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Pore-forming alpha-hemolysin efficiently improves the immunogenicity and protective efficacy of protein antigens

['Jin-Tao Zou', 'National Engineering Research Center Of Immunological Products', 'Department Of Microbiology', 'Biochemical Pharmacy', 'College Of Pharmacy', 'Third Military Medical University', 'Chongqing', 'Pr China', 'Hai-Ming Jing', 'Yue Yuan']

Date: None

Highly immunogenic exotoxins are used as carrier proteins because they efficiently improve the immunogenicity of polysaccharides. However, their efficiency with protein antigens remains unclear. In the current study, the candidate antigen PA0833 from Pseudomonas aeruginosa was fused to the α-hemolysin mutant Hla H35A from Staphylococcus aureus to form a Hla H35A -PA0833 fusion protein (HPF). Immunization with HPF resulted in increased PA0833-specific antibody titers, higher protective efficacy, and decreased bacterial burden and pro-inflammatory cytokine secretion compared with PA0833 immunization alone. Using fluorescently labeled antigens to track antigen uptake and delivery, we found that Hla H35A fusion significantly improved antigen uptake in injected muscles and antigen delivery to draining lymph nodes. Both in vivo and in vitro studies demonstrated that the increased antigen uptake after immunization with HPF was mainly due to monocyte- and macrophage-dependent macropinocytosis, which was probably the result of HPF binding to ADAM10, the Hla host receptor. Furthermore, a transcriptome analysis showed that several immune signaling pathways were activated by HPF, shedding light on the mechanism whereby Hla H35A fusion improves immunogenicity. Finally, the improvement in immunogenicity by Hla H35A fusion was also confirmed with two other antigens, GlnH from Klebsiella pneumoniae and the model antigen OVA, indicating that Hla H35A could serve as a universal carrier protein to improve the immunogenicity of protein antigens.

Pore-forming toxins, a kind of exotoxins utilized by many pathogens as immune escaping weapons that targeting the immune cells and disturbing the immune system, are conventionally deemed as perfect antigens for vaccine development against infectious diseases. In this study, we reported that fusion of Hla H35A , a typical pore-forming toxin toxoid, to candidate protein antigens from different species resulted in improved immunogenicity and protective efficacy. The improvement was mainly due to the increased antigen uptake and activating of immune-associated signaling pathways, probably by targeting ADAM10, the receptor of Hla on host immune cells. The importance of this work was to demonstrate the possible mechanisms of that pore-forming toxin function as atypical carrier protein to improve the immunogenicity of other proteins and confirm the potential of non-conservative but highly immunogenic exotoxins derived from hyper-virulent clinical strains for application in rational antigen design and vaccines development.

Funding: This work was funded by the National Natural Science Foundation of China (grant number 31970138) to JYZ. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability: Raw data files for RNA-seq have been deposited in the NCBI Gene Expression Omnibus under accession number GEO: GSE167077. All other relevant data are within the manuscript and its Supporting Information files.

Copyright: © 2021 Zou et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

In this study, to avoid the interference in immune protection of antigens from the same species, PA0833 from Pseudomonas aeruginosa (P. aeruginosa), which we previously identified as an effective candidate antigen [ 17 ], was chosen to construct a fusion protein with Hla H35A . The immunogenicity and protective efficacy of the Hla H35A -PA0833 fusion protein, termed HPF, were evaluated in a P. aeruginosa lethal pneumonia model. The possible mechanism of action was assessed in in vivo and in vitro experiments. Finally, the universal efficacy of Hla H35A as a carrier protein for antigen design was determined using the model antigen ovalbumin (OVA), and a newly identified vaccine candidate, GlnH, from Klebsiella pneumoniae (K. pneumoniae).

