All proteome information was obtained from the WormBase ParaSite database WBPS10 version. The proteome information includes 17 species of helminths from the genera Ancylostoma (Ancylostoma caninum, Ancylostoma ceylanicum, and Ancylostoma duodenale), Ascaris (Ascaris suum and Ascaris lumbricoides), Schistosoma (Schistosoma mansoni), Strongyloides (Strongyloides papillosus, Strongyloides ratti, Strongyloides stercoralis, and Strongyloides venezuelensis), Toxocara (Toxocara canis), Trichuris (Trichuris trichiura), and Wuchereria (Wuchereria bancrofti). The encoded proteins from each genome were filtered by excluding those lacking an initial codon for methionine or a final stop-codon, or a complete reading frame, or those with sequences smaller than 100 amino acids. Proteins without a signal peptide predicted by SignalP 3.0, Markov’s hidden model, and the neural network’s method were also excluded. B-cell epitopes were predicted from the remaining proteins from each worm genome using Bepipred and IUpred predictors. For each helminth, 600 predicted B-cell epitopes with the highest scores were selected. Identical epitopes present in the same helminth were removed. Only B cell-predicted epitopes conserved among all 17 helminths were finally selected. After these analyses, the samples of the same genus were analyzed, and epitopes were recovered with a coverage of 85% and identity of 100% as evaluated by BlastP. In all stages, the highest scores were obtained for each of the epitopes. For duplicated epitopes, the highest prediction scores were considered. Some epitopes were discarded, taking into account the size, being accepted only 9 amino acids (maximum size generated by the predictor), and with the presence of greater than 7 of the same amino acid residues in the sequence. Finally, a table was generated with all epitopes by gender and with their respective scores. Finally, 480 B-cell predicted peptides that were conserved among the 17 helminth parasites were selected and screened by immunoblot for immunogenicity using helminth-reactive sera ( Figure 1 ).

The 480 B-cell predicted peptides (30 nmole each) conserved among 17 helminth parasites were each synthesized by the “Spot-Synthesis” method on a cellulose membrane with a 4.5 mm diameter. Peptides were tested for their specific reactivities to the sera from mice infected with Ascaris infective eggs three times (hyperimmune sera) or immunized with Ascaris adult worm crude antigens. Sera from normal mice were used as a negative control. Briefly, the membrane dotted with individual synthesized peptides was blocked with blocking solution (1xPBS-BSA 3%, Sucrose 4%) for 12–16 h at room temperature under agitation. On the next day, the membrane was washed three times using washing solution (1xPBS-0.1% Tween 20) for 10 min each wash, then incubated with Ascaris-immune-sera from mice (infected three times or immunized with adult crude antigens) or normal mouse control sera diluted 1:1,000 in 50 ml of washing solution. The membrane was washed three times and then incubated with anti-mouse IgG/HRP secondary antibody at 1:10,000 dilution in 50 ml of washing solution for 1 h at room temperature. After being washed three times, the membrane was visualized by Luminata revealing solution (Millipore/Merck, USA) and automatically exposed using the ImageQuant LAS4000 instrument. Our focus was to locate those peptides recognized strongly by A. suum immunized mouse sera or Ascaris crude antigen immunized mouse sera compared to the sera of negative control mice.

The recognition intensity of individual peptides by the Ascaris-specific sera was quantitatively measured by densitometry scanning analysis using the Software Image J- Protein Array Analyzer. Through the densitometry analysis, we were able to characterize the reactivity of each peptide to Ascaris-immune sera compared to the normal mouse sera control. The Ascaris-immune sera–recognized positive peptides were selected based on the following selection criteria (1): Cutoff value (four spots with the lowest densitometry value were selected to calculate the mean as the cutoff value) (2); exclusion of negative peptides (cutoff value + 2× standard deviation, SD) (3); exclusion of barely positive peptides (cutoff value + 3× SD) (4); the densitometry value ≥ cutoff value 3× SD were selected as positive peptides. Based on these criteria, 35 peptides with the highest reaction to Ascaris-immune sera were selected as Ascaris-reactive peptides for the following experiments.

