1.
Cryptococcal meningitis
The Cryptococcus neoformans and Cryptococcus gattii species complexes are the common causative agents of life-threatening cryptococcal meningitis (CM) among the immunocompromised, especially human immunodeficiency virus/ acquired immune deficiency syndrome (HIV/AIDS) patients, other immunosuppressed patients and to a small extent immunocompetent individuals [1],[2]. If available, timely diagnosis, specific treatment (immunotherapy), and vaccine can help convert the fatality recorded against CM in human populations. After many molecular studies, the C. neoformans/C. gattii species complex was divided into seven species haploids and four inter-species diploid/aneuploid hybrids. The species exhibit varying epidemiology, pathogenicity, and chemotherapeutic susceptibility, but all C. neoformans/C. gattii species complex members cause pneumonia and meningoencephalitis [3]. Generally, in this review, we use C. neoformans in the write-up and figures to refer to C. neoformans/C. gattii species complex.
Classification of the C. neoformans/C. gattii species complex summarized in Table 1 below were as follows, under C. neoformans we have C. neoformans sensu stricto formally serotype A (VNI/AFLP1 and VNII/VNB/AFLP1A, VNII/AFLP1B) and C. deneoformans (VNIV/AFLP2) previously serotype D. While the C. gattii had five species under it namely, C. gattii sensu lato (VGI/AFLP4: serotype B), C. bacillisporus (VGIII/AFLP5: serotype B & C), C. deuterogattii (VGII/AFLP6: serotype B), C. tetragattii (VGIV/AFLP7: serotype C), and C. decagattii (VGIV and VGIIIc/AFLP10: serotype B). Further, the diploid/aneuploid species hybrids between C. neoformans and C. gattii are C. neoformans × C. deneoformans hybrid (AFLP3/VNIII:serotype AD), C. deneoformans × C. gattii hybrid (AFLP8:serotype DB), C. neoformans × C. gattii hybrid (AFLP9:serotype AB) and C. neoformans × C. deuterogattii hybrid (AFLP11:serotype AB) [3]–[5].
The respiratory tract is the primary route of entry for Cryptococcus; the lungs and central nervous system (CNS) are the common sites of the invasion, causing a spectrum of infections from simple colonization to systemic/invasive fungemia and meningitis [6]. Figure 1 below outlines the pathogenesis of CM. Cryptococcal screening is recommended in HIV patients with a CD4 count of 100 cells/µL or less [7]. Conventionally, cryptococcosis is diagnosed in the laboratory using Indian ink staining, this procedure is retarded by reduced sensitivity. The fungal culture is considered the gold standard, however, a definitive diagnosis can be reached in one to two weeks. Serological detection of fungal capsular polysaccharides plays a significant role in CM diagnosis [8]. Enzyme immunoassay (EIA) and latex agglutination assays have been employed successfully in targeting the cryptococcal capsular antigen in body fluids to diagnose cryptococcosis [8],[9].
The immunochromatographic dipstick assay detects the cryptococcal antigen, principally glucuronoxylomannan (GXM), in serum by the fourth week of infection before meningism [10]. The cryptococcal capsular antigen (CrAg) lateral flow assay (LFA) being more sensitive than the CrAg latex detects 1 ng of CrAg/mL, while the latter detects 19 ng of CrAg/mL of the sample. The CrAg LFA employs simple technology that is convenient for mass production at affordable cost, for rapid testing, and for easy interpretation on a portable device for testing many parameters in a sample. Contrastingly, CrAg LFA has some setbacks as the chemical reporters can fail and the test is inappropriate for assays requiring high analytical sensitivity and reproducibility [11]. A study reported a false-negative serum CrAg LFA in a patient with disseminated cryptococcosis and positive C. neoformans blood culture result [12].
The cryptococcal antigen latex agglutination system (CALAS) is a quantitative/semiquantitative assay that detects cryptococcal polysaccharide capsular antigens. Despite CALAS accuracy in the diagnosis of CM using CSF, it had been substantially replaced by CrAg LFA due to the former's unfavorable requirement of laboratory expertise, long incubation, and interpretation dexterity. The FDA-approved Immuno-Mycologics (IMMY) CrAg LFA is considered superior among CrAg LFA platforms, however, needle prick-sample proved less sensitive compared to blood collected with a pipette and read after 20 minutes [13].
