proteins from the tryptophan-rich antigen (PvTRAg) family (Zeeshan et al., 2014), merozoite surface protein-1 (PvMSP-1) (RodríGuez et al., 2002) and apical membrane antigen-1 (AMA-1) (Arévalo-Pinzón et al., 2017). Important mediators in AMA-1 erythrocyte binding have also been identified, such as rhoptry neck proteins (RONs), including PvRON5 (Arévalo-Pinzón et al., 2015), PvRON4 (Arévalo-Pinzón et al., 2013) and PvRON2 (Arévalo-Pinzón et al., 2011). RONs have been strongly associated with MJ formation, thereby helping parasite entry into erythrocytes; RON2 is a vaccine candidate since anti-AMA1 and anti-RON2 antibodies can block erythrocyte invasion (Arévalo-Pinzón et al., 2011; Lamarque et al., 2011; Srinivasan et al., 2011; Tyler et al., 2011; Vulliez-Le Normand et al., 2017).

The data presented here has described naturally-acquired Tcell immune responses to PvRON2 high-affinity binding-MHC-II DRB1∗ peptides in P. vivax-exposed individuals from two endemic areas of Colombia. Certain requirements are involved in protection-inducing vaccine design; for example, the proper antigen presentation by MHC-II and MHC-I molecules to Tcell receptors, so as to induce strong immune responses. MHC class II and I molecules are critical in host–pathogen interactions as they determine host immune response quality. High-affinity peptide-MHCII-TCR binding time or peptide-MHCII complex amount is critical for mounting protective T-cell responses (Blum et al., 2013; Tubo et al., 2013). Post-genomic era bioinformatics tools and reverse vaccinology approaches have drawn scientists' attention, since an individual antigen can be screened from one or several microorganisms and the highest affinity epitopes be determined from these might induce protective immune responses (Sette and Rappuoli, 2010).

This study has analyzed the PvRON2 sequence aa to determine high-binding HLA-DRB1∗ T-cell epitopes in silico. NetMHCIIpan 3.0 (Andreatta et al., 2015) predicted eleven high-affinity HLA-DRB1∗ epitopes where at least one epitope bound to one HLADRB1∗ molecule; this was confirmed by in vitro competition assays using biotin-control peptides. However, some predicted epitopes have not bound to HLA-DRB1∗ , according to other studies (Bergmann-Leitner et al., 2013), while HLA-DRB1∗ 04 bound to 10/11 peptides here. A previous study has shown a strong naturally-acquired humoral response in HLA-DRB1∗ 04 people living in the Brazilian Amazon against 5/9 recombinant P. vivax proteins (Lima-Junior et al., 2012). HLA-DRB1∗ 04 is one of the most frequently occurring alleles in Colombian Amerindian groups, accompanied by DR2 (DRB1∗ 1602), DR6 (DRB1∗ 1402) and DR8 (DRBl∗ 0802). The patients' blood samples used in this study were mostly taken from Amerindians from Córdoba and Chocó. Tule (5 HLA-DRB1∗ 04 alleles) is the main Amerindian population in Córdoba and the Waunana (4 HLA-DRB1∗ 04 alleles) in the Chocó region, having more diverse DRBl alleles than other groups (Trachtenberg et al., 1996).

Previous studies had suggested that some alleles' over dominance in a population could be due to the spread of advantageous alleles (positive selection) after pathogen-driven selection. This has been shown by Hill et al., in a study of West African alleles (HLA-DRB1∗ 1302-DQB1∗ 0501) which were not

present in other racial groups and were associated with protection against severe malaria, i.e., directional selection (Hill et al., 1991).

Several factors (e.g., parasite evasion mechanisms and immune molecule polymorphism) can affect protection-inducing immune responses to malarial parasites and, consequently, malaria vaccine development. Parasite evasion mechanisms include immunodominant antigen polymorphism, antigenic variation and diversion, epitope masking and the smoke-screen strategy. P. vivax and P. ovale have additional escape mechanisms which are mediated by long-lasting hypnozoites, as well as using different erythrocyte invasion pathways (Rénia and Goh, 2016). MHC Class I and II have enormous allele polymorphism and aa sequence variation in the peptide binding region, thereby enabling peptides to bind to different alleles (Blum et al., 2013). Such variability hampers a single epitope-based vaccine against Plasmodium parasites being developed; however, in silico analysis of T- and B-cell epitopes could be useful for identifying vaccine candidates represented by different epitopes which could provide coverage of the whole target population. The exposed patients studied here were grouped according to their HLA-DRB1∗ to cover the most frequently occurring alleles in the endemic population worldwide, such as HLA-DRB1∗ 04, HLA-DRB1∗ 07, HLA-DRB1∗ 11, and HLA- DRB1∗ 13. Cell-mediated immune

(n = 30). Seropositive samples were those above the cut-off point (0.218, dotted line), calculated as control group's mean plus two standard deviations. The Kruskal-Wallis test was used for analyzing differences between each B-epitopes response in P. vivax-exposed individuals' samples. \*p < 0.05.

responses were investigated based on MHC class II peptide binding specificity and humoral immune responses by detecting antibody levels against linear B-cell epitopes.

Antibodies against P. vivax blood-stage proteins are important elements for blocking RBC invasion (Wipasa et al., 2002); such antibodies thus play an important role in identifying and validating P. vivax vaccine candidates (Soares et al., 1999; Lima-Junior et al., 2008; Storti-Melo et al., 2012; Changrob et al., 2017; Rodrigues-Da-Silva et al., 2017). We confirmed the presence of naturally-acquired humoral responses against four PvRON2 Bepitopes which were recognized by IgG antibodies and subclasses. Low individual PvRON2 B-epitope responder frequency was observed (20% 39041, 10% 39042, 6.6% 39043 and 39044); such low responses have previously been reported for other bloodstage antigens such as PvMSP8. A loss of mean response to a target protein has been observed as time has elapsed when average response has been about ten times greater to recombinant protein than linear epitopes in acute-infection patients (Cheng et al., 2017). This has been associated with short-lived antibodies, due to short-lasting memory responses or parasite-induced Bcell dysregulation (Rénia and Goh, 2016) and parasite genetic variations or in exposed populations.

Antibody response differences against PvRON2 B-cell epitopes between exposed individuals from Chocó (83%) and Córdoba (17%), could be attributed to several factors, including the level of parasitemia and the number of episodes (Druilhe and Pérignon, 1994). The last 3 SIVIGILA reports (2015–2017), showed a higher incidence of P. vivax infection in Chocó regarding Córdoba (Instituto-Nacional-De-Salud, 2017). Other intrinsic factors from the responders such as their HLA, sex, age, psychological stress, nutrition and other infectious diseases could also be involved in such differences (Van Loveren et al., 2001).