Thirty-five positive peptides with the highest reaction to the Ascaris-immune sera were selected for constructing a chimeric vaccine candidate against Ascaris infection. The sequence of the 35 peptides was arranged based on their best conformation predicted by Immunorank Bioinformatics Analysis using 10,000 possible sequence arrangements. The best sequence arrangement of 35 peptides was further chosen based on its immunogenicity index. The final 35 selected linear B-cell epitopes were connected by lysine (KK) linkers as flexible connectors to generate the peptide-based chimera vaccine: ASCVac-1. It has been reported that KK linkers preserve independent immune responses when they are inserted between epitopes (17). The DNA coding for the chimera was codon-optimized based on E. coli codon usage using Graphical Codon Usage Analyser, with a TAA stop codon added to the end of the sequence, was synthesized by GenScript and cloned into the E. coli expression vector pET28a-TEV using NdeI and HindIII restriction sites.

The chimeric protein was subcloned into the bacterial expression vector pET28a-TEV. The recombinant plasmid was transformed into E. coli BL21 by electroporation and selected on LB agar plate containing 50 µg/ml kanamycin. The transformants with the correct insert were confirmed by PCR with vector flanking primers T7 and T7t. The small-scale expression of recombinant proteins in each transformant was induced with 1 mM IPTG. One of the transformants with the highest expression yield was used for large-scale expression. Briefly, 3 L of 2XYT medium containing 50 µg/ml kanamycin was inoculated with 150 ml overnight culture of the selected clone. When the culture reached an OD of 0.6, IPTG was added to a final concentration of 1 mM to induce the expression of the recombinant protein at 37°C with 180 rpm shaking overnight. Subsequently, the culture was centrifuged at 2,000 × g for 30 min at 4°C. The pellet was suspended in PBS buffer containing 30 mM imidazole and 10 mg/ml lysozyme. The complete lysis of the bacteria was achieved using an EmulsiFlex-C3 homogenizer. The expressed recombinant protein with a His-Tag at its N-terminus was purified by immobilized metal affinity chromatography using the AKTA System. Western blot with anti-His antibodies was performed to confirm the expressed chimeric protein containing His-tag. The recognition of the expressed chimeric protein by serum from mice infected with A. suum was also confirmed by ELISA.

A. suum adult worms were collected from the intestines of infected pigs from a slaughterhouse located in the city of Belo Horizonte, Minas Gerais, Brazil. Adult worms were kept in 0.4 M NaCl and 10 mM NaPO₄, pH7.4, and taken to the Laboratory of Immunology and Genomics of Parasites of the Federal University of Minas Gerais to be processed. The eggs were isolated from the uteri of female adult worms by mechanical maceration, filtrated through a 100 μm nylon strainer, and placed in 50 ml of 0.2 M sulfuric acid in a culture flask at a concentration of 25 eggs/μl. The flask was stored in a BOD incubator at 26°C. On the 150^th day of culture, the eggs were fully embryonated and had reached the peak of larvae infectivity and were ready for experimental infections (18).

BALB/c mice (female, 7 weeks old) were obtained from the Central Animal Facility–Federal University of Minas Gerais, Brazil, and divided into four experimental groups with 16 mice per group. The first group was the control group without immunization and infection (CT NI). The mice in the second group received PBS only (PBS). Following our immunization protocol described in the previous study (16), the mice in the third group each received 25 μg of the adjuvant BpMPLA (Monofosforil lipid A from Bordetella pertussis, Butantan Institute) as adjuvant control (MPLA). The mice in the 4^th group were each immunized subcutaneously with 25 μg of the chimera ASCVac-1 formulated with 25 μg BpMPLA (ASCVac-1+MPLA) in a total volume of 130 µl. The mice were boosted twice with the same amount of antigen or adjuvant at 10 days intervals. Ten days after the last immunization, mice in groups 2, 3, and 4 were challenged with 2,500 Ascaris fully embryonated infective eggs in a total volume of 0.2 ml by oral gavage. Just before challenge, the fully embryonated eggs of A. suum were treated with 5% (v/v) sodium hypochlorite in an incubator (37°C and 5% CO₂) for 2 h to disrupt the outer layer to facilitate in vivo larval hatching. After the incubation, the eggs were washed in PBS five times and counted under a microscope. Blood was collected from each mouse before each immunization and challenge, and serum was isolated for antibody measurement. The immunization regime is described in Figure 2 .