Other CrAg LFA available in the market include Dynamiker [14], CryptoPS (Biosynex) [15], StrongStep (Liming Bio) [16], and FungiXpert cryptococcal capsular polysaccharide K-Set (Genobio) [17]. Scientists have reported extensively on the properties of these CrAg LFA tests and their comparison with IMMY or CALAS [15],[16],[18]–[20]. Recently, researchers compared the four CrAg LFA certified in Europe and reported that IMMY and FungiXpert LFAs were able to detect all the species in the C. neoformans/gattii complex, however, Dynamiker and CryptoPS failed to diagnose C. tetragattii and C. bacillisporus, also C. deuterogattii is a blind spot on CrptoPS. Importantly C. tetragattii is prevalent in Subsaharan Africa and the Indo-Pakistani subcontinent; accounting for one-fifth of CM among HIV/AIDs patients [21].
2.
The GXM
The C. neoformans polysaccharide capsule (CNPS); principal virulent factor, salient serological target, and critical immune evasion factor that is shed in culture and body fluids is composed of GXM (90%), galactoxylomannan (GalXM)—recently reviewed as GXMGal, and mannoproteins [22]. The intact capsular and the secreted GXM are both utilized for cryptococcal studies [23]. However, the duo possesses distinct chemical, physical, and antigenic properties [24]. The GXM is common to all the species in the Cryptococcus neoformans/gattii complex [25].
In the early 1990s, advancement was recorded in the identification of the GXM part of CNPS, which is specific for C. neoformans as such murine monoclonal antibodies were produced and characterized, which showed the upper hand in the diagnosis. Moreover, studies to uncover the role of anti-GXM for possible immunotherapy of C. neoformans commenced in pari-passu [26]. The cryptococcal GXM, unlike proteinaceous antigens, provoked an inadequate immune response. Previous studies coupled GXM to carrier proteins and use adjuvants to boost humoral immune response [27],[28].
2.1. GXM structure
The component carbohydrate residues of GXM are mannose, xylose, and glucuronic acid in decreasing order. The mannan moieties provide GXM backbone through their lineal linkage to each other with α-(1→3) bond, while two xylose molecules occupy positions β-(1→2) and β-(1→4), and a glucuronic acid moiety takes up position β-(1→2) of the mannose molecule forming the octasaccharide structure of GXM as shown in Figure 2 below [29]. The GXM mannose backbone is substantially acylated at position 6 [30]. The GXM polysaccharide expressed on C. neoformans exercises six motifs triad, present in different motif combinations in any given GXM; a complexity superior to bacterial single oligosaccharide motif [31].
The C. neoformans sensu stricto (serotype A) dominant triad has a six-residue repeat unit bearing two xylose side chains at β-(1→2) [32]. Glucuronic acid position confers structural variability of GXM having different motifs within the same isogenic C. neoformans phenotypes, yielding considerable structural and antigenic variations [33]. GXM provides the framework for the serotype classification of Cryptococcus, based on structural diversification of the GXM molecule. As each GXM possesses six differing triads (motifs) of oligosaccharides that account for its diversity making the molecule highly heterogeneous [32].
2.2. Immune response to GXM
The cryptococcal GXM is tolerogenic, poorly immunogenic, and provoked T cell-independent humoral immune response in humans and experimental animals [28],[34]. Only 40% of CM patients produce detectable antibodies against cryptococcal polysaccharide capsular antigens [35]. The dendritic cells (DCs) recognize the C. neoformans GXM using toll-like receptors (TLR)-2 and TLR4, leading to the fungal agent's phagocytosis with no role in cytotoxicity. Bedsides, the protective immune response follows recognition of GXM from ingested fungus by nod-like receptors (NLR) family pyrin domain containing 3 (NLRP3) that promote expression of IL-1β [36], as depicted in Figure 3b below. TLR4 is essential for GXM recognition and subsequent activation of macrophages [37].
Other pattern recognition receptors utilized by monocyte-derived macrophages to recognize GXM for subsequent uptake include CD14, CD18, and FcγRII, in addition to TLR2 and TLR4. Exposure of these macrophages to GXM demonstrates the immune system's downregulation through decreased expression of MHC I and MHC II and increased secretion of IL-10 which is counterproductive to cryptococcal immunity, as shown in Figure 3a below [38]. Phagocytic cells: macrophages and neutrophils form the first-line innate defense against cryptococcosis [39].