Two of the selected B-epitopes (39042 and 39044) were located on an α-helical coiled motif protein and other studies have shown that selected in silico peptides related to these motifs have been recognized by naturally-acquired antibodies and have been immunogenic in mice (Villard et al., 2007; Arévalo-Pinzón et al., 2011); however, 39042 (10%) and 39044 (6.6%) epitopes had the lowest recognition values in our study. 39041 had significant differences in the IgG subclasses analyzed; IgG2 predominated while low IgG4 and IgG1 levels were observed. A predominant IgG2 response and low IgG4 reactivity in previous studies has been associated with P. falciparum infection resistance and clearance, IgG2/IgG4 relationship being associated with a protective role (Aucan et al., 2000). Similar results have been found in PvMSP8 studies recording IgG2 noncytophilic antibody predominance which has been associated with resistance to P. vivax malaria (Cheng et al., 2017). 39041 has been seen to be immunogenic in mice (Arévalo-Pinzón et al., 2011) and its potentially protective role makes this peptide a pivotal PvRON2 epitope for inclusion in a subunit-based vaccine.

It has been thought that antibodies would be enough to protect against malaria and that T-cells do not play an important role during the erythrocyte stage. Advances in immunology-related knowledge have demonstrated that B-cells must be activated by CD4+ T-helper cells to prompt good humoral responses, thereby inducing cytokine, memory cell and antibody production (Batista and Harwood, 2009; Tubo et al., 2013; Yuseff et al., 2013). The role of exposed patients' T-cell response against PvRON2 high-affinity binding peptides was studied in cytokine proliferation and production assays. Only exposed individuals' PBMC cultures showed proliferation induced by universal and specific binding peptides, suggesting that PvRON2 induced memory T-cells against high-affinity peptides. However, Th1 and Th2 cytokine responses were low, except for IL-6. Low cytokine responses/production have been observed in other studies; Silva-Flannery et al., found that immunization with monomeric peptide did not result in peptide-specific IFN-γ-secreting cell expansion and was not protective. They also reported that the monomeric peptide was less taken up by antigen-presenting cells and was not going through the phagolysosome (Silva-Flannery et al., 2009).

Cytokine production by unexposed individuals' PBMCs against PvRON2 synthetic peptides may have been due to dendritic or macrophages cells priming naïve T-cells and inducing effector T-cell cytokine production; nevertheless, secretion was very low for some of them.

Unlike the other cytokines tested here, IL6 was highly secreted by exposed patients' PBMCs; this was not surprising, since one of IL6's multi-functions is to stimulate hybridoma and plasmacytoma cell growth and help antibody production (Matsuda et al., 1988). IL6, together with IL12 and VDR, have been associated with reduced parasitemia, its severity and

gametocytemia clearance in P. vivax-exposed individuals (Sortica et al., 2014). P. vivax lysate-induced cytokine responses in unexposed individuals' PBMCs could be explained by innate immune cell cytokine production, i.e., macrophages, dendritic cells, NK, NKT and naïve T-cells which become effector cells (Stevenson and Riley, 2004). High cytokine induction in healthy individuals compared to P. vivax-exposed individuals might be related to a parasite evasion mechanism for inhibiting effective immune responses able to eliminate the parasite (Rénia and Goh, 2016). Nonetheless, the healthy individuals had not been exposed to P. vivax since immunofluorescence assays did not show their antibodies' reactivity to the parasite.

Taken together, the in silico T-cell and B-cell epitope selection results highlighted two T-cell epitopes (39047 and 39154) and one B-cell epitope (39041) as promising vaccine candidates. Despite the significant differences observed in immune responses evoked in the exposed individuals compared to the control group, overall the responses were relatively low. All selected peptides were conserved among the 11 P. vivax strains which may also explain such low immune responses. This has been demonstrated in P. falciparum studies where conserved high activity binding peptides (HABPs) were poorly antigenic and poorly immunogenic (Patiño et al., 1997; Lougovskoi et al., 1999; Ocampo et al., 2000; Parra et al., 2000; Hensmann et al., 2004). It should be stressed that caution must be taken, since using these 3 promising peptides in a multi-epitope vaccine in their unmodified state would probably mean that they could induce low immunogenicity and not provide longlasting protection; however, proven approaches have shown that modifying their critical residues should induce a strong and longlasting protection-inducing immune response (Patarroyo et al., 2010).

HABPs have been seen to be P. falciparum vaccine candidates during the last two decades (Rodriguez et al., 2008); however, they must be modified to make them antigenic and protectioninducing by replacing critical aa with others having the same mass but different polarity (Cifuentes et al., 2008; Patarroyo et al., 2011). Such HABPs can only be used in a tailor-made vaccine targeting a specific HLA-DRB1∗ endemic population; however, a universal protection-inducing vaccine will require studying other peptides which can bind to other HLA-DRB1∗ alleles. Future studies should be carried out using modified peptides aimed at assessing immunogenicity and protectioninducing ability in the Aotus experimental model to confirm their suitability as P. vivax vaccine candidates. Likewise, additional peptides should be included to cover all parasite stages, aiming at a 100% protection-inducing, multistage, multi-epitope, minimal subunit-based vaccine.

# AUTHOR CONTRIBUTIONS

CL: designed and performed the experiments, analyzed the data, drafted the manuscript. YY-P: designed and performed the experiments, analyzed the data, drafted the manuscript, designed the figures. DD-A: drafted the manuscript. MEP: critical suggestions regarding the manuscript. MAP: conceiving the work and drafting all versions of the manuscript. All authors have revised the manuscript and approved the version to be submitted.

# FUNDING

This work was financed by the Departamento Administrativo de Ciencia, Tecnología e Innovación (COLCIENCIAS) through grant RC # 0309-2013. CL received support from Colciencias within the framework of the Convocatoria Nacional para Estudios de Doctorado en Colombia (call for candidates # 6172).

# ACKNOWLEDGMENTS

We would like to thank all the people from Bahía Solano (Chocó) and Tierra Alta (Córdoba) who participated in this study, as well as all the people from healthcare institutions who coordinated sample collection, especially the Centro Médico Cubis in Bahía Solano, Chocó. We would like to thank Jason Garry for translating the manuscript, Natalia Hincapié-Escobar for her technical support, Alejandro Giraldo Escobar for his support with the native community, the Immunotoxicology group (UNAL) for their experimental support, Carlos Fernando Suárez for his critical suggestions and Luis Alfredo Baquero for donating rPvGAMA.

# SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcimb. 2018.00156/full#supplementary-material

Supplementary Figure 1 | Control group supernatant culture in vitro cytokine production. Data individual show the mean value of unstimulated and stimulated PBMC (n = 8) with universal epitope (39153), specific epitopes (39047, 39152,

and 39154) and P. vivax lysate. IFN-γ, TNF, IL-10, and IL-6 levels were measured by CBA kit and cytokine concentration is expressed in pg/mL. Statistically significant differences (p ≤ 0.05) are shown and data is the means ± SEM of all values.

Supplementary Figure 2 | Immunofluorescence patterns for exposed individuals from Colombia's P. vivax-endemic areas and the control group. The upper panels show one Bahía Solano's exposed individual serum recognition of pRBC. Nuclei were stained with DAPI, the parasite with anti-parasite FITC and then, both were merged. The bottom panels show serum from one non-exposed individual.

#### REFERENCES

Supplementary Figure 3 | IgG antibody response against PvGAMA control recombinant protein. Significant differences (calculated by Mann-Whitney test) are shown between samples from exposed individuals and control. The dashed line indicates the cut-off point for seropositive samples.

Supplementary Table 1 | Exposed-individuals' HLA-DRB1∗ allele frequency

Supplementary Table 2 | Non-exposed individuals' lymphoproliferation assay using PvRON2 peptides.

erythrocyte invasion are blocked by naturally acquired human antibodies. J. Exp. Med. 186, 1689–1699. doi: 10.1084/jem.186.10.1689

against virulent Plasmodium falciparum infection. NPJ Vaccines 2:14. doi: 10.1038/s41541-017-0015-7

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 López, Yepes-Pérez, Díaz-Arévalo, Patarroyo and Patarroyo. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Chitosan Microsphere Used as an Effective System to Deliver a Linked Antigenic Peptides Vaccine Protect Mice Against Acute and Chronic *Toxoplasmosis*

Jingjing Guo† , Xiahui Sun† , Huiquan Yin, Ting Wang, Yan Li, Chunxue Zhou, Huaiyu Zhou, Shenyi He\* and Hua Cong\*

*Department of Human Parasitology, Shandong University, School of Medicine, Jinan, China*

#### *Edited by:*

*Kamal El Bissati, University of Chicago, United States*

#### *Reviewed by:*

*Fiona Luisa Henriquez, University of the West of Scotland, United Kingdom Hetron Mweemba Munang'andu, Norwegian University of Life Sciences, Norway*

#### *\*Correspondence:*

*Shenyi He<EMAIL_ADDRESS>Hua Cong<EMAIL_ADDRESS>
*†These authors have contributed equally to this work.*

> *Received: 24 January 2018 Accepted: 30 April 2018 Published: 23 May 2018*

#### *Citation:*

*Guo J, Sun X, Yin H, Wang T, Li Y, Zhou C, Zhou H, He S and Cong H (2018) Chitosan Microsphere Used as an Effective System to Deliver a Linked Antigenic Peptides Vaccine Protect Mice Against Acute and Chronic Toxoplasmosis. Front. Cell. Infect. Microbiol. 8:163. doi: 10.3389/fcimb.2018.00163* Multiple antigenic peptide (MAP) vaccines have advantages over traditional *Toxoplasma gondii* vaccines, but are more susceptible to enzymatic degradation. As an effective delivery system, chitosan microspheres (CS) can overcome this obstacle and act as a natural adjuvant to promote T helper 1 (Th1) cellular immune responses. In this study, we use chitosan microparticles to deliver multiple antigenic epitopes from GRA10 (G10E), containing three dominant epitopes. When G10E was entrapped within chitosan microparticles (G10E-CS), adequate peptides for eliciting immune response were loaded in the microsphere core and this complex released G10E peptides stably. The efficiency of G10E-CS was detected both *in vitro*, via cell culture, and through *in vivo* mouse immunization. *In vitro*, G10E-CS activated Dendritic Cells (DC) and T lymphocytes by upregulating the secretion of costimulatory molecules (CD40 and CD86). *In vivo*, Th1 biased cellular and humoral immune responses were activated in mice vaccinated with G10E-CS, accompanied by significantly increased production of IFN-γ, IL-2, and IgG, and decreases in IL-4, IL-10, and IgG1. Immunization with G10E-CS conferred significant protection with prolonged survival in mice model of acute toxoplasmosis and statistically significant decreases in cyst burden in murine chronic toxoplasmosis. The results from this study indicate that chitosan microspheres used as an effective system to deliver a linked antigenic peptides is a promising strategy for the development of efficient vaccine against *T. gondii*.

Keywords: *Toxoplasma gondii*, chitosan microspheres, peptides, epitopes, vaccine

# INTRODUCTION

Toxoplasma gondii is an important medical pathogen that infects approximately 30% of the global population. Generally, toxoplasmosis is asymptomatic in immune-competent hosts, however, it can result in severe symptoms in immunocompromised individuals due to cerebral cyst reactivation. Another potentially fatal presentation is vertical transmission in the fetus, which can result in encephalitis, neonatal malformations, or spontaneous abortion (Blader et al., 2015; Dimier-Poisson et al., 2015). Current drug treatment cannot control this disease completely because of the inability of drugs to kill bradyzoites (Henriquez et al., 2010). Therefore, the advantages of a preemptive vaccine for preventing toxoplasmosis are obvious (Innes, 2010). Traditional vaccine development strategies against T. gondii mainly focused on subunit and DNA vaccines. Their use raises several issues, since subunit vaccines have poor stability and may cause undesired immune responses (Skwarczynski and Toth, 2014), and DNA vaccines have the theoretical risk of genomic integration into host cells (Kofler et al., 2004).

Peptide-based vaccines could overcome these weaknesses. They use minimal antigenic epitopes to induce desired immune responses, and are less likely to trigger allergic or autoimmune responses (Skwarczynski and Toth, 2016). In recent years, there has been increasing interest in the study of peptide-based vaccines (Dudek et al., 2010). T. gondii is an intracellular parasite with a complex life cycle, so synthetic multiple antigenic peptide (MAP) vaccines containing different epitopes may prove a highly efficacious strategy in the development of T. gondii vaccines (Henriquez et al., 2010).

Successful T. gondii infection requires active invasion and the formation of the parasitophorous vacuole (PV) (Braun et al., 2008). Dense granules are secretory organelles involved in maturation and modification of both the PV and its membrane (PVM) (Nam, 2009). Dense granules proteins (GRAs) are major components of the vacuole surrounding tachyzoites and encysted bradyzoites, and are related to host-parasite interactions and immune responses (Cesbron-Delauw and Capron, 1993). Dense granule protein 10 (GRA10) which is released into the PV shortly after invasion and then localizes to the PVM (Ahn et al., 2005), is essential for parasite growth with potential immunogenic capability. Researchers found that there is severe inhibition of T. gondii growth in human fibroblasts cells when GRA10 is knocked out (Witola et al., 2014). Of note, immunogenic peptides from GRA10 in a vaccine formulation have not been previously explored.