ELISAs were performed to measure the vaccine-specific antibodies in the sera of mice after each immunization with the chimera ASCVac-1 and the controls (CT NI, PBS, and MPLA). Briefly, ELISA plates (Greiner-Bio-One, USA) were coated with 1 µg of purified chimera protein ASCVac-1 in a total volume of 100 µl per well overnight at 4°C. On the following day, plates were washed five times with the washing buffer (PBS-0,05% Tween20) and then blocked with 250 μl of PBS containing 3% BSA for 1 h at 37°C. The antigen-coated plates were incubated with 100 μl of the sera of mice from each group diluted at 1:1,000 in PBS-BSA 3% per well for 2 h at room temperature. After being washed five times with washing solution, 100 μl of HRP conjugated anti-mouse IgG diluted at 1:2,000 in PBS-BSA 3% was added into each well. After 1 h of incubation at 37°C, the plates were washed and developed with 100 μl of the developing solution containing 0.1 M citric acid, 0.2 M Na₂PO, 0.05% OPD (o-phenylenediamine dihydrochloride), and 0.1% H₂O₂. The plates were incubated at 37°C for 20 min, and the reaction was stopped by the addition of 50 μl 0.2 M sulfuric acid. The resulting absorbance was read on an ELISA reader (VersaMax ELISA Microplate Reader/Molecular Devices, USA) using Softmax Pro 5.3 software at 492 nm. All assays were performed in duplicates.

For measuring IgG subclasses, the same ELISA protocol was used except for using HPR conjugated anti-mouse IgG1, IgG2a, and IgG3 (Southern Biotech, USA) diluted at 1:1,000 in PBS-BSA 3%. Sera from the vaccinated groups were diluted at 1:500 for IgG1, IgG3, and 1:100 for IgG2a. To determine IgE levels, serum samples were diluted 1:10 and the HRP-conjugated anti-mouse IgE (Southern Biotech, USA) was diluted 1:500 in PBS-BSA (3%).

Eight days post-challenge with 2,500 A. suum infective eggs, each mouse in Groups 2, 3, and 4 was euthanized and lung tissue was collected. The tissue was minced into small pieces with surgical scissors, and then placed in a modified Baermann apparatus and incubated in PBS at 37°C and 5% CO₂ for 4 h. The larvae precipitating to the bottom of the apparatus were collected and fixed in 4% formalin. The number of larvae from each mouse lung was counted under a microscope, and larval reduction in the vaccinated group was calculated compared to the adjuvant control group. To collect Ascaris larvae in the bronchoalveolar compartment of infected mice, eight animals from each group were anesthetized. BAL fluid was collected from each mouse by inserting a 1.7 mm catheter into the trachea and washing it with 1 ml of PBS two times. The collected BAL fluid was filtered using a cell strainer of 40 µm to purify A. suum larvae, and the remaining flowthrough fluid was centrifuged at 3,000 × g for 10 min. The larvae in the pellet were counted using a microscope, and BAL fluid supernatants were used to quantify total proteins, hemoglobin, and inflammatory mediators.

The concentration of total proteins present in BAL was determined by the bicinchoninic acid (BCA) method using a commercial kit (Pierce, USA) following the manufacturer’s instructions. A standard curve was performed using BSA, and the optical density (O.D.) value of the samples was aligned with the standard curve to determine the protein concentration. The results were expressed as μg of total protein per ml of BAL.