The proinflammatory Th1 subtypes of CD4+ T cells that produce IFNγ as its signature cytokine was shown to be a key player in immunity to cryptococcosis, through its ability to polarize macrophages to M1 phenotype: fungicidal [40],[41]. This fact was further supported by a higher prevalence of CM among HIV/AIDs patients with severely low CD4 cell count [41],[42], a similar phenomenon was observed in idiopathic CD4 lymphocytopenia [43]. CD4+ T cells are essential for cryptococcal clearance, a property that was shown to be transferred in the murine study, in addition to macrophages and neutrophils attraction [43]. In a murine study, it was reported that the presence of CD4+ or CD8+ T cells is indispensable for cryptococcal immunity [44]. Another study found IL-17A secreted by tissue-resident memory T cells to be a requisite towards immunity to cryptococcal infection in the study mice [34]. To further showcase the significance of Th1 and Th17 CD4+ T cells, scientists reported consistently low levels of IFNγ and IL-17 secreting cells among HIV/AIDs patients with CM complicated by immune reconstitution inflammatory syndrome (IRIS) [45]. IFNγ, IL-17A and TNFα production by CD4+ T cells function in immune cells chemotaxis and granuloma formation at the local site of cryptococcal infection; hence IL-17A(−/−) mice failed to form granuloma after dendritic cell vaccination for cryptococcal infection [34]. In sum, cell-mediated immunity through Th1 and M1 macrophages, in addition to humoral immunity with associated complement activation are salient towards cryptococcal elimination [46].
In an in vitro study, Rocha and coworkers reported that C. neoformans GXM blocks the release of neutrophil extracellular traps (NETs) and inhibits the generation of reactive oxygen species (ROS) needed for fungal killing by human neutrophils [47]. O-acetyl groups present in GXM are essential to immune evasion by the Cryptococcus [30]. Increased serum levels of GXM in patients with CM were found to be associated with monocyte anergy: reduced monocytes expression of HLA-DR and TNF-α, reduce Th1 response, increase expression of IL-6 and IP-10, and increase neutrophils count. Cumulatively, labeled as a mortal immune signature in CM patients [48]. Cell-mediated immunity through the expression of Th1 cytokines is a prerequisite to anti-cryptococcal immunity, while CD8+ T cells provide anti-cryptococcal immunity in the absence of CD4+ T cells [49]. GXM Immune evasion to phagocytes was outlined in Figure 4 below.
Scientists found a direct association between a low serum GXM IgM and the development of cryptococcosis-associated immune reconstitution inflammatory syndrome (C-IRIS) among patients with CM. Further, GXM-specific IgM deficiency was shown to be associated with disseminated cryptococcosis among the study mice [50]. No association was found between C-IRIS and B cell populations [51]. Mice immunization with GXM conjugated to protein carrier yields different monoclonal antibodies (mAbs), some with protective properties, others with non-protective specificity [52]. GXM-specific IgM was shown to confer protective immunity to cryptococcosis [53].
The C. neoformans express GXM of varying molecular masses that were recognized by murine bone marrow-derived macrophages, but have a differing effect on the expression of IL-10, IL-6, TNF-α, and regulated upon activation, normal T cell expressed and secreted (RANTES:CCL5) [54]. The CSF secretion of GXM is positively correlated with levels of IL-6, IL-7, IL-8, and TNF-α expression among patients with CM [55]. Extracellular vesicles containing GXM and sterylglucosides were shown to protect against secondary cryptococcosis [56]. C. neoformans display varying cell morphology types during infection to evade the host's immune system [39].
A complex of GXM and GaLXM: glucuronoxylomanogalactan (GXMGal) released during infection evades the immune system through apoptosis induction on immune cells, thereby conferring fungal survival within the host [57],[58]. Hence, the characteristic cytopenia observed in CM, further compounds immunosuppression among the patients [59]. GXM is antiphagocytic and resists respiratory burst stress [22],[57]. Excessive production of GXM was directly related to decreasing survival of severe combined immunodeficiency (SCID) mice models infected with C. neoformans [60].
