal or no such reaction [106,107,131,157]. Several serum markers, such as β-D-glucan (BDG), anti-P. insidiosum antibodies, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP), have been used to monitor the clinical course of some vascular pythiosis patients [90,151,164]. A declining level of BDG [90,151], ESR [164], or CRP [164] links with a recovery condition in the course of pythiosis treatment. Lower serum BDG and higher anti-P. insidiosum antibodies are associated with a better prognosis [151].
Similar to vascular pythiosis, surgical intervention is also the primary option for treating ocular pythiosis [132,150,167,177,180,181,184,185]. Topical or systemic antimicrobial agents (including linezolid, azithromycin, itraconazole, terbinafine, amphotericin B, natamycin, moxifloxacin, and minocycline) were administered in the ocular cases [97,106,174,[180][181][182]186,187]. In addition, many ocular pythiosis patients underwent penetrating keratoplasty to save the affected eye [53,54,91,165,166,168,170,173,176,183]. Post-keratoplasty recurrent infection resulted in eye removal by evisceration or enucleation [66,91,150,176,184]. In addition to the surgical and medical treatments, PIA was administered to modulate the immune response in 35 out of 168 ocular pythiosis patients (20.8%; one of which had bilateral ocular infections) [1,106,127,132,150,180] and 2 out of 5 periorbital pythiosis patients (40.0%) [43,160] (Table 2). The affected eye was removed in 17 out of 35 PIA-immunized (48.6%) and 47 out of 133 PIA-unimmunized (35.3%) patients (Table 2). Regarding the patients with periorbital pythiosis (n = 5), one of the two cases with, and all three cases without, PIA immunotherapy survived the disease. One each of the ocular and periorbital pythiosis patients, who obtained PIA immunotherapy, died due to an invasive infection [1,43]. Based on these reports, the PIA immunotherapy may not provide an advantage in a local P. insidiosum infection of the eye.
Administration of the PIA immunotherapy for pythiosis patients (i.e., antigen concentration, injection site, number of doses, and duration between shots) can differ based on the clinician's judgment and PIA availability. The immunotherapeutic PIA is generally prepared to the final concentration of 2 mg/mL [90,106,107,127,131,132,151,180]. Either 0.1-0.2 mL [43,106,107,131,132] or 1.0 mL [90,106,109,151,180] of PIA per dose is injected subcutaneously at least 2 times: initial and subsequent time points (i.e., 0.5, 1, 1.5, 3, 6, and 12 months) [13,43,61,106,107,109,127,131,132,149,151,180]. In some cases, the first PIA dose was provided intradermally [106,131]. Several patients with an aggressive P. insidiosum infection obtained a PIA injection once a week up to 7 weeks [7,106,156]. Sermsathanasawadi et al. describe the immunotherapy outcome of some patients with vascular pythiosis and recommend to provide an affected patient 3 PIA injections at days 0, 7, and 21 [109].
Regardless of the clinical condition and management, the horses receiving PIA immunotherapy showed a slightly lower cure rate than those not receiving such treatment (69.9% vs. 73.5%) ( Table 5). In contrast, the PIA-immunized dogs exhibited a markedly higher cure rate than the PIA-unimmunized cases (40.0% vs. 22.7%) ( Table 5). Some animal species (i.e., sheep, camel, cat, and donkey), those that appeared to be less affected by P. insidiosum, had a relatively low favorable response (from none to 33.3%) to PIA immunotherapy. From 130 recruited cows with pythiosis, only 2 were treated with PIA, resulting in 1 cured case (Table 5). Notably, most infected cows (127 from 128 cases; 99.2%) were spontaneously recovered from pythiosis, suggesting a potent host immunity against P. insidiosum generated in this particular animal species. A question has arisen concerning the immunotherapy efficacy since the overall cure rate for the infected animals who received PIA (as a part of their treatments) was lower than those who did not receive the antigen: 65.2% (176 in 270 cases) vs. 71.9% (271 in 377 cases) (Table 5). However, it is uncertain whether the clinical conditions of the affected animals with PIA immunotherapy were more severe than those without it, resulting in a relatively worse prognosis. Nevertheless, among the horses who were cured of pythiosis, it appears that the surgical interventions took place in 75.2% of the PIA-unimmunized cases. The rate of such interventions dropped to 22.8% in the PIA-immunized horses, suggesting PIA immunotherapy could reduce disease morbidity. As with human pythiosis, unless there is a case-control study, the favorable efficacy of PIA immunotherapy in animals cannot be directly assessed due to the different clinical settings among the cases, such as underlying condition, disease onset, severity, lesion size, pathologic location, and choices of treatment.