Pore-forming toxins (PFTs) are produced by numerous pathogenic bacteria to promote their growth and dissemination [ 8 ]. Alpha hemolysin (Hla) is a member of the PFT family secreted by Staphylococcus aureus (S. aureus), and harbors the ability to activate the NLRP3 inflammasome, which subsequently increases IL-1β and IL-18 secretion by activating procaspase-1 [ 9 ]. Hla has been identified as a critical virulence factor in a murine model of S. aureus infection, and inflammation was observed in the lungs of rats and rabbits treated with this protein [ 10 , 11 ]. Hla targets alveolar epithelial cells via the protein receptor, a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) [ 12 ], and the lipid receptor phosphocholine [ 13 ]. Hla has been tested for use in numerous vaccines against S. aureus. Inactivated Hla forms, including Hla H35A , Hla H35L [ 14 ], and Hla AT62 [ 15 ], which lack the transmembrane domain, showed no hemolytic capacity but strong antigenicity. Immunization with serotype 5 capsular polysaccharide conjugated to Hla H35L reduced the dissemination of S. aureus into the blood in a murine model [ 16 ]. However, to the best of our knowledge, few studies have focused on the impact of Hla toxoid fusion on the immunogenicity of other proteins, and the underlying mechanisms whereby Hla improves immunogenicity remain unclear.

Antigens are the most important components within a vaccine, and immunogenic antigens are crucial for the development of a successful vaccine [ 3 ]. Pure proteins or polysaccharides are generally not well recognized by the immune system, and require the addition of carrier systems or adjuvants to improve immunogenicity. Numerous substances with acceptable adverse effects have been tested to improve the immunogenicity of proteins or polysaccharides, but few of them have been clinically approved as adjuvants [ 4 , 5 ]. Among them, alum has been the most successful and widely used adjuvant during the past century [ 6 ]. The mechanisms whereby alum improves immunogenicity include, but are not limited to, antigen uptake, antigen depot, and pyrin domain containing 3 protein (NLRP3) inflammasome activation [ 7 ]. The identification of substances with adjuvant activity and the elucidation of their mechanism of action are essential for vaccine development.

Antibiotic-resistant infections have become an urgent global threat to public health [ 1 ]. In 2017, the World Health Organization (WHO) released a list of drug-resistant bacteria, which consists of 12 ‘priority pathogens’ that pose the greatest threat to human health, calling for the development of more effective therapeutic strategies [ 2 ]. Vaccination has proven to be the most cost-effective preventative measure against infectious diseases, and functional vaccines against these ‘priority pathogens’ are critical for preventing or halting the escalation of antibiotic resistance.

Results

Immunization with HPF resulted in improved protective efficacy against P. aeruginosa pneumonia To test whether HlaH35A fusion improves the immunogenicity of protein antigens, PA0833 and the fusion protein HPF were constructed, purified, and used to immunize BALB/c mice. Immunization was carried out via intramuscular route three times with each of the proteins, which were formulated with or without alum adjuvant. The PA0833-specific immunoglobulin gamma (IgG) titers and their subtypes were determined. Seven days after the first immunization, PA0833-specific IgGs were detectable in the sera of antigen-immunized mice, with no significant difference. After the second immunization, the PA0833-specific IgG titers were significantly increased in all immunized mice. In particular, mice immunized with antigens formulated with alum showed higher IgG titers than those immunized with antigen alone. HPF elicited a higher PA0833-specific IgG titer than PA0833, both with and without alum adjuvant. The PA0833-specific IgG titer induced by HPF was comparable to that induced by PA0833 with alum, which was approximately 15.8-fold higher than that induced by PA0833 alone. However, the third immunization did not further increase the IgG titers, except for HPF with alum (Fig 1A). PPT PowerPoint slide