The hemoglobin concentration present in BAL fluid was measured by a colorimetric assay using the cyanometahemoglobin method. The reading was performed in a spectrophotometer at a wavelength of 540 nm using a modified Drabkin solution (Bioclin Quibasa, Brazil). The hemoglobin reagent (Bioclin Quibasa, Brazil) was used to prepare a standard curve in which the O.D. values of the samples were aligned to determine their concentration. Hemoglobin content was expressed in g of Hb per dl of BAL.

To determine the tissue cytokine profile, the right lobe of the lung was removed from eight mice from each group, and 100 mg of tissue was homogenized by the Power-Gen 125 tissue homogenizer (Fisher Scientific, USA) in 1 ml of PBS supplemented with protease inhibitors (0.1 mM of methyl sulfonyl phenyl fluoride; 0.1 mM benzethonium chloride; 10 mM EDTA and 20 KI of aprotinin A) and 0.05% of Tween20. The homogenate was centrifuged at 800 × g for 10 min at 4°C, and the supernatant was used to determine the cytokines IL-4, IL-5, IL-6, IL-10, IL-13, and IL-33 by ELISA (R&D, USA) according to the manufacturer’s instruction. The absorbance of the samples was determined by VersaMax ELISA microplates (Molecular Devices, USA) at a wavelength of 492 nm.

The n-acetylglucosaminidase activity of macrophages (NAG), neutrophil myeloperoxidase (MPO), and eosinophil peroxidase (EPO) was evaluated in the homogenates of the lungs. After being homogenized using the Power-Gen 125 tissue homogenizer (Fisher Scientific, USA), the lung homogenate was centrifuged at 1,500 × g for 10 min at 4°C, and the resulting sediment was examined to determine the activity of EPO, MPO, and NAG.

The left lobes of the lungs were removed from the eight mice of each group. The lungs were fixed in 4% formalin solution and gradually dehydrated in ethanol, then embedded in paraffin. The paraffin blocks were cut at 4–5 microns thick and stained with hematoxylin and eosin for evaluating tissue damage.

For the histopathological analysis in the lungs of animals after the migration of larvae through the organ, three analysis tools were used. The histopathological description in order to evaluate the lesions caused by larval migration in the pulmonary parenchyma of mice, and for that the infiltration of inflammatory cells and exudative phenomena were observed. In addition, semiquantitative and quantitative analysis was used to assess degree of airway inflammation, peribronchial inflammation, and presence of hemorrhage of the lungs. The difference between these two analyzes is based on the characterization of inflammation through an inflammation score previously described in the cited work, and the quantitative analysis is only to corroborate the semi-quantitative result, but this time analyzing the exact areas of each inflammation and its magnitude (16).

For quantitative analysis of inflammation in larva migrated lungs, peribronchial or perivascular inflammatory cells were captured and counted on a Motic 2.0 microcamera. The areas of peribronchial or perivascular inflammation were delimited with the aid of a cursor, segmented, processed, and the values of the respective areas in µm² by using the Image J plus program.

Pulmonary dysfunction was measured as previously described (19). Mice were anesthetized by subcutaneous injection of ketamine and xylazine (8.5 mg/kg xylazine and 130 mg/kg ketamine), tracheostomized, and connected to a computer-controlled ventilator (Forced Pulmonary Maneuver System R, Buxco Research Systems, Wilmington, NC, USA). Mice were ventilated at a rate of 160 breaths per minute under anesthesia. Under mechanical respiration, the Lung Resistance (Rl) was determined by Resistance and Compliance (RC) test. A fast-flow volume maneuver was performed, and the lungs were first inflated to +30 cm H₂O and immediately afterward submitted to a high negative pressure to force the expiration until −30 cm H₂O. The Flow-Volume curves were recorded during this maneuver. Suboptimal maneuvers were discarded, and for each test in every single mouse, at least three acceptable maneuvers were conducted to obtain a reliable mean for all numeric parameters.