Proposed Mechanism of P. insidiosum Antigen-Based Immunotherapy
During the P. insidiosum infection, an antigen-presenting cell (APC), such as a dendritic cell, could process and present a pathogen antigenic epitope (via major histocompatibility complex or MHC) to a naïve T cell (through T-cell receptor or TCR). Such APC-T cell interaction induces differentiation and clonal proliferation of a naïve CD4+ T (Th0) cell to T helper-2 (Th2) cells [142,[242][243][244]. An elevated level of IL-4 is responsible for the differentiation and proliferation of Th2 cells, which, in turn, secretes IL-5 for activating eosinophils [131,142,245,246]. This process leads to the non-protective Th2-mediated immunity, where eosinophils are predominantly recruited, together with other cell types, such as mast cells, neutrophils, giant cells, and plasma cells, into the infection area [18,28,66,160,199,217,247]. The eosinophils surround the P. insidiosum hyphae inside necrotic tissues, producing the histological phenomenon called "Splendore-Hoeppli" [49,105,[248][249][250]. P. insidiosum might employ the accumulated eosinophilic materials as a protective shield against host immunity [122].
The PIA immunotherapy shows a favorable response in some humans and animals with pythiosis [46,106,[129][130][131]180]. The mechanism of PIA action in pythiosis treatment is not clearly understood. However, recovery of the PIA-immunized humans and animals from pythiosis is associated with switching Th2 to T helper-1 (Th1) mediated immunity [131,142,244,251,252]. An APC should process an immunomodulating antigen of P. insidiosum, such as (1,3)(1,6)-β-glucan, which is a main cell wall component [251,253,254], before priming a resulting antigenic epitope to a naïve T cell (via MHC-TCR complex) [242,243]. This cellular interaction might trigger the release of IFNγ and IL-2 for enhancing the differentiation of Th0 to Th1 cell and Th0 to regulatory T (Treg) cell, respectively [242,246,251,252,255,256]. The Th1 cell produces IFN-γ to activate the macrophages and cytotoxic T lymphocytes [131,142,242,244,246,251,253,256]. The PIA raises the T helper-17 (Th17) cells, which secrete IL-17A to recruit neutrophils and macrophages into the infected tissue [242,243,246,[251][252][253]. The PIA could also promote the release of the IL-10 cytokine, which relates to the anti-inflammatory and immunoregulation activity of Treg cells [242,246,[251][252][253]256]. In the rabbit model of pythiosis, the PIA elevates ecto-adenosine deaminase (E-ADA), which could stimulate the purinergic signaling system and promote the Th1-mediated immunity [257][258][259]. After PIA immunotherapy, histological findings at the infection site include the recruitment of lymphocytes, the gradual absence of eosinophils, and the reduced tissue burden of P. insidiosum hyphae [121,122,250,257]. The immunoglobulin E (IgE), which is increased during the P. insidiosum infection, is then decreased following the immunotherapy [121,131,244]. Taken together, the proposed P. insidiosum clearance mechanism of PIA immunotherapy is summarized in Figure 1 [131,142,242,244,251,253,259]. The in-depth mechanism of the immunomodulating PIA action in pythiosis treatment needs further investigation.
