The Pathogenesis of Aspergillus fumigatus, Host Defense Mechanisms, and the Development of AFMP4 Antigen as a Vaccine

As a thermophilic fungus, A. fumigatus can grow at 55°C and survive at temperatures above 75°C. This ability facilitates to thrive in dead or decayed organic matters and to infect mammalian host cells. Thus, the genes related to thermotolerance contribute to the virulence of A. fumigatus. Five genes have been proved to be associated with the thermotolerance of A. fumigatus (thtA, cgrA, afpmt1, kre2/afmnt1, and hsp1/asp f 12). The thtA gene is necessary for the growth of A. fumigatus at 48°C, but it is not involved in the pathogenicity of A. fumigatus. The afpmt1 gene encodes for one mannosyl transferase, which is essential for the growth of A. fumigatus over 37°C. It was found that the Δafmnt1 mutant was attenuated in a mouse infection model and more sensitive to azoles (Wagener et al. 2008).

A. fumigatus produce toxins for protection against predators and competitors, and they can directly attack the host and contribute to the pathogenesis of the fungus. Many toxins are secondary metabolites of fungi. They can affect the synthesis of DNA, RNA, and proteins, or alter cell membrane and impair cellular functions. Many toxins and relevant genes of A. fumigatus have been studied, such as diffusible toxic substances from conidia, gliotoxin (gliP and gliZ), mitogillin (res/mitF/aspf1), hemolysin (aspHS), verruculogen, fumagillin, and the transcription factor laeA. Gliotoxin is the most potent toxin produced by A. fumigatus (Zhang et al. 2019), which can suppress macrophage phagocytosis, T cell proliferation, cytotoxic T cell response, and monocyte apoptosis (Schlam et al. 2016; Schmidt et al. 2017; Fraga-Silva et al. 2019). Gliotoxin can also inhibit the NADPH of neutrophils (Tsunawaki et al. 2004), suppress ROS production, and impair the neutrophil’s phagocytic capacity (Orciuolo et al. 2007). In addition, it should be noted that the transcription factor laeA is a crucial regulator for secondary metabolite biosynthesis (Pfannenstiel et al. 2017), and it has been proved that laeA deletion in A. fumigatus inhibited the production of almost all secondary metabolites containing gliotoxin (Arias et al. 2018).

Moreover, A. fumigatus can produce a large number of allergens; among them 23 have their official names ranging from Asp f1 to Asp f34 (available at: http://www.allergen.org/, updated on July 11, 2019). Some allergens show toxic or enzymatic activities, which are related to the virulence. Other allergenic molecules have no virulence functions. All Aspergillus allergens are likely to trigger a Type I hypersensitivity response in patients and induce a high-affinity IgE antibody production. Aspergillus allergens can cause hypersensitivities in immunocompetent patients, such as ABPA, allergic rhinosinusitis, asthma, and aspergilloma. In immunocompromised patients, these allergenic molecules can significantly increase the risk of aspergillosis. Many Aspergillus allergens have been explored and developed for diagnostic purposes (Masaki et al. 2017).

So far, none of the pathogenic factors is unique to A. fumigatus, and it is necessary to further investigate why A. fumigatus is more pathogenic than other common conditional pathogens. Some scholars believed that, unlike other human pathogenic bacteria such as Candida and Cryptococcus, the pathogenicity of A. fumigatus is not caused by one or several pathogenic factors but caused by the result of its unique biological characteristics such as growth and metabolism and the joint action of multiple pathogenic factors.

Recently, based on the homology between fungal endoether glucokinase and AnmK kinase of bacterial cell wall circulatory metabolism, we proposed the hypothesis that fungal cell wall has a mechanism similar to that of bacterial cell wall circulatory metabolism, which plays a role in the growth and reproduction of fungi. Whether this hypothesis is correct and related to the pathogenicity of A. fumigatus needs to be further confirmed by research.

Anatomical barriers. At the entry point of airborne conidia, the upper respiratory tract’s airway epithelium is the first defensive line of innate host immunity against A. fumigatus. As an airway epithelium cell, the mucous secreting cell can secrete mucus to trap inhaled conidia. Another airway epithelium cell, a ciliated cell, can drive the trapped conidia to the oropharyngeal junction (van de Veerdonk et al. 2017). In this way, a significant number of A. fumigatus are expulsed from the lung. The respiratory epithelium can also secrete some peptides or enzymes to combat A. fumigatus, indicating that chitinase produced by epithelium can damage chitin on the cell wall of A. fumigatus (Garth et al. 2018).