Statistical analyses of the data were performed using the GraphPad Prism 7 software (GraphPad Inc, USA). To verify the distribution of the data, the Kolmogorov-Smirnov and Shapiro-Wilk tests were used. For analysis between two groups, the Student Test T was used for parametric data and Mann-Whitney U for non-parametric data. For analysis of variances between three or more groups, with only one qualitative variable, the One-way ANOVA tests followed by the Tukey and Bonferroni post-test (parametric data), or the Kruskal Wallis tests followed by Dunn’s post-test (non-parametric data). For analysis of variances between three or more groups, with two or more qualitative variables, the Two-way ANOVA tests followed by Tukey’s post-test were used. The test was considered significant when the p-value was ≤0.05.

The maintenance and use of animals were carried out following the recommendations of the Brazilian College of Animal Experimentation (COBEA). The present study was approved by the Ethics Committee for Animal Experimentation (CEUA) of the Federal University of Minas Gerais, Brazil, through protocol #61/2018. All efforts were made to minimize animal suffering.

In silico prediction for helminth B-cell epitopes using a bioinformatic pipeline in 17-helminth proteome identified 480 conserved epitopes. These peptides were synthesized by the “Spot-synthesis” method and dotted on a cellulose membrane ( Figure 3A ). The peptide dots were probed with pooled sera from mice immunized with A. suum adult crude antigens (Immunized-Sera) (16), or mice infected with A. suum infective eggs three times (Reinfected-Sera), as well as the serum from normal control mice (Negative Control Serum). The peptide spots were scanned using densitometry to evaluate the signal as weak, medium, or strong after incubating with the Ascaris-immune sera in comparison with the negative sera. The intensity was converted to different colors by Image J- Protein Array Analyzer software allowing the characterization of the reactivity intensity of the peptides recognized by different immune sera compared to the normal mouse sera ( Figure 3B ). Those peptides below the cutoff value (background), or weak positive peptides (< cutoff + 3 SD) were excluded. Only those with intensity value ≥ cutoff + 3SD, without recognition by normal mouse serum, were selected as potential Ascaris-specific vaccine candidate peptides ( Figure 3C ). A total of 157 Ascaris-reactive peptides met the selection criteria. From these, the top 35 peptides with the highest reaction to Ascaris-immune sera with recognition intensity 20 times greater than the control sera were finally selected to be linked together by a KK linker in a sequence arrangement with optimal conformation and solubility to construct a multiepitope-based chimera vaccine antigen against Ascaris infection named ASCVac-1 (patent number: BR 10 2020 026968 2).

The sequence of ASCVac-1 is shown in Figure 4A . The 3D conformational structure of ASCVac-1 was predicted by I-TASSER. The model with the highest prediction score was selected and presented in Figure 4B .

The DNA encoding the chimeric protein ASCVac-1 was codon-optimized based on E. coli codon preference and synthesized by GenScript. The synthesized DNA was subcloned into the bacterial expression vector pET28a-TEV using NdeI and HindIII restriction sites. In the first lane of the gel, the plasmid pUC57+ASCVac-1 is found in the form of a closed plasmid without digestion by the enzymes. In the second lane, the plasmid pUC57/ASCVac-1 is digested with NdeI and HindIII, releasing the 1,164 bp ASCVac-1 insert (blue frame), and the 2,710 bp pUC57 vector. In lane 3, the expression vector pET28a-TEV was likewise digested with NdeI and HindIII ( Figure 5A ). After ligation of the insert and the linearized vector, the recombinant plasmid was transformed into E. coli BL21, and the transformants with the correct insert were confirmed by PCR ( Figure 5B ), and one of the positive colonies was used for large-scale expression. The expressed recombinant protein with a His-Tag at N-terminus was purified by affinity chromatography using the AKTA System ( Figure 5C ); it was successfully expressed as a soluble recombinant protein. The purified recombinant ASCVac-1 protein was recognized by the anti-His antibodies ( Figure 5D ).

The antigenicity of ASCVac-1 chimera against both Ascaris-immune sera (re-infected or crude Ag-immunized) and negative control (CN) sera were assessed by ELISA ( Figure 5E ). Ascaris crude antigens (ExAD) were used as a positive control of the assay. The ELISA results revealed that the chimeric ASCVac-1 protein was strongly recognized by both immune sera from mice immunized with Ascaris crude antigen (Immunized Serum) and infected with A. suum infective eggs (Reinfected Serum), but not by the sera from negative control mice (CN Serum). The ExAD has higher recognition better though by both mouse immune sera than the ASCVac-1 antigen.