A clinical study reported a direct relationship between C. neoformans that produced large capsules in vivo with raised intracranial pressure (ICP), inadequate CNS immune response, reduced cryptococcal clearance, dwindle expression of pro-inflammatory cytokines, and low leukocyte count among patients with CM [61]. The GXM possesses several immunomodulatory effects during cryptococcal infections, which include increasing Fas Ligand expression, subduing LPS-induced signaling, blocking T cell proliferation, immune cells apoptosis induction, and interference of antigen presentation by professional antigen-presenting cells (pAPCs), cumulatively favoring cryptococcal survival [62]. Furthermore, GXM disrupts the healthy balance of pro-inflammatory to anti-inflammatory cytokines, facilitating anti-inflammatory cytokines expression in the presence of Cryptococcus pathogen [63].
2.3. GXM: diagnostic target
GXM is an essential component of Cryptococcus that is required for survival, pathogenicity, and immune evasion. GXM is continuously shed in the serum and CSF during infection. The body fluid level of GXM correlates directly with a fungal burden in CM [64]. As a formidable cryptococcal biomarker, scientists target GXM to diagnose cryptococcosis in the last four decades. Of note, low immunogenicity inherent to polysaccharide antigens seems to stand in the way of developing anti-GXM for immunoassays. So, Cryptococcal serological diagnostic immunoassays evolve in specificity from targeting the whole CNPS, narrowing it down to GXM [65]. Others developed polyclonal or monoclonal antibodies by coupling GXM to carrier proteins, to boost the polysaccharide's immune response [34],[66],[67].
Scientists describe a monoclonal antibody of IgG1 subclass to CNPS after immunizing mice with extracted soluble capsular polysaccharide of C. neoformans serotype A, obtained through ultrafiltration of grown culture. The discovered IgG1 cross-reacts mildly with other C. neoformans serotypes and Trichosporon beigelii. The anti-CNPS also successfully recognized the GXM of C. neoformans [68]. Devi and colleagues discover a superior monoclonal antibody response secondary to immunization of laboratory mice with a vaccine they synthesize from conjugation of +GXM to tetanus toxoid (TT) [28]. The same study's highest antibody response for both IgG and IgM in the mice was recorded when the initial immunization dose was followed by monophosphoryl lipid A (MPL) adjuvant injection on the second day [28]. In another study, the GXM was precipitated with cetyltrimethylammonium bromide (CTBA) and conjugated to TT using adipic acid as a spacer; the vaccine formed was then used for intraperitoneal immunization of Balb/c mice yielding monoclonal antibodies specific for all the four serotypes of C. neoformans [69].
2.4. GXM: a therapeutic target
The emergence of widespread antibiotic resistance has pushed the scientific world to search for alternative therapies for infectious diseases, which are affordable and effective, especially those that stimulate appropriate host responses [70]. The well-studied anti-GXM (18B7) was shown to hydrolyze the fungal oligosaccharide and increase GXM release from Cryptococcus [71], a therapeutic potential that needed to be elaborated. Interestingly, another study uncovered a mechanism by which GXM activates macrophages through TLR4 signaling to secrete pro-inflammatory cytokines providing protective immunity to the host [37].
Researchers reported promising preliminary studies of CD8+T cells expressing GXM-specific chimeric antigen receptor (GXM-CAR) as a form of immunotherapeutic modality. Treatment of immunocompromised NSG mice infected with C. neoformans yielded a significantly lower fungal burden than the control [72]. The CD40L expressed by activated T cells interacts with CD40 molecules expressed by myeloid cells for successful co-stimulation that is tightly linked to NF-κβ activation: proinflammatory response. Hence, CD40L/CD40 immune checkpoint provides an interesting target for immunotherapy. [73]. The mutation of CD40L in humans and CD40L(−/−) among mice both leads to disseminated cryptococcosis, emphasizing the critical role of Th1 cells in cryptococcal immunity. Again combination therapy of CD40L and IL-2 in systemic murine cryptococcosis reveals extended survival of the study mice compared to the control [46]. Recombinant IFNγ therapy in a clinical study yielded a promising result, however, limited by side effects, and as such advocated as an adjuvant for systemic cryptococcal therapy [46],[74].
Radiolabeled anti-GXM therapy shows promising results [75]. Radioimmunotherapy was conducted using 18B7 mAb labeled with alpha emitter bismuth-213 (213Bi) or a powerful beta emitter rhenium-188 (188Re) to target GXM in a mice model of C. neoformans infection. 18B7-213Bi and 18B7-188Re radiolabeled mAbs extend mice survival by 60% and 40% on the eleventh week of therapy, respectively [76]. Scytovirin, a lectin from cyanobacteria, has activity against mannose molecules found on microbial agents; has antiviral and antifungal effects. Scytovirin proves promising in inhibiting C. neoformans with lesser effect than C. gattii [77].