The PIA immunotherapy can cure many affected human and animal patients with pythiosis [13,106,149]. However, several pieces of evidence indicate that the host immunity induced by PIA could not prevent the P. insidiosum infection [46,121,122,144]. For example, Santurio et al. demonstrate no difference in the incidences of pythiosis between PIA-immunized and PIA-unimmunized horses [144]. Besides, several reports show that pythiosis can recur in recovered patients with or without PIA immunotherapy [45,123,125,188,206]. These findings suggest that the host immunity, induced by either immunotherapy or natural infection, may be inadequate in preventing another episode of P. insidiosum infection. Nevertheless, P. insidiosum reinfection could induce a stronger host immune response (i.e., a higher level of IgG antibodies) than the previous infection [206]. In patients with a persistent infection, the immunotherapy-induced antibody level could be affected by several factors, including host immune status, underlying diseases, and severity of the infection [2,106,201,204]. Although a high level of the anti-P. insidiosum antibodies generated by immunotherapy or natural infection is associated with the ability to eliminate the pathogen, the antibodies can be gradually decreased over time to a low or undetectable level [107,131,134,206], explaining the limited protective immunity against reinfection. lymphocytes, the gradual absence of eosinophils, and the reduced tissue burden of P. insidiosum hyphae [121,122,250,257]. The immunoglobulin E (IgE), which is increased during the P. insidiosum infection, is then decreased following the immunotherapy [121,131,244]. Taken together, the proposed P. insidiosum clearance mechanism of PIA immunotherapy is summarized in Figure 1 [131,142,242,244,251,253,259]. The in-depth mechanism of the immunomodulating PIA action in pythiosis treatment needs further investigation. Figure 1. Proposed mechanism of P. insidiosum antigen (PIA) immunotherapy. (A) P. insidiosum's zoospores (asexual stage) attach and germinate as hyphae into the host tissue during natural infection. Antigen-presenting cells (APC) process and present the P. insidiosum antigens to naïve T lymphocytes via major histocompatibility complex-antigen-T cell receptor complex (MHC-Ag-TCR). This interaction elevates some cytokines (mainly IL-4) to differentiate and clonally proliferate a naïve CD4+ T (Th0) to T helper-2 (Th2) cell, which, in turn, produces IL-4 and IL-5 to attract and activate eosinophils and mast cells. The eosinophils surround the P. insidiosum, creating the histological phenomenon called "Splendore-Hoeppli". (B) Processed antigens (prepared from the crude extract of P. insidiosum) lead to forming the MHC-Ag-TCR complex that induces the release of some cytokines, mainly IFN-ϒ and IL-2. IFN-ϒ promotes differentiation and clonal proliferation of a Th0 to T helper-1 (Th1) cell. Th1 cell-produced IFN-ϒ attracts macrophages and cytotoxic T lymphocytes (CTL) to the infection site for eliminating the pathogen. The MHC-Ag-TCR complex also facilitates the differentiation of a Th0 to T helper 17 (Th17) cell to produce IL-17A and accumulate more macrophages and neutrophils at the infection area. On the other hand, IL-2 promotes the differentiation of a Th0 to regulatory T (Treg) cell to regulate or suppress an excessive immune response through the release of IL-10. See the text for the details and the references to the proposed mechanism of PIA immunotherapy.
The PIA immunotherapy can cure many affected human and animal patients with pythiosis [13,106,149]. However, several pieces of evidence indicate that the host Figure 1. Proposed mechanism of P. insidiosum antigen (PIA) immunotherapy. (A) P. insidiosum's zoospores (asexual stage) attach and germinate as hyphae into the host tissue during natural infection. Antigen-presenting cells (APC) process and present the P. insidiosum antigens to naïve T lymphocytes via major histocompatibility complex-antigen-T cell receptor complex (MHC-Ag-TCR). This interaction elevates some cytokines (mainly IL-4) to differentiate and clonally proliferate a naïve CD4+ T (Th0) to T helper-2 (Th2) cell, which, in turn, produces IL-4 and IL-5 to attract and activate eosinophils and mast cells. The eosinophils surround the P. insidiosum, creating the histological phenomenon called "Splendore-Hoeppli". (B) Processed antigens (prepared from the crude extract of P. insidiosum) lead to forming the MHC-Ag-TCR complex that induces the release of some