To determine the immunogenicity of recombinant ASCVac-1 multiepitope chimeric protein, mice were subcutaneously immunized with 25 µg of rASCVac-1 formulated with 25 µg BpMPLA three times. Sera were collected from each mouse 10 days after the final immunization. The ASCVac-1-specific IgG level in sera started to increase after the 2^nd immunization and reached a high level after the 3^rd immunization. No anti-ASCVac-1 IgG antibody was detected in the sera of mice receiving PBS or MPLA adjuvant only ( Figure 6A ). The antigen-specific IgG subclasses and IgE levels were detected in the sera of each group after the 3^rd immunization. The ASCVac-1 immunization induced a marked IgG1 dominant response in the group of ASCVac-1+MPLA ( Figure 6B ). The IgG3 response was also significantly induced ( Figure 6C ), but with a relatively low IgG2a response ( Figure 6D ) in this immunization group compared with groups receiving PBS or MPLA. Results suggest that the ASCVac-1 antigen induces Th-2 polarized immune responses. Interestingly, immunization with ASCVac-1 three times did not induce antigen-specific IgE response ( Figure 6E ).

To investigate the preclinical efficacy of the chimeric ASCVac-1 vaccine and its capacity to protect against or reduce Ascaris infection burden, we counted the larvae collected from lungs and bronchoalveolar lavage fluid (BAL) of mice immunized with ASCVac-1+MPLA at day 8 of infection (peak of larval migration in lungs). Notably, ASCVac-1+MPLA-vaccinated mice showed a significant reduction in the larva burden in both BAL fluid ( Figure 7A ) and lung tissue ( Figure 7B ) when compared to PBS or adjuvant controls. Combining the number of larvae collected from BAL fluid and lung tissue, mice immunized with ASCVac-1+MPLA produced a significant larval reduction of 50.15% compared to the PBS control group (p<0.01) or 33.7% reduction compared to the adjuvant MPLA control group (p<0.05) ( Figure 7C ).

After observing a marked reduction of the parasite burden in animals vaccinated with the chimeric ASCVac-1, the next aim was to identify the potential mechanisms involved in this protection. Different arms of the immune response were characterized after immunization followed by infection, including the leukocyte influx profile into the airways, cytokine production, lung tissue histopathology, and lung mechanical function.

To characterize the leukocyte influx into the airways, as well as the presence of hemoglobin (a marker for hemorrhage) and total proteins, the BAL fluid was collected from mice in different groups at day 8 of infection. Overall, during the infection, Ascaris larval migration not only caused lung damage and serious hemorrhage (increased hemoglobin and total protein in BAL fluid, Figures 8F, G ) but also drove an intense influx of inflammatory cells to the lung tissue and airways, characterized by a marked increase of leukocyte numbers in the BAL ( Figure 8A ). However, mice immunized with ASCVac-1 significantly reduced the larva migration-caused lung hemorrhage ( Figures 8F, G ), associated with a significant reduction in the presence of these leukocytes in the BAL when compared to the infected PBS control group (20, 31 ± 11, 37 cells × 10⁵ vs. 43, 02 ± 12, 48 cells × 10⁵, p<0.01). After being classified, we found that the reduced leukocytes in the ASCVac-1 group were mostly inflammatory cells such as neutrophils (p<0.001) ( Figure 8C ) and macrophages (p<0.01) ( Figure 8D ) compared to the non-vaccine control groups. However, lymphocytes and eosinophils were significantly increased in the BAL fluid of mice immunized with ASCVac-1 compared to mice in PBS or MPLA control groups (p<0.01) ( Figures 8B, E ). Taken together, vaccination with ASCVac-1 not only reduced larval burden in the lungs but also reduced lung damage and inflammation of immunized mice. Increased eosinophils and lymphocytes may be related to protective immunity.