However, peptides are very susceptible to enzymatic degradation. Thus, a delivery system is needed to protect protease-sensitive epitopes from degradation (van Riet et al., 2014; Skwarczynski and Toth, 2016). Recently, microparticles as a delivery system to load antigens has emerged as one of the most promising strategies to induce a strong immune response (Kwon et al., 2005; Reddy et al., 2007). In this way, the peptides are delivered by microspheres, thereby inducing enhanced recognition by the immune system compared with naked easy degradation peptides (van der Lubben et al., 2001). Thus far, many types of microparticles have been tested, and chitosan has attracted particular interest. Chitosan is the deacetylated form of chitin, a naturally occurring and abundantly available biocompatible polysaccharide (Shrestha et al., 2014). Chitosan microspheres have many advantages over traditional vaccine microsphere formulations with starch, gelatin, or albumin. They are easy to load with peptides, thereby circumventing protein denaturation (Lin et al., 2013). Compared to other biodegradable polymers, chitosan is the only one that has a cationic (Bernkop-Schnürch and Dünnhaupt, 2012) and mucoadhesive character, increasing residual time at the site of absorption, therefore prolonging the release time of protein antigens (Agnihotri et al., 2004). When chitosan degrades, the amino sugars produced are nontoxic and easily removable from the body without causing side reactions (Wang et al., 2016). More importantly, chitosan is a demonstrated natural adjuvant to promote dendritic cell (DC) activation and T helper 1 (Th1) cellular-associated immune responses via cGAS-STING signaling pathway (Carroll et al., 2016; Riteau and Sher, 2016). Collectively, as an attractive carrier and adjuvant, chitosan has been used extensively in vaccine applications (Islam and Ferro, 2016).

Herein, we present a novel attempt to combine highly immunogenic MAP from GRA10 with chitosan microspheres in the design of a vaccine against T. gondii. In this study, three dominant epitopes from GRA10 were screened from nine potential epitopes and were used to synthesize the MAP of GRA10 (G10E). The release efficiency of these chitosan microparticles containing the MAP of GRA10 (G10E-CS) was detected in terms of peptide loading and releasing efficiency. As an adjuvant, the capacity of chitosan to activate dendritic cells and T lymphocytes was tested in vitro. Furthermore, the protective effect of G10E-CS vaccination on acute and chronic toxoplasmosis murine models was evaluated. This study aims to make a contribution to peptides vaccines against T. gondii by exploring chitosan microspheres, an effective delivery system, to enhance MAP vaccine immunogenicity.

# MATERIALS AND METHODS

#### Animals

Female 6–8weeks-old BALB/c and C57BL/6 mice were purchased from Shandong University Laboratory Animal Centre (Jinan, China) and housed in SPF conditions. All the mice were housed 5 per cage under pathogen-free conditions and were adequately supplied with sterilized water and food.

### Ethical Approval

Experiments were carried out in accordance with animal ethics approved by the Institutional Animal Care and Use Committee of Shandong University under Contract LL201602044. Humane endpoints were chosen to terminate the pain or distress of experimental animals via euthanasia.

#### Parasite

Tachyzoites of the high-virulence RH strain (type I) (Howe and Sibley, 1995) of T. gondii were propagated in human foreskin fibroblast (HFF) cells in Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich, USA) supplemented with 10% fetal bovine serum (FBS) (Clark Biosciences,USA) as previously described (Shen and Sibley, 2014). Pru (Prugniaud) strain (type II), a low-virulence strain of T. gondii, (Saeij et al., 2005)was a kind gift from Professor Xingquan Zhu at Lanzhou Veterinary Research Institute, China. The cysts of the type II Pru strain were maintained in Kunming mice by oral passage of infectious cysts in mice as previously described (Zhou et al., 2016).

# Screening of Candidate Epitopes From GRA10

The PROTEAN subroutine in the DNASTAR software package was used to predict the B cell epitopes of GRA10 (TGME49\_268900, ToxoDB) as described previously (Wang et al., 2014). Three B cell candidate epitopes, GRA10192−215 (P1), GRA10221−244 (P2), and GRA10310−332 (P3), were selected with good hydrophilicity, high conformational flexibility, surface accessibility and strong antigenicity.

To predict the CD8+ and CD4+ T cell epitopes of GRA10, the complete amino acid sequence of the protein was analyzed using consensus algorithms available at the Immune Epitope Database and Analysis Resource (IEDB, http://www.iedb.org/) as described previously (Cong et al., 2010). GRA1049−57 (P4), GRA10161−169 (P5), and GRA10300−308 (P6) were chosen based on their predicted binding affinity to MHC I supertype molecules (those with a percentile rank lower than 10 were selected; Moutaftsi et al., 2006). GRA1080−94(P7), GRA10170−184(P8), and GRA10180−194(P9) were chosen based on their predicted binding affinity to MHC II (Wang et al., 2008). The peptides (P1-P9) sequences are shown in **Table 1** and were synthesized (>95% purity) by Synpeptide Co., Ltd. (Shanghai, China) and stored at −80◦C.

ELISA analysis of serum samples from T. gondii infected mice was used to screen three GRA10 B cell epitopes as previously described (Cardona et al., 2009). IgG OD value was recorded as the absorbance at 450 nm detected by a Thermo Scientific Multiskan FC Microplate Photometer (Thermo Scientific, USA). Meanwhile, lymphocyte proliferation assays of infected mice were used to screen GRA10 CD8+ T cell epitopes and CD4+ T cell epitopes as previously described (Wallace et al., 2008).

### Construction of Multiple Antigenic Peptide (MAP) of GRA10 (G10E)

T. gondii GRA10 MAP (G10E) was designed by linking the selected epitopes with the spacer sequence Gly-Ser (**Figure 1**) and

TABLE 1 | Candidates of B cell or T cell epitopes predicted from GRA10 of *T. gondii*.

*aThree B cell candidate epitopes, GRA10192-215 (P1), GRA10221-244 (P2), and GRA10310-332 (P3 with good hydrophilicity, high conformational flexibility, surface accessibility and strong antigenicity, were selected via DNASTAR software.*

*b*,*cCD8*+ *and CD4*+ *T cell epitopes of GRA10 were analyzed using consensus algorithms available at the Immune Epitope Database and Analysis Resource (IEDB, http://www.iedb. org/).*

then synthesized by Synpeptide Co., Ltd. (Shanghai, China) and stored at −80◦C.