cytokines, mainly IFN-PIA immunotherapy, histological findings at the infection site include the recruitment of lymphocytes, the gradual absence of eosinophils, and the reduced tissue burden of P. insidiosum hyphae [121,122,250,257]. The immunoglobulin E (IgE), which is increased during the P. insidiosum infection, is then decreased following the immunotherapy [121,131,244]. Taken together, the proposed P. insidiosum clearance mechanism of PIA immunotherapy is summarized in Figure 1 [131,142,242,244,251,253,259]. The in-depth mechanism of the immunomodulating PIA action in pythiosis treatment needs further investigation. igure 1. Proposed mechanism of P. insidiosum antigen (PIA) immunotherapy. (A) P. insidiosum's zoospores (asexual stage) ttach and germinate as hyphae into the host tissue during natural infection. Antigen-presenting cells (APC) process and resent the P. insidiosum antigens to naïve T lymphocytes via major histocompatibility complex-antigen-T cell receptor omplex (MHC-Ag-TCR). This interaction elevates some cytokines (mainly IL-4) to differentiate and clonally proliferate a aïve CD4+ T (Th0) to T helper-2 (Th2) cell, which, in turn, produces IL-4 and IL-5 to attract and activate eosinophils and ast cells. The eosinophils surround the P. insidiosum, creating the histological phenomenon called "Splendore-Hoeppli". ) Processed antigens (prepared from the crude extract of P. insidiosum) lead to forming the MHC-Ag-TCR complex that duces the release of some cytokines, mainly IFN-ϒ and IL-2. IFN-ϒ promotes differentiation and clonal proliferation of Th0 to T helper-1 (Th1) cell. Th1 cell-produced IFN-ϒ attracts macrophages and cytotoxic T lymphocytes (CTL) to the fection site for eliminating the pathogen. The MHC-Ag-TCR complex also facilitates the differentiation of a Th0 to T elper 17 (Th17) cell to produce IL-17A and accumulate more macrophages and neutrophils at the infection area. On the ther hand, IL-2 promotes the differentiation of a Th0 to regulatory T (Treg) cell to regulate or suppress an excessive mune response through the release of IL-10. See the text for the details and the references to the proposed mechanism f PIA immunotherapy.
The PIA immunotherapy can cure many affected human and animal patients with pythiosis [13,106,149]. However, several pieces of evidence indicate that the host and IL-2. IFN-PIA immunotherapy, histological findings at the infection site include the recruitment of lymphocytes, the gradual absence of eosinophils, and the reduced tissue burden of P. insidiosum hyphae [121,122,250,257]. The immunoglobulin E (IgE), which is increased during the P. insidiosum infection, is then decreased following the immunotherapy [121,131,244]. Taken together, the proposed P. insidiosum clearance mechanism of PIA immunotherapy is summarized in Figure 1 [131,142,242,244,251,253,259]. The in-depth mechanism of the immunomodulating PIA action in pythiosis treatment needs further investigation. Figure 1. Proposed mechanism of P. insidiosum antigen (PIA) immunotherapy. (A) P. insidiosum's zoospores (asexual stage) attach and germinate as hyphae into the host tissue during natural infection. Antigen-presenting cells (APC) process and present the P. insidiosum antigens to naïve T lymphocytes via major histocompatibility complex-antigen-T cell receptor complex (MHC-Ag-TCR). This interaction elevates some cytokines (mainly IL-4) to differentiate and clonally proliferate a naïve CD4+ T (Th0) to T helper-2 (Th2) cell, which, in turn, produces IL-4 and IL-5 to attract and activate eosinophils and mast cells. The eosinophils surround the P. insidiosum, creating the histological phenomenon called "Splendore-Hoeppli". (B) Processed antigens (prepared from the crude extract of P. insidiosum) lead to forming the MHC-Ag-TCR complex that induces the release of some cytokines, mainly IFN-ϒ and IL-2. IFN-ϒ promotes differentiation and clonal proliferation of a Th0 to T helper-1 (Th1) cell. Th1 cell-produced IFN-ϒ attracts macrophages and cytotoxic T lymphocytes (CTL) to the infection site for eliminating the pathogen. The MHC-Ag-TCR complex also facilitates the differentiation of a Th0 to T helper 17 (Th17) cell to produce IL-17A and accumulate more macrophages and neutrophils at the infection area. On the other hand, IL-2 promotes the differentiation of a Th0 to regulatory T (Treg) cell to regulate or suppress an excessive immune response through the release of IL-10. See the text for the details and the references to the proposed mechanism of PIA immunotherapy.