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larger image TIFF original image Download: Fig 1. HlaH35A fusion resulted in improved immunogenicity and protection efficacy of PA0833 against P. aeruginosa lethal pneumonia. Mice were immunized with His buffer, PA0833 with or without alum, or HPF with or without alum. (A) The PA0833-specific IgG titers in sera of immunized mice were determined by ELISA one week after the first, second, and third immunizations. (B) The IgG1 and IgG2a titers were assessed one week after the last immunization. Two-way ANOVA, Tukey’s multiple comparison test, ***P<0.001, ****P<0.0001. Data are presented as the mean ± SEM. (C) One week after the last immunization, mice were challenged with 1 × 107 CFUs of P. aeruginosa strain XN-1 and monitored continuously for 14 days (n = 15). Gehan-Breslow-Wilcoxon test, *P<0.05, ***P<0.001. (D) Bacterial loads in the lungs were determined 24 h after challenge with a sub-lethal dose of PA XN-1 (n = 10). One-way ANOVA, Tukey’s multiple comparison test, ***P<0.001, ****P<0.0001. (G) HE staining of lungs from immunized mice challenged with a sub-lethal dose of PA XN-1. Photomicrographs were taken at 400× magnification. Analysis of the pro-inflammatory cytokines, TNF-α (E), IL-1β (F), IL-6 (H), and IL-12 (I) in the lungs of immunized mice 24 h after challenge with 2 × 106 CFUs of XN-1 (n = 10). One-way ANOVA, Tukey’s multiple comparison test, ***P<0.001, ****P<0.0001. Data are presented as the mean ± SEM. https://doi.org/10.1371/journal.ppat.1009752.g001 Isotype switch to IgG2a and IgG1 is mediated by Th1 and Th2 immune responses in mice, respectively [18]. IgG1 and IgG2a titers were detected one week after the last immunization. Notably, the IgG1 titer in HPF-immunized mice was significantly higher than that in PA0833-immunized mice, and the addition of alum was also able to increase the IgG1 titer. In contrast, the IgG2 titers were improved by the addition of alum, but HlaH35A fusion did not show such an effect. This result demonstrated that alum and HlaH35A fusion synergistically increased the PA0833-specific IgG1 titers, while the IgG2a titers were increased by alum but not the HlaH35A fusion (Fig 1B). One week after the last immunization, mice were challenged with 1 × 107 CFUs of the P. aeruginosa strain XN-1, and their survival was monitored continuously for 2 weeks. The result showed that 100% of mice were survived in HPF with alum group, while the PA0833 with alum group showed a survival rate of 33.3%. Meanwhile, the PA0833 and HPF groups showed 20% and 40% of survival, respectively (Fig 1C). These results were consistent with the antigen-specific IgG titers elicited by each of the preparations. The lungs from immunized and control mice were harvested 24 h after sub-lethal infection. Bacterial loads (Fig 1D) in the PA0833 with alum and HPF groups showed no difference, but were significantly lower than those in the control group, while the bacterial burden in the HPF with alum group was dramatically decreased when compared to all other groups. In addition, the histological (Fig 1G) and pro-inflammatory cytokine secretion (Fig 1E, 1F, 1H and 1I) examination of the lungs of immunized mice was consistent with the data on survival and bacterial burden. To further confirm the efficacy of HlaH35A, the same assays were repeated in a pneumonia model in male C57BL/6 mice. The results showed improved protective efficacy (S1B Fig), immunogenicity (S1C Fig), complement functional activity (S1D Fig) and Th2 responses (S1E, S1F and S1G Fig), consistent with that obtained from BALB/c mice. Collectively, these results demonstrated that HlaH35A fusion and alum formulation enhance the protective efficacy of PA0833, both separately and synergistically.

Antigen uptake and migration of antigen-positive cells were promoted by the HlaH35A fusion After revealing that HlaH35A fusion improves the immunogenicity and protective efficacy of PA0833, the impact of HlaH35A fusion on antigen uptake, which is essential for initiating an immune response, was further investigated. PA0833 and HPF were labeled with Alexa Fluor 488 to enable antigen tracking after immunization. The flow cytometric analysis pipeline is shown in S2 Fig. Antigen-positive cells were determined in muscles and draining lymph nodes (LNs) from all groups (Fig 2A), and antigen-positive cell subsets were gated against the His buffer control group. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 2. Antigen-positive cells in muscles and draining lymph nodes (LNs). (A) Alexa Fluor 488-labeled antigen uptake in the muscle and draining LNs 24 and 72 hours after injection with His buffer, PA0833, HPF, PA0833 + Alum, or HPF + Alum (n = 5). Antigen-positive cells in the muscles (B) and draining LNs (inguinal lymph nodes) (C) were detected using a flow cytometer 24 h and 72 h after immunization. Data are shown as the mean ± SEM. Two-way ANOVA, Tukey’s multiple comparison test, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. https://doi.org/10.1371/journal.ppat.1009752.g002 In muscles, the number of antigen-positive non-leukocyte (CD45-) cells induced with HPF was 4.78 times higher than that with PA0833 alone, and became 13.2 times higher when the antigens were formulated with alum. Absorption of antigens by alum led to a reduction in antigen uptake at 24 h post injection (hpi) but an increase at 72 hpi (Fig 2A and 2B), which suggests that the large size of alum (2–9 μm) [19] halted antigen uptake and degradation by non-leukocyte cells. Formulation with alum resulted in a 2-fold increase in the number of antigen-positive leukocytes (CD45+), while HlaH35A fusion resulted in an approximately 4-fold increase at 24 hpi. Thus, the highest number of antigen-positive leukocytes was observed in the HPF with alum group at 72 hpi. Elevation of antigen uptake by leukocytes was observed over time in all immunized groups except for the PA0833 group (Fig 2A and 2B). These results indicated that both HlaH35A fusion and alum promoted antigen uptake at the site of infection. In draining LNs, the antigen-positive cells homing from the site of injection present the antigens to T or B cells. As shown in Fig 2B and 2C, neither PA0833-positive leukocyte (CD45+) cells nor antigen-positive non-leukocyte (CD45-) cells were detected at 24 hpi and 72 hpi. In contrast, HPF-positive leukocyte (CD45+) cells were detected at 24 hpi, and the frequency of these cells was significantly higher at 72 hpi. This result confirmed that only HPF-positive cells were delivered to draining LNs, which was likely due to HlaH35A fusion.