Professional phagocytes and classical signaling pathways. The dominant role of phagocytes defense against A. fumigatus in vivo and in vitro has been reported (Liu et al. 2017; Almeida et al. 2019; Mackel and Steele 2019). The primary phagocytes responsible for the phagocytosis of A. fumigatus are alveolar macrophages (AM) and neutrophils.

In the lung of the immunocompetent host, certain soluble recognition receptors produced by alveolar macrophages such as Pentraxin 3 (PTX3) and surface protein-D (SP-D) can immediately bind to the inhaled conidia of A. fumigatus, and enhance the phagocytosis of alveolar macrophages (Smole et al. 2020). Alveolar macrophages then recognize and swallow the conidia through the TLR2/4 and Dectin-1. Toll-like receptors (TLRs) are type I membrane receptors that function in recognition of PAMPs and an intracellular TLR domain required for downstream signaling. TLRs can recognize pathogens and activate transcription factors such as NF-κB, which mediate the expression of inflammatory cytokines and chemokines (Anthoney et al. 2018). Some studies implicated the membrane receptors TLR2 and TLR4 were the crucial recognition components for host defense against A. fumigatus (Dai et al. 2019). An essential role for TLR2 and TLR4 in cytokine production against A. fumigatus has been established in many in vitro studies (Briard et al. 2019; Gupta et al. 2019). Dectin-1 is a type II transmembrane protein, which is highly expressed in macrophages, neutrophils, and DCs. Dectin-1 can recognize β-glucan on germinating conidian but cannot identify the resting conidia, allowing macrophages to differentiate the various forms of A. fumigatus (Li et al. 2019; Dutta et al. 2020). Alveolar macrophages capture conidia leading to a proinflammatory response accompanied by the secretion of many cytokines and chemokines, including TNF-α and CXCL2 (Chemokine (C-X-C motif) ligand 2), which are essential activators for neutrophil recruitment (Guo et al. 2020). The conidia escaped from the phagocytosis by alveolar macrophages continue to germinate and spread. Proinflammatory factors derived by alveolar macrophages and epithelial cells recruit neutrophils to the infection site. Neutrophils perform an effective elimination of the germinating conidia and hyphae.

In the immunocompetent lung, conidia are immediately trapped by the soluble recognition receptors (PTX3, SP-D), promoting conidial phagocytosis by alveolar macrophage (AM). AM also captures conidia through TLRs and Dectin-1, leading to a proinflammatory response. The escaped conidia continue to germinate and penetrate through the alveolar epithelial cells. Neutrophils employ the processes of NET formation, degranulation, and lactoferrin production to inactivate germinating conidia and hyphae. Dendritic cells phagocytose, and process germinated conidia for antigens presentation to T cells, and finally activate an adaptive immune response to A. fumigatus (Garth and Steele 2017).

Mechanism of innate immune cells removing A. fumigatus. The mechanism includes phagocytosis, reactive oxygen species (ROS) generations mediated by nicotinamide adenine dinucleotide phosphate-oxidase (NADPH), lactoferrin production, and neutrophil extracellular traps (NETs) formation (Schoen et al. 2019; Souza et al. 2019; Shopova et al. 2020). ROS production responds to swollen conidia, but not resting conidia, through NADPH oxidase activation (Ferling et al. 2020; Khani et al. 2020). It has been demonstrated that the ROS-producing complex plays a crucial fungicidal role during A. fumigatus infection (Shen et al. 2016). NETs are networks of extracellular fibers, mainly composed of DNA from neutrophils. The NET formation is induced by a variety of proinflammatory mediators such as IL-8. It is significant for defense against large pathogens, such as hyphae of A. fumigatus (Li et al. 2020). It has been demonstrated both in vitro and in vivo, NET formation is dependent on NADPH oxidase and ROS generation (Khan et al. 2019; Ravindran et al. 2019). In addition, dendritic cells (DCs) play a well-established role in the host defense against A. fumigatus. Immature DCs can phagocytose conidia and hyphae through PRRs and present the processed antigens of A. fumigatus to host T cells, leading to the activation of adaptive immune responses (Wang et al. 2017).

The elimination of the daily-inhaled conidia mainly depends on the innate immune response, but the treatment for serious Aspergillus infections relies on the cooperation of the adaptive immune system, which responds to the signaling generated by innate immunity. Lymphocytes T and B represent the two main parts of the adaptive defensive system.