No major differences were found in the levels of neutrophil myeloperoxidase (MPO) ( Figure 9A ), eosinophil peroxidase (EPO) ( Figure 9B ), and macrophage N-acetylglucosaminidase (NAG) ( Figure 9C ) when we compared the vaccinated with the unvaccinated mice upon Ascaris infection. Marked differences were observed in the groups infected with Ascaris when compared to the non-infected control group (CT NI).

To help characterize the immune response in the lung tissue and to compare the pulmonary cytokine signatures of ASCVac-1+MPLA-immunized mice followed by infection with PBS- or adjuvant-immunized mice followed by infection, we quantified the cytokines IL-4, IL-5, IL-6, IL-10, IL-13, and IL-33 in the lung homogenate. Overall, Ascaris infection in all groups induced a marked type-2 dominated immune response, with a significant increase in all cytokines assessed when compared with non-infected mice (CT NI) ( Figures 9D–I ). However, ASCVac-1+MPLA-immunized mice followed by Ascaris infection showed a marked reduction in the IL-33 levels in the lung tissue (similar to non-infected control mice) when compared with PBS-immunized mice followed by Ascaris (0, 6745 ± 0, 09743 pg/100 mg vs. 1, 102 ± 0, 09670 pg/100 mg, p<0.001).

To evaluate the effect of ASCVac-1 vaccination on the reduction of pathology in the lung caused by the A suum larva infection, the histopathological analysis of the pulmonary parenchyma including topography, inflammatory infiltrate, presence or absence of larvae, and vascular and exudative phenomena was performed in the sections of lung tissues collected from different groups of mice ( Figure 10A ). After being infected with A. suum infective eggs for 8 days, lung tissues demonstrated significant exudative phenomena characterized by perivascular edema, hemorrhage, and mixed inflammatory cells infiltration around vessels, bronchi, and bronchioles in mice of PBS and MPLA groups. Hypertrophy and hyperplasia of the epithelium cells of the bronchi and bronchioles were frequently observed. The presence of pulmonary L3 larvae in or near hemorrhagic areas and the lumen of bronchi and bronchioles was also observed. However, in mice immunized with ASCVac-1+MPLA, the lung inflammation and damage were significantly reduced with less inflammatory cell infiltration, less exudative phenomena, and hemorrhage. The most infiltrated cells were lymphocytes and macrophages rather than eosinophils and neutrophils. In addition, few infected larvae in lung tissue were observed compared to non-immunized groups.

A semiquantitative analysis based on the pathological score identified that scores based on the total inflammation, peribronchial inflammation, and perivascular inflammation were significantly reduced in the group of mice immunized with ASCVac-1 + MPLA compared to PBS or MPLA groups (p<0.01), and the score of hemorrhage reduced only compared to PBS group ( Figure 10B ). The perivascular lesion area was also significantly reduced in the immunized group compared to the MPLA control group, but peribronchial lesion reduction was only observed between the immunized group and PBS control group ( Figure 10C ).

Dysfunction of pulmonary mechanics is used as a marker of tissue injury. Thus, we evaluated the impact of immunization with ASCVac-1 on the improvement of lung function compared to control mice. The analysis of the Flow-Volume curve revealed that airflow through airways was significantly reduced in mice infected with A. suum infective eggs (PBS or MPLA groups) ( Figure 10D ). Immunization with ASCVac-1 greatly improved the airflow of mice compared to mice receiving PBS or MPLA only ( Figure 10D ). The Lung Resistance evaluation also demonstrated that mice infected with A. suum infective eggs (PBS or MPLA) increased the airway resistance and reduced the elasticity of infected lungs, and immunization with ASCVac-1 significantly improved the lung elasticity affected by Ascaris larva infection ( Figure 10E ). The improved lung function results correlate with our findings of improved tissue pathology in ASCVac-1 immunized mice ( Figures 10A –C ). Collectively, mice immunized with ASCVac-1 experienced improved lung function by increasing airflow and reducing airway resistance correlating with reduced lung pathology.