The antigen uptake promoted in muscles by HlaH35A fusion and alum depended on different subsets of infiltrated cells In view of the elevated level of antigen-specific leukocytes in injected muscles after immunization, the infiltration and antigen uptake efficacy of different leukocyte subsets were further evaluated. CD11c-positive cells were seldom detected in the injected muscles (S2 Fig), which indicated that dendritic cells did not infiltrate into the site of injection or differentiated from infiltrated monocytes. Thus, infiltrated monocytes, macrophages, and neutrophils were further investigated (Fig 3A, 3B and 3C, left panels). PPT PowerPoint slide

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larger image TIFF original image Download: Fig 3. Monocytes, macrophages, and neutrophils in muscles. Bars represent the mean and SEM of infiltrated and antigen-positive monocytes (A), macrophages (B), and neutrophils (C) in the muscles 24 h and 72 h after immunization. Two-way ANOVA, Tukey’s multiple comparison test, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. https://doi.org/10.1371/journal.ppat.1009752.g003 Recruitment of monocytes rapidly vanished in His buffer and peaked in the HPF group; a 3.916-, 1.95-, 1.33-, and 1.88-fold increase in monocytes was detected at 72 hpi, compared to 24 hpi, in the PA0833, HPF, PA0833 with alum, and HPF with alum groups, respectively (Fig 3A). In contrast, antigen-positive monocytes, regardless of the formulation with alum, were dramatically higher in the HPF groups than in the PA0833 groups, both at 24 hpi and 72 hpi, which indicated that the enhancement of antigen uptake with the HlaH35A fusion was mediated by monocytes (Fig 3A). Notably, the percentages of antigen-positive monocytes were similar between HPF with alum and HPF alone at 72 hpi (S3 Fig). The difference in the quantity of antigen-positive cells may be due to a lower recruitment of monocytes induced by alum compared to other types of cells. The total number of recruited macrophages in each group was similar to that of monocytes. Antigen-positive macrophages reached a peak in the HPF group at 72 hpi, which was remarkably reduced by formulation with alum. Macrophages are mostly derived from infiltrated monocytes at the immunization sites [20]. Hence, the significantly fewer antigen-positive macrophages in alum groups at 72 hpi suggested that the formulation with alum did not induce the differentiation of monocytes into macrophages in BALB/c mice (Fig 3B). Neutrophil recruitment was observed in all immunized mice at 24 hpi, and both HlaH35A and alum increased the frequency of neutrophil recruitment. Since neutrophils die within a few hours after the antigen is engulfed [21], neutrophils in the muscles at 72 hpi were newly recruited. The number of neutrophils decreased to normal levels in mice immunized with antigen alone and increased in mice immunized with antigen plus alum, suggesting that alum, but not HlaH35A, is required for continuous neutrophil recruitment (Fig 3C). The percentage of antigen-positive monocytes and macrophages increased over time, while that of neutrophils did not differ (S3 Fig). Together, these results suggested that HlaH35A fusion mainly improved antigen uptake by monocytes and macrophages, whereas alum improved antigen uptake by neutrophils.