The PIA immunotherapy can cure many affected human and animal patients with pythiosis [13,106,149]. However, several pieces of evidence indicate that the host promotes differentiation and clonal proliferation of a Th0 to T helper-1 (Th1) cell. Th1 cell-produced IFN-purinergic signaling system and promote the Th1-mediated immunity [257][258][259]. After PIA immunotherapy, histological findings at the infection site include the recruitment of lymphocytes, the gradual absence of eosinophils, and the reduced tissue burden of P. insidiosum hyphae [121,122,250,257]. The immunoglobulin E (IgE), which is increased during the P. insidiosum infection, is then decreased following the immunotherapy [121,131,244]. Taken together, the proposed P. insidiosum clearance mechanism of PIA immunotherapy is summarized in Figure 1 [131,142,242,244,251,253,259]. The in-depth mechanism of the immunomodulating PIA action in pythiosis treatment needs further investigation. Figure 1. Proposed mechanism of P. insidiosum antigen (PIA) immunotherapy. (A) P. insidiosum's zoospores (asexual stage) attach and germinate as hyphae into the host tissue during natural infection. Antigen-presenting cells (APC) process and present the P. insidiosum antigens to naïve T lymphocytes via major histocompatibility complex-antigen-T cell receptor complex (MHC-Ag-TCR). This interaction elevates some cytokines (mainly IL-4) to differentiate and clonally proliferate a naïve CD4+ T (Th0) to T helper-2 (Th2) cell, which, in turn, produces IL-4 and IL-5 to attract and activate eosinophils and mast cells. The eosinophils surround the P. insidiosum, creating the histological phenomenon called "Splendore-Hoeppli". (B) Processed antigens (prepared from the crude extract of P. insidiosum) lead to forming the MHC-Ag-TCR complex that induces the release of some cytokines, mainly IFN-ϒ and IL-2. IFN-ϒ promotes differentiation and clonal proliferation of a Th0 to T helper-1 (Th1) cell. Th1 cell-produced IFN-ϒ attracts macrophages and cytotoxic T lymphocytes (CTL) to the infection site for eliminating the pathogen. The MHC-Ag-TCR complex also facilitates the differentiation of a Th0 to T helper 17 (Th17) cell to produce IL-17A and accumulate more macrophages and neutrophils at the infection area. On the other hand, IL-2 promotes the differentiation of a Th0 to regulatory T (Treg) cell to regulate or suppress an excessive immune response through the release of IL-10. See the text for the details and the references to the proposed mechanism of PIA immunotherapy.
The PIA immunotherapy can cure many affected human and animal patients with pythiosis [13,106,149]. However, several pieces of evidence indicate that the host attracts macrophages and cytotoxic T lymphocytes (CTL) to the infection site for eliminating the pathogen. The MHC-Ag-TCR complex also facilitates the differentiation of a Th0 to T helper 17 (Th17) cell to produce IL-17A and accumulate more macrophages and neutrophils at the infection area. On the other hand, IL-2 promotes the differentiation of a Th0 to regulatory T (Treg) cell to regulate or suppress an excessive immune response through the release of IL-10. See the text for the details and the references to the proposed mechanism of PIA immunotherapy.

Future Perspective
Pythiosis has high mortality and morbidity. For decades several immunotherapeutic antigen formulations, prepared from crude extracts of P. insidiosum, have been used in the management of human and animal pythiosis [1,25,29,106,107,109,119,[129][130][131][132]149,180,[188][189][190]193,239,260]. However, a prophylactic approach to prevent the infection has yet to be developed. Clinical pieces of evidence show that the PIA immunotherapy can cure some, but not all, humans and animals with pythiosis [13,131,149,151]. The PIA immunotherapy, mostly in conjunction with surgery and antimicrobial drugs, demonstrates different clinical outcomes from study to study, likely depending on the severity of the P. insidiosum infection, host immune status, PIA formulations, the strain used for antigen preparation, and affected host species (i.e., human, horse, dog, cat, and cattle). Understanding how the PIA modulates the host immune responses to eliminate P. insidiosum could lead to developing more effective immunotherapy and, therefore, improving pythiosis patients' clinical outcomes.