#### Preparation of Chitosan Microspheres Loaded With G10E (G10E-CS)

Chitosan microspheres loaded with G10E were prepared by emulsion crosslinking method as previously described (Jose et al., 2013; Pichayakorn and Boonme, 2013) with minor modifications. Briefly, chitosan solution (3% w/w) was prepared by dissolving chitosan in 2% aqueous glacial acetic acid as aqueous phase. G10E peptides were mixed in this solution. Oil phase was paraffin oil containing 5% Span80. The aqueous phase was gradually added into the oil phase and continuously mixed at 1,000 rpm for 2h to form water-in-oil (w/o) emulsion. Subsequently, genipin was added into the mixture and mixed further for 2 h. The obtained microspheres were then separated by centrifugation at 3,000 rpm and the sediment was washed thrice with petroleum ether, isopropanol and deionized water and finally dried using a freeze dryer (CHRIST ALPHA 1-4 LSC, Germany) (**Figure 1**).

G10E-CS microspheres were characterized for morphological shape by a scanning electron microscope (JSM6610LV SEM, JEOL Ltd., Japan) as previously described (Wang et al., 2005). The samples were dusted on an adhesive carbon tape and coated with a thin layer of gold. Samples were then imaged at magnification of 15,000. The particle size and zeta potential of chitosan microspheres was determined by dynamic light scattering on a Zetasizer NanoZS (Malvern, UK). Measurements were carried out in triplicate.

#### Loading Capacity and Encapsulation Efficiency

1–5 mg/ml G10E peptides were mixed with 3% (w/v) chitosan microspheres and crosslinked by genipin as above described. The suspension was centrifuged (3,000 rpm for 30 min) to collect the supernatant. Then, the non-bound G10E concentration in the supernatant was determined by Protein Quantitative Reagent Kit-BCA Method (Beyotime, China) (Wang et al., 2016). Loading capacity (LC) and encapsulation efficiency (EE) in chitosan microspheres were determined by applying the following equations:

$$\begin{aligned} \text{Loading capacity (\%LC)} &= \\ \frac{\text{(total amount G10E)} - \text{(free G10E)}}{\text{weight of microparticles}} &\times 100\% \\ \text{Encapsulation efficiency (\%EE)} &= \\ \frac{\text{(total amount G10E)} - \text{(free G10E)}}{\text{total G10E}} &\times 100\% \end{aligned}$$

#### Evaluation of Release Characteristics of G10E *in Vitro*

G10E release from chitosan microparticles in vitro was determined in PBS (pH 7.4) as previously described (Skop et al., 2013) with minor modifications. Genipin-crosslinked G10E-CS microspheres and G10E peptides were dispersed in PBS (pH 7.4) at 37◦C under mild shaking (100 rpm). At specific time

intervals, 250 µl of the supernatant containing the released G10E was taken by centrifugation and stored at −20◦C in tubes. The chitosan microspheres that settled at the bottom of the tube were resuspended in PBS (pH 7.4) after each collection. After the last time point, the protein concentration of all the collected G10E was analyzed using the Protein Quantitative Reagent Kit-BCA Method.

The degradation of released G10E was analyzed by gel electrophoresis. Briefly, G10E-CS microspheres were dispersed in PBS (pH 7.4) using a shaking air bath (37◦C, 100 rpm) for 15 d. After centrifuging (5,000 rpm, 20 min), the microsphere precipitate at the bottom of the tube was collected to be analyzed by gel electrophoresis.

# The Expression of Costimulatory Molecules on the Surface of Stimulated Dendritic Cells

Bone-marrow derived dendritic cells (DCs) were prepared as previously described (Molavi et al., 2010). Briefly, C57BL/6 mice were euthanized, intact femurs were removed, and purified of surrounding muscle tissue. The bones were washed with sterile PBS, the bone marrow cells were harvested by repeated flushing with RPMI 1640 using a syringe with a 0.45 mm diameter needle. To derive conventional DCs, bone marrow cells (2 × 106 cells/ml) were cultured for 7 days in 6-well plate (Corning Inc., USA) in RPMI 1640 medium (Hyclone, USA) supplemented with 10% FBS (Hyclone, USA), 50µM penicillin and streptomycin, and 20 ng/ml GM-CSF and IL-4 (Sigma-Aldrich, USA). Half of the medium was replaced with fresh complete medium on day 3 and 5. At day 7, non-adherent cells were harvested and ready for use as mature DCs.

To analyse costimulatory molecule expression, dendritic cells (2 × 106 cells/ml) were seeded in 96-well plate (Corning Inc., USA) in the same medium as described above (including GM-CSF and IL-4) and cultured with CpG (4µg/mL), CS, G10E or G10E-CS (10µg/mL each) for 48 h. CpG, a TLR agonist that generally enhances CD40 and CD86 surface expression, served as a positive control. Cell suspension was subsequently stained with 0.3µg/ml of anti-CD11c, 1.25µg/ml of anti-CD86 and 2.5µg/ml of anti-CD40 antibody (BD Biosciences, USA) (Carroll et al., 2016). Samples were detected by Cytoflex S Flow Cytometer (Beckman Coulter, USA) and the data were analyzed by CytExpert software.

# Activation of Dendritic Cells and Lymphocyte *in Vitro* Induced by G10E-CS Microspheres

Dendritic cells (DCs) were plated at 2 × 105 cells per well and stimulated as described above. At the same time, lymphocytes isolated from the spleen of C57BL/6 mice were plated in 96-well plates, at a density of 2 × 106 cells/ml, in 100 µL RPMI-1640 medium supplemented with 10% FBS, 50µM penicillin and streptomycin. Then, induced DCs were cocultured with these T cells (1:10 ratio) for 72 h (Höpken et al., 2005), and lymphocyte proliferation was measured by CCK-8 assay. In the meantime, supernatants of mixed cells were assayed for IL-2 and IFN-γ using a commercial ELISA Kit (R&D Systems, USA) following the procedure recommended by the manufacturer.

## Cytotoxicity of G10E-CS Microspheres

The cytotoxicity of the G10E-CS microspheres in vitro was evaluated by CCK-8 assay (Dojindo, Japan) conducted on the aforementioned dendritic cells (DCs) and T lymphocytes as previously described (Shrestha et al., 2014). Briefly, these cells were added to 96-well plates of 2 × 106 cells/ml at 100 µL per well and incubated overnight. The culture medium was then replaced by serum-free medium with different concentrations (125, 250µg/mL and 500µg/mL) of G10E-CS microspheres and incubated for 48 and 72 h at 37◦C. Ten micro liters CCK-8 was added to the wells and the absorbance of the solution was measured at 450 nm in a Thermo Scientific Multiskan FC Microplate Photometer (Thermo Scientific, USA). Cell viability (%) was defined as the OD ratio in test group relative to controls. All experiments were carried out five times.