Role of T cells in adaptive immunity. The T cell immunity system interacts with the innate immune response in many ways. For instance, DCs recruit at the infection sites can load and migrate the A. fumigatus antigen to lymph nodes, leading to T lymphocytes’ activation (Wang et al. 2017). CD4+ T cells are the dominating organizer, which play a major role in antifungal immunity. These activated T cells are able to invoke phagocytes or restrict the immune response. The initiating of CD4+ T cell immunity occurred between TCR and its cognate antigens on the DC cells. The excessive inflammatory response secreted by the activated T cells can induce the naïve T cells to differentiate into CD4 T help (Th) subsets while damaging the incident tissues and contributing to the invasive infections of A. fumigatus. Th1 response was the predominant cell type that involved the protective immune response to the host through the production of pro-inflammatory cytokines such as IFN-γ, IL-2, IL-12, and TNF-α. In contrast, Th2 immune response was associated with the germination of fungal and exacerbation of disease as well as induced the alternatively activated macrophages in the defense against A. fumigatus. The balance between Th1 and Th2 subset determined the quality and outcome of host immune responses. It was also found that Th17 played multiple roles in clearing infections by participating in the production of proinflammatory genes and antimicrobial peptides, recruitment, and activation of neutrophils (Pathakumari et al. 2020). T cell immunity against fungal infections primes Th1 type response (Shenoy et al. 2017). However, in patients with aspergillosis, the predominance of Th2 T cells’ immune response was conducted to exacerbate disease (Dewi et al. 2017). Besides, DCs contribute to a damaging inflammatory response by stimulating the Th17 cells and producing IL-23 (Movahed et al. 2018).

Humoral immunity to A. fumigatus. The function of Aspergillus-specific antibodies in immunocompromised patients with IA has been investigated (Boniche et al. 2020). Early researches indicated that the antibody responses failed to provide effective protection against IA or played only a minor role in fighting aspergillosis (Cutler et al. 2007). However, recent research reported that β-1,3-glucan specific antibodies could not only inhibit Aspergillus hyphae but also protect CD2F1 mice against Aspergillus challenge (Matveev et al. 2019). Although it is generally acknowledged that T cells immunity plays a vital role in combating fungal aspergillosis (Diaz-Arevalo and Kalkum 2017), there are a variety of ways, such as opsonization, complement activation, and virulence factors neutralization, in which antibodies affect T cells response and suppress the growth, adherence, and germination of fungi (Liedke et al. 2017; Ulrich and Ebel 2020).

Although recently there is some progress published on the study of vaccines against A. fumigatus (Chauvin et al. 2019; Khani et al. 2020), several significant obstacles remain to be overcome for producing effective vaccine (Levitz 2017). First of all, since A. fumigatus is an opportunistic pathogen and most invasive infections appear in the immunocompromised population, the induction of an effective adaptive immunity in such individuals is a real challenge. One of the feasible measures is the prophylactic vaccination, especially in target population, such as the patients waiting for bone marrow transplant and the patients before the treatment with immunosuppressive agents. Another major problem in developing a vaccine against A. fumigatus is related to the fungus’ molecular complexity. The extract of A. fumigatus is a mixture containing up to 200 different proteins, glycoproteins, and compounds of low molecular weights. In addition, safety issues should also be well addressed because a wide range of allergic diseases correlates with Aspergillus allergens. The potential of activating an adverse immunoreaction is also an intractable problem (Dewi et al. 2017).

Currently, some A. fumigatus allergens were identified based on their reactivity to patients’ antibodies. Some of these allergens are not effective antigens for vaccine development because they primarily induce Th2 cell response but cannot provide adequate protection against Aspergillus infection. Nevertheless, a recombinant allergen of A. fumigatus – Aspf3 was confirmed to induce protective response when presented mixed with TM adjuvant in a murine inhalation model (Namvar et al. 2015). It was also reported that various yeast genera could activate innate CD8+ T lymphocyte response and generate broad antifungal protection (Bazan et al. 2018). It suggested yeasts as a potential antifungal vaccine candidate to activate antigen-specific CD8+ T cell responses effectively.

For a long time, most antifungal vaccine research intended to activate memory T lymphocytes and raise a Th1 type immune response that would produce some favorable cytokines to enhance phagocytosis or T cell killing (Upadhya et al. 2016). Most studies suggested that the protective antifungal reactions induced by vaccines are cell-mediated immune responses. However, some recent investigations focused on protective antibodies. One β-glucan conjugate vaccine was shown to provide good protection against Candida albicans and A. fumigatus (Catellani et al. 2020). The serum of vaccinated mice significantly inhibited the growth of fungus hyphae. The mechanism of the β-glucan conjugate vaccine might involve specific anti-β-glucan antibody (Matveev et al. 2019).