Information on the efficacy of PIA immunotherapy against pythiosis has been obtained, based mainly on clinical observation of the affected patients. However, no case-control clinical trial study has been conducted to comprehensively evaluate the effectiveness of such a treatment modality, partly because pythiosis is a relatively rare disease. Therefore, a multicentric prospective clinical study should be performed to gain insights into the pharmacologic properties of PIA, such as efficacy, adverse effect, optimal dose, and antigen administration. The rabbit model of pythiosis has been established to investigate PIA immunotherapeutic properties [122,250,255,261]. Such an animal model shows atypical manifestations (i.e., cutaneous nodules) compared with pythiosis in the natural hosts (i.e., humans and various animals) [50,97,122,216,224,[262][263][264]. Besides, housing the experimental rabbits comes at a high cost, demanding space, facilities, and skilled personnel. These drawbacks impede the use of this model in the evaluation of PIA immunotherapy. On the other hand, the mouse is a well-studied model for the immunological study of many infectious diseases [265][266][267]. Reproduction of the P. insidiosum infection in mice is possible but requires pre-treatment with an immunosuppressive agent (i.e., cyclophosphamide) [251,268,269], making this versatile animal model less suitable for the PIA assessment. A murine model of Leishmaniasis (rather than pythiosis) has been developed to evaluate the immunomodulatory properties of PIA, but it does not demonstrate a direct immunological effect of PIA on P. insidiosum clearance [256]. Finding a clinically relevant, and cost-effective, animal model would advance our understanding of the properties of the immunotherapeutic PIA.
Because pythiosis has been increasingly reported worldwide, the disease has become a global healthcare concern. The causative agent, P. insidiosum, colonizes ubiquitously on a water plant in the environment [36,37,41]. Once an individual comes in contact with the organism in its habitat, the infection can be initiated, causing a difficult-to-treat disease. The pathological structure called "kunker" formed in P. insidiosum-infected animal tissue can give rise to a propagating organism upon exposure to water [42]. Outbreaks of pythiosis have been reported in animals [118,239,240,274] due to the interplay between humans, animals, plants, and their environment. One Health-based approach (as described by the Centers for Disease Control and Prevention; https://www.cdc.gov/onehealth/; accessed on 22 August 2021) should be incorporated into preventive and control measures to promote an optimal health outcome for patients with pythiosis. * CFA (culture filtrate antigens) represents extracellular proteins; SABH (soluble antigens from broken hyphae) represents intracellular proteins. ** Number and geographic distribution (countries) of P. insidiosum isolated from humans and animals with pythiosis and used to prepare CFA and SABH for Western blot and ELISA analyses. *** Number and geographic distribution (countries) of pythiosis sera, derived from humans and animals with pythiosis, and used for Western blot and ELISA analyses.
Pythiosis is a neglected tropical disease with high morbidity and mortality, in which clinical information on the disease and availability of the preventive, diagnostic, and therapeutic tools are limited. The disease can be considered a part of the Sustainable Developments Goals (SDGs) defined in the United Nations Development Program (https: //www.undp.org/sustainable-development-goals/; accessed on 22 August 2021). This review article presents the up-to-date information of the immunotherapeutic PIA for the proper use and future development of this treatment measure, aiming to promote good health and well-being of affected patients. The current formulation of PIA can mitigate morbidity and mortality in humans and animals with pythiosis by reducing surgical intervention and increasing the cure rate. However, more work needs to be done to improve the PIA efficacy in the prevention and treatment of pythiosis.

Conclusions
The fungus-like organism P. insidiosum causes pythiosis, a high morbidity and mortality disease, in humans and animals worldwide. As a part of the treatment, many pythiosis patients received the immunotherapeutic PIA. Only the PIA formulation containing the crude antigenic extracts of P. insidiosum has been clinically used over the past decades. Based on 967 documented human and animal pythiosis cases, PIA immunotherapy reduced disease morbidity and mortality. As the final clinical outcomes, 19.4% of PIA-immunized human patients succumbed to vascular pythiosis instead of 41.0% in unimmunized cases. PIA immunotherapy may not provide an advantage in a local P. insidiosum infection of the eye. Both PIA-immunized and unimmunized horses with pythiosis showed a similar survival rate of~70%; however, demands for surgical intervention were much lesser in the immunized cases who were cured (22.8% vs. 75.2%). A case-control clinical trial study should be conducted to evaluate the effectiveness of immunotherapy for pythiosis. The proposed mechanism of the PIA action involves switching the non-protective Th2 to curative Th1 mediated immunity against P. insidiosum. Finding a clinically relevant, and cost-effective, animal model of pythiosis is necessary to advance our understanding of the underlying mechanism and the required component of the immunotherapeutic PIA. By exploring the available P. insidiosum genome data, synthetic peptides, recombinant proteins, and nucleic acids are potential sources of the immunomodulating antigens worth investigating. The PIA therapeutic property needs improvement for a better prognosis of pythiosis patients.

Conflicts of Interest:
The authors declare no conflict of interest.