### Immunization and Challenge Infection in Mice

BALB/c mice were randomly divided into four groups (n = 23 per group) and were immunized thrice via intramuscular injection at 2-week intervals with G10E (100 µg), G10E-CS microspheres containing 100 µg of G10E (667 µg) (LC = 15%) or empty CS microsphere (667 µg) (Zhou et al., 2007; Jose et al., 2012). Mice that received 100 µl PBS were used as non-immunized control.

Two weeks after the last immunization, the protection of G10E-CS microspheres against acute and chronic T. gondii infection was investigated as previously described (Wang J. L. et al., 2017). Ten mice per immunized group were infected intraperitoneally with a lethal dose of RH strain (1 × 103 tachyzoites). The challenged mice were monitored over a period of 21 days and observed daily for mortality.

At the same time, 10 mice per group were challenged orally with a sublethal dose of cysts (30 cysts) of the PRU strain (type II) to evaluate the effect of vaccination in mice with chronic toxoplasmosis. All surviving mice were euthanized at 45 days after challenge. Brains were collected and homogenized in 1 ml PBS. The number of cysts per brain was confirmed by counting eight 10 µl samples of each cerebral homogenate under an optical microscope.

# Detection of Total IgG and Antibody Isotypes in Vaccinated Mice

Serum samples were collected from the mice by retro-orbital bleeding at 0, 2, 4, and 6 weeks after immunization. Standard ELISA assay was used to determine the levels of T. gondiispecific IgG, IgG1 and IgG2a antibodies in the serum samples as previously described (Tang et al., 2016). Briefly, 96-well plates were coated with the 100 µL (10µg/mL) G10E peptides in 50 mM carbonate-bicarbonate buffer (pH 9.6) and were incubated at 4 ◦C overnight. After washing three times with PBS plus 0.05% Tween 20 (PBST), the coated plates were blocked with 1% low fat milk in PBST for 1 h at 37◦C. The mouse serum was diluted in PBS (1:25) and incubated at 37◦C for 1 h. Then plates were washed, and anti-mouse-IgG, IgG1 or IgG2a HRPconjugated antibodies (Sigma-Aldrich, USA) were added to each well. Peroxidase activity was revealed by 10 mg/ml 3, 3′ , 5, 5′ tetramethylbenzidine (TMB, Sigma–Aldrich, USA) and stopped by adding 50 µl of 2 M H2SO4. The results were recorded as the absorbance at 450 nm and detected by a Thermo Scientific Multiskan FC Microplate Photometer (Thermo Scientific, USA). For each serum sample the assay was done in triplicate and average values were calculated.

### Lymphocyte Proliferation Assay

Two weeks after the last immunization, three mice per group were euthanized to obtain single splenocyte suspensions as described previously (Luo et al., 2017). After erythrocytes were lysed, the splenocytes were adjusted to a concentration of 2 × 106 cells/ml in RPMI-1640 medium with 10% FBS and plated in 96-well plates in presence of the G10E peptides (10µg/mL). Plates were incubated for 72 h at 37 ◦C. Lymphocyte proliferative activity was measured by a Cell Counting Kit-8 (CCK-8, Dojindo; Japan) according to manufacturer's instructions. The absorbance was detected at 450 nm and the stimulation index (SI) was calculated.

#### Flow Cytometry Analysis of T Cell Subsets and Cytokine Production

The percentages of CD4+ and CD8+ T lymphocytes subsets and the production of cytokine in the splenocytes of immunized mice were determined using flow cytometry techniques as described previously (Zhang et al., 2015b; Wang S. et al., 2017). Briefly, splenocytes (2 × 106 cells/ml) of vaccinated mice were stimulated with G10E peptides (10µg/mL) for 72 h. Then the suspensions were stained with anti-mouse CD3-APC, anti- mouse CD4-FITC and anti-mouse CD8-PE (eBiosciences, USA) for 30 min at 4 ◦C in the dark.

Splenocytes (2 × 106 cells/ml) were also stimulated with G10E peptides for 72 h in the presence of Cell Stimulation Cocktail (eBiosciences, USA) containing Phorbol myristate acetate (PMA, 20 ng/ml), Ionomycin (2µg/ml), Brefeldin A (1µg/ml), and Monensin (1µg/ml) to inhibit the secretion of cytokine into the extracellular space. The cells were fixed using an Intracellular Fixation & Permeabilization Buffer Set Kit in accordance with the manufacturer's protocol (eBiosciences, USA) and then stained directly with anti-mouse CD4-FITC, anti-mouse CD8-PE, antimouse IL-2 (APC), anti-mouse IFN-γ (PerCP-Cyanine 5.5), antimouse IL-4 (APC), and anti-mouse IL-10 (PerCP-Cyanine5.5) (eBiosciences, USA) for 30 min at 4◦C. All these cell population were analyzed by Cytoflex S Flow Cytometer (Beckman Coulter, USA) and the data were analyzed by CytExpert software.

#### Statistical Analysis

Statistical analysis was carried out using SPSS19.0 (SPSS Inc., Chicago, IL, USA) and GraphPad Prism 7.0 (GraphPad Software Inc., San Diego, USA). Differences between groups were analyzed using one-way ANOVA analysis. Student's t-test was used for comparison between levels of IgG1 and IgG2a isotypes. Survival percentage was analyzed by the Kaplan-Meier test and survival curves were compared using the Log-rank test. Data are expressed as means ± SD. Values of p < 0.05 were considered statistically significant.

# RESULTS

# Epitopes Screened From *Tg*GRA10 and Construction of Multiple Antigenic Peptide

The peptide sequences of candidate epitopes from GRA10 showing high affinity in the prediction algorithm in **Table 1** were further tested in mice. The IgG test showed that all the B cell epitopes from GRA10 could recognize the IgG in the serum of BALB/c mice infected by T. gondii, in which GRA10192−215 (P1) was demonstrated to induce highest level of IgG (**Figure 2Aa**). Lymphocyte proliferation testing showed that GRA10161−169 (P5) and GRA1080−94 (P7) stimulated the highest level of proliferation of lymphocytes in infected mice (**Figure 2Ab**).

Thus, GRA10192−215 (P1), GRA10161−169 (P5), and GRA1080−94 (P7) were chosen to construct the MAP G10E with linker GS (**Figure 1**). Mice immunized with P1(B cell epitope), P5(CD4+ T cell epitope), P7(CD8+ T cell epitope) or G10E were tested for B cell antibody level and T cell proliferation ability. The results showed that significant enhanced B cell and T cell immune responses were achieved in G10E vaccinated group (**Figures 2Ba,b**).