HlaH35A fusion improved antigen uptake in vitro Since monocytes and macrophages were mainly responsible for the HlaH35A-dependent improved antigen uptake, RAW264.7 cells, which are typical antigen-presenting cells, and human alveolar epithelial A549 cells (non-professional antigen-presenting cells) were used for antigen uptake analysis in vitro. Considering that antigen uptake is affected by precipitated alum, only antigens without alum were used in in vitro experiments. After incubating with the indicated antigens and washing three times with PBS, the cells were lysed and analyzed by immunoblotting. HPF, but not PA0833, was detected in total cell lysates (Fig 4A). A similar phenotype was observed in THP-1 cells, a human monocyte cell line (S4B Fig). However, HPF ceased to increase in the lysates over time, indicating that HPF was attached to the membrane and quickly enzymolysed into polypeptides (Fig 4C). Immunoblotting confirmed that HPF was able to interact with ADAM10, which was identified as the Hla receptor in RAW264.7 and A549 cells, but not in human embryonic kidney-derived 293T cells (Fig 4B). To better detect antigen uptake, PA0833 and HPF were labeled with Alexa Fluor 488. Confocal images showed that the uptake of HPF and PA0833 was time-dependent. In addition, HPF uptake by RAW264.7 cells was significantly higher than that of PA0833. Interestingly, most of the bound HPF was not located at the plasma membrane, but in vesicles (Fig 4D). Similar results were obtained for A549 cells (S4A Fig). The degradation of all the HPF taken by RAW264.7 cells was next examined and found to be a rapid process (Fig 4E). GI254023X, an ADAM10 specific inhibitor, decreased the uptake of HPF by RAW 264.7 cells in a concentration dependent manner (Fig 4F), which suggested that the interaction between ADAM10 and Hla is essential for improved uptake of HPF. These in vitro results were comparable to those from the in vivo experiments, and suggested that HPF may sequentially be attached to the cell membrane, engulfed into vesicles, and degraded in the cells. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 4. Antigen uptake by monocytes in vitro. Immunoblotting examining PA0833 and HPF uptake by RAW264.7 cells at different antigen concentrations (A) and incubation times (B). (C) Co-immunoprecipitation and immunoblotting of 293T, A549, and RAW264.7 cells treated with 128 μg/ml of Flag-HPF afterwashing three times with PBS, 0.5 mM 3,3’-dithiobis (sulfosuccinimidyl propionate) was added. (D) Confocal imaging of Alexa Fluor 488-labeled PA0833 and HPF uptake by RAW264.7 cells. (E) After 3 h of incubation, degradation of HPF was determined by immunoblotting. (F) After 16 h pre-treatment of indicated GI254023X, HPF uptake by RAW264.7 cells was measured by immunoblotting. https://doi.org/10.1371/journal.ppat.1009752.g004

HPF uptake was macropinocytosis-dependent Given the efficient uptake of HPF, the host cell machinery implicated in this process was screened using several inhibitors. As shown in Fig 5A, a significant decrease in whole HPF was observed in total cell lysates from RAW264.7 cells treated with amiloride before incubation with HPF, and this inhibition was concentration-dependent. This result was further confirmed by imaging AF488-labeled using confocal microscopy, which showed that the uptake of PA0833 was also inhibited by amiloride (Fig 5B). Amiloride is a specific inhibitor of macropinocytosis, which internalizes a large quantity of plasma membrane and non-selectively uptakes soluble antigens. In contrast, ATP depletion by 2-DG, inhibition of dynamin by dynasore, inhibition of ATPases by vanadate, and inhibition of cholesterol synthesis by lovastatin did not affect whole HPF uptake (S5 Fig). Together, these data demonstrated that HlaH35A fusion improved antigen uptake mainly through the concurrent internalization of large amounts of plasma cell membrane via macropinocytosis. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 5. Antigen uptake of HPF is micropinocytosis-dependent. HPF (A) and Alexa Fluor 488-labeled PA0833 and HPF (B) uptake by RAW264.7 cells pretreated with 0, 0.5, and 1 mM amiloride for 1 h. Complete HPF levels were determined by immunoblotting. Alexa Fluor 488-labeled PA0833 and HPF appear green in the confocal images. https://doi.org/10.1371/journal.ppat.1009752.g005

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