A glycopeptide isolated from sheep red blood cells (SRBCs) membrane termed T11TS was a CD2 receptor agonist, promoting T cell proliferation, activation, and survival [78]. In a rat model of CM, T11TS therapy facilitates fungal recognition through upregulated TLRs expression, increases antigen presentation by microglial cells, and promotes proinflammatory cytokines secretion, in sum promoting fungal immunity [79]. T11TS immunotherapy in a rodent model of C. neoformans infection was shown to induce nuclear retention of NFAT, a mechanism that causes upregulation of IL-2 expression [80]. Similarly, T11TS counters molecular mechanisms of C. neoformans immune evasion and facilitates immune recognition and effector immune response against the microbe, which cumulatively, favor C. neoformans clearance from the study rats [81]. Figure 5 summarizes various immunotherapeutics that target GXM discussed above.
2.5. GXM: vaccine target
Researchers respond exponentially to the dire need to develop a vaccine for cryptococcosis, considering the morbidity and mortality associated with the systemic disease, which worsened with the advent of HIV infection. The cryptococcal vaccine is intended to protect individuals prone to severe cryptococcosis due to genetic predisposition or compromised immune system. Initial cryptococcal vaccines explore the immunization modality from the first principle perspective: using live attenuated and heat-killed Cryptococcus protocols [49]. A preclinical immunization study was conducted to compare the immune response between heat-killed C. neoformans and culture filtrate of C. neoformans. The culture filtrate immunization gives way to the robust immune system that is CD4+ T cell-dependent, associated with copious secretion of IFN-γ and IL-2, optimal fungal clearance, and increased mice survival. However, no anti-GXM was discovered in the studied mice sera. Of note, the heat-killed vaccine displayed an indifferent immune response [82]. Th17 polarization is a prerequisite to vaccine activity against cryptococcal pathogens [83]. Further, the lack of IFNγ, IL-17, and TNFα cytokines production in the study mice cumulatively lead to cryptococcal vaccine failure [34], which signifies the role of CD4+ T cells in cryptococcal immunity.
GXM conjugated to tetanus toxoid (GXM-TT) used to immunize mice in the presence of monophosphoryl lipid A (MPL) as an adjuvant, recorded promising protection against systemic cryptococcosis through antibodies induction [84]. Researchers studied GXM mimotope's immunogenicity: P13 conjugated to either TT (P13-TT) or bovine serum albumin (P13-BSA) adjuvanted with aluminum hydroxide. Both vaccines yielded IgM and IgG to GXM and P13 that confer mice survival with decreased serum levels of GXM than controls [85]. Another study of P13 on chronically infected mice prolongs the study animals' survival through immunomodulation by IL-10 expression. However, the protective immune response recorded depends on the conjugate protein type, mice strain, and infection route [86].
Scientists cultured bone marrow DCs with a heat-killed clinical isolate of C. gattii species complex (C. bacillisporus:VGIII/AFLP5) and transferred them to mice, which were later infected with the clinical isolate. They reported prominent Th1 and Th17 cytokines expression that lowered the fungal load, decreased the lung GXM levels, improved the associated pathology, and increased mice survival [87]. Pulsed DC vaccines of C. gattii induce IL-17A secreting resident memory Th17 cells in the lung, which also potentiate the activity of neutrophils and giant immune cells in the study animals. The increased mice survival and the decreased fungal levels are deficient in IL-17A knock-out mice [88]. Other cryptococcal antigenic compounds (mannoprotein and MP98), synthetic cryptococcal proteins, GXM peptide mimetic, and fungal extract are being extensively studied to provide potent and safe vaccines [49] not covered in this review. Besides, with ongoing studies, it is hoped that there will be light at the end of the tunnel in the treatment and prevention of CM. Figure 6 below summarized the salient role played by GXM in immunity, pathogenicity, and vaccine development in cryptococcal infection.
3.
Conclusions
The global morbidity and mortality indices associated with CM, especially among HIV/AIDs patients, further complicated by widespread antifungal resistance, translate into the pressing need for immunotherapy and vaccine to curtail this severe infection's menace. Of note, C. neoformans evolves escape mechanisms to evade the immune system and persist in a strong or weak immune response. Both immunotherapy and vaccine development are still ongoing, and substantial literature targets GXM as a promising biomarker.