FIGURE 2 | Screening of dominant epitopes from GRA10 and characteristic of G10E-CS. (A) Screening of the dominant B cell epitopes (a) and T cell epitopes (b) from TgGRA10. Positive serum samples from *T. gondii* infected mice were used to screen dominant B cell epitopes by ELISA analysis. Lymphocyte proliferation test was used to identify dominant CD8+ T and CD4+ T cell epitopes from spleen cells of infected mice by CCK-8 assay. P1(TQSPPESRKKRRRSGKKKRGKRSV)(B cell epitope), P5(LGYCALLPL) (CD8+ T cell epitope) and P7(SGFSLSSGSGVSVVE) (CD4+ T cell epitope) were selected to construct the multiple antigenic peptide (MAP) of *T. gondii* GRA10 (G10E). Values represent mean ±SD (*n* = 5). \**p* < 0.05. (B) Immunogenicity of P1, P5, P,7 and G10E peptides. Mice immunized with P1(B cell epitope), P5(CD4+ T cell epitope), P7(CD8+ T cell epitope) or G10E (P5-P7-P1) were tested for B cell antibody (a) and T cell proliferation ability (b).\*\**p* < 0.01. (C) The purity of G10E peptides detected by high performance liquid chromatography (HPLC) (a) and the molecular weight of G10E peptides analyzed by electrospray ionization mass spectrometry (ESI-MS) (b). (D) Scanning electron microscopy (SEM) images of G10E-CS microspheres prepared by emulsion crosslinking method. G10E-CS microspheres were then imaged at magnification of ×15,000 (bar represents 1µm). (E) The diameter (a) and zeta of G10E-CS (b) microparticles was detected by Malvern particle size potentiometer. (F) Loading capacity (%LC) and encapsulation efficiency (%EE) of G10E-CS microspheres amongst 1–5 mg/ml concentration of total G10E peptides (a). Release profile of G10E peptides from free G10E peptides and crosslinked chitosan microparticles (G10E-CS) *in vitro* over a 15-day period (b). The amount of G10E in the supernatant was measured using BCA assay. Values represent mean ± SD (*n* = 3).

# Physical Characterization and Release of Chitosan Microspheres Loaded With G10E

Chitosan microspheres loaded with G10E peptides (G10E-CS) were prepared by emulsion cross-linking method. The purity of G10E was 98.4% detected by high performance liquid chromatography (HPLC) (**Figure 2Ca**), and its molecular weight was 5450.9 daltons, analyzed by electrospray ionization mass spectrometry (ESI-MS) (**Figure 2Cb**). The morphology of G10E-CS microspheres formed using 3% chitosan was spherical in shape with a smooth surface, as observed by scanning electron microscopy (SEM) (**Figure 2D**). The average diameter of G10E-CS microparticles was 1283 nm with the low dpi detected by Malvern particle size potentiometer, suggesting that G10E-CS were uniform in size (**Figure 2Ea**).

The zeta potential of the CS microspheres was positive 37.1 ± 5.2 mV while the G10E peptides was negative 31.7 ± 9.1 mV (**Table S1**). After loading G10E into chitosan microspheres, the G10E-CS's zeta potential were positive 36.0 ± 5.48 mV (**Figure 2Eb**). Size and zeta analysis of G10E-CS formulations confirmed that G10E was completely loaded into the core of the CS microspheres, and not simply adherent to their surfaces.

The loading capacity and encapsulation efficiency of the chitosan microparticles were analyzed. When the concentration of G10E reached 4 mg/ml, a high encapsulation efficiency (EE) of 71% was achieved, and the peptide loading was almost saturated (15.3%). Therefore, 4 mg/ml G10E peptides were selected as the optimal concentration (**Figure 2Fa**).

We further evaluated the release of the entrapped G10E peptides from the chitosan microparticles. The crosslinked G10E-CS microspheres showed the more favorable and steady release profile (**Figure 2Fb**). Only 22.4% of the total encapsulated G10E peptides were released within the first day and slowly released 2.4–6.9% peptides daily over the next 14 days. While G10E peptides released 80% in the first 2 days in the freedom style This was further supported by PAGE and SDS-PAGE analysis of released G10E (**Figure S1**).

### G10E-CS Microspheres Induce the Activation of Dendritic Cells and Lymphocyte *in Vitro*

In order to determine if chitosan can induce dendritic cells (DC) activation in vitro, the expression of the co-stimulatory molecules CD40 and CD86 was detected by flow cytometry in stimulated DCs. Dramatic upregulation of CD40 and CD86 in DCs was observed after incubation with G10E-CS, G10E, CS, CpG (positive control) compared to PBS groups (p < 0.05; **Figure 3A**). G10E-CS microspheres achieved the highest level of the co-stimulatory molecules in dendritic cells (p < 0.01).

We further confirmed that CD86 and CD40 could provide activate lymphocytes. Hence, we assessed T cell proliferation and cytokines levels in supernatant of C57BL/6 mice lymphocytes after coculture with the above stimulated DC cells in vitro. As shown in **Figure 3B**, stimulated DCs provided more efficient co-stimulation of T cell proliferation, especially for G10E-CS stimulated cells (p < 0.01).

Supernatants of lymphocytes cocultured with the DCs showed that the CpG, G10E-CS, G10E microparticles could induce substantial production of IFN-γ and IL-2 compared to other groups (p < 0.05). The level of IFN-γ induced by G10E-CS microspheres (p < 0.01) was particularly noteworthy (**Figure 3C**).

Further, we studied the cytotoxic activity and biocompatibility of G10E-CS. Using CCK-8 assay, the viability of dendritic cells and T cells was analyzed by exposing them to different concentrations of G10E-CS (125, 250, and 500µg/ml) for different incubation times (48 or 72 h). For DCs and lymphocytes, the results demonstrated that all the G10E-CS microspheres showed cell viabilities higher than 90% of the negative control (**Figure 3D**). Hence, G10E-CS microspheres were suggested to be safe for the mouse vaccine.

#### G10E-CS Induced Robust *T. gondii* Specific Humoral Immune Responses

Specific IgG in the sera of mice vaccinated intramuscularly with G10E-CS, G10E, CS, was measured every 2 weeks. A dramatically higher level of total IgG was observed in the sera of mice immunized with G10E-CS and G10E after the third immunization, in contrast to the other groups. G10E-CS immunized mice had the highest level of T. gondii specific IgG (p < 0.05) (**Figure 4A**).

Furthermore, the level of specific IgG1 and IgG2a sub-classes against G10E peptides was measured. As shown in **Figure 4B**, significant production of IgG2a and IgG1 was observed in the sera of mice immunized with G10E-CS (p < 0.01) compared with G10E group. These results indicated that mice immunized with G10E-CS microspheres generated Th1 polarize immune response with more IgG2a than IgG1.

# G10E-CS Induced Robust *T. gondii* Specific Cellular Immune Response

Antigen-specific proliferative responses of lymphocytes from immunized mice were detected by CCK-8 2 weeks after the third immunization and represented by the SI value as illustrated in **Figure 5A**. The level of lymphocyte proliferation in the mice immunized with G10E-CS (p < 0.01) and G10E (p < 0.05) was significantly higher than those of other groups. This result indicated that the T lymphocytes proliferation of mice vaccinated with G10E-CS was successfully stimulated.

To determine CD8+ or CD4+ T cells numbers after vaccination, the percentages of CD4+ and CD8+ T lymphocytes subsets in the spleen of immunized mice were evaluated by flow cytometry analysis. As shown in **Figures 5Ba,b**. the percentages of CD8+ and CD4+ T cells were both increased in mice immunized with G10E-CS and G10E compared to other groups (p < 0.05). G10E-CS-immunized mice also had the highest ratio of CD8+/CD4+ (p < 0.05). G10E-CS microspheres, therefore, could elicit more activation of CD8+ and CD4+ T cells in immunized mice than other vaccine formulations.

The production of cytokines in splenocytes supernatants of immunized mice was evaluated by Flow Cytometry. Significantly higher levels of IFN-γ and IL-2 were secreted by lymphocytes of

the G10E-CS (p < 0.01) and G10E (p < 0.05) immunized mice compared to that of CS and PBS (**Figures 5Ca,b**). Interestingly, spleen cells from mice immunized with G10E-CS produced more IL-10 and IL-4 compared to other groups (p < 0.05; **Figures 5Da,b**).

#### G10E-CS Immunization Against Acute and Chronic Toxoplasmosis

In order to evaluate if G10E-CS could confer effective protection against T. gondii acute and chronic infection, immunized mice were challenged with RH tachyzoites, 10 mice per group were challenge with 1 × 103 RH tachyzoites 2 weeks after the last immunization. The survival curves of immunized mice challenged with 1 × 103 RH tachyzoites 2 weeks after the last immunization were illustrated in **Figure 6A**. All mice vaccinated with PBS, G10E, or CS succumbed within 10 days of infection. In contrast, a significantly prolonged survival time (21 days) was observed in mice vaccinated with G10E-CS microspheres compared to other groups (p < 0.01). While only 3 days survival extension when mice immunized with only G10E without CS (10 days) compared to control group (p > 0.05).

The efficacy of this vaccine formulation in the context of chronic infection with T. gondii was also evaluated by challenging the immunized mice with 30 cysts of the PRU strain orally. On day 45 post challenge, as shown in **Figure 6B**, G10E-CS immunized mice had fewer cysts (882 ± 194 cysts) in brain than G10E (1852 ± 245 cysts), CS (1966 ± 183 cysts) and PBS (2091 ± 215 cysts) treated mice (p < 0.01). This result suggests that the G10-CS microsphere vaccine could significantly reduce the cyst form in mice chronically infected with T. gondii.

### DISCUSSION

In this study, we first screened and constructed a MAP encoding three dominant B, CD8+ and CD4+ cell epitopes from T. gondii GRA10 (G10E). Chitosan microspheres were loaded with adequate G10E in an effort to protect the peptides from enzymatic degradation and release the G10E peptides stably. This complex was referred to as G10E-CS. In vitro, G10E-CS microspheres induced DC maturation and T cell activation with enhanced expression of costimulatory molecules (CD40 and CD86). In vivo, significantly prolonged survival and

decreased brain cyst burdern were observed in mice vaccinated with G10E-CS, accompanied by strong humoral and cellular responses.

To control outbreaks of toxoplasmosis, caused by this intracellular protozoan parasite with its complex life cycle, the MAP vaccine, containing multiple epitopes, has many advantages over traditional vaccines (Henriquez et al., 2010; Skwarczynski and Toth, 2016). Currently, individual B (Darcy et al., 1992), CD4+ T (Grover et al., 2012), CD8+ T (Blanchard et al., 2008) cell epitopes have been able to induce partially protective immune responses against toxoplasmosis, and MAP vaccines are more likely to confer more robust protection (Bastos et al., 2016). GRA proteins of T. gondii, major components of both tachyzoites and bradyzoites, are related to host-parasite interaction and immune response. Dense granule protein 10 (GRA10) is essential for parasite growth with potential immunogenic capability, and it had not been studied as a candidate vaccine antigen prior to the work presented herein. In this study, we used bioinformatics and immunological methods to select nine B cell or T cell mimotopes of GRA10. Among them, three strongest immunogenic B and T cell epitopes, namely B cell epitope (TQSPPESRKKRRRSGKKKRGKRSV), CD8+ T cell epitope (LGYCALLPL), and CD4+ T cell epitope(SGFSLSSGSGVSVVE), were selected to construct the MAP, G10E. Peptide-based vaccines have some weaknesses. For example, they are poor immunogens, in and of themselves, and very susceptible to enzymatic degradation. Therefore, they need the assistance of an effective delivery system (Salvador et al., 2011; Skwarczynski and Toth, 2016).

Recently, a variety of delivery systems have been used for the formulation of peptide vaccines against T. gondii. Of particular interest, usage of nano- and micro-particles for vaccine delivery has rapidly become popular (Skwarczynski and Toth, 2016). Poly(lactide-co-glycolide) (PLG) has been tested as safe delivery systems to encapsulate recombinant SAG1 protein (Chuang et al., 2013) or rhoptry proteins (ROP18 and ROP38) (Xu et al., 2015) as controlled-release microparticle vaccines. Nanosized carriers have also been used to deliver parasite antigens to immune competent cells. Assembled GRA7 derived HLA-B∗ 0702 -restricted epitopes into nanospheres (SAPN) (El Bissati et al., 2014) or loaded T. gondii extracts protein in porous nanoparticles (DGNP) (Dimier-Poisson et al., 2015) have been developed. All these delivery systems could protect protein epitopes against enzymatic degradation, and microspheres can sustain the stable release of antigen. Chitosan has particular advantages because easily assumes a spherical conformation, and is positively charged. It has been widely studied for vaccine delivery systems to increase the response to immunization with protein antigens, DNA plasmids, and bacterial derived toxins (Arca et al., 2009; Chua et al., 2012). There is comparatively little assessment of the utility of chitosan-based microparticles as vaccine carriers for peptides.

In our study, G10E-CS microspheres prepared by emulsion cross-linking were spherical in shape and had uniform diameter around 1,283nm. Size and zeta analysis of G10E-CS formulations confirmed that G10E was completely loaded into the core of the CS microspheres, and not simply adherent to their surfaces. These properties ensured no obvious degradation of G10E peptides during loading and release processes. This was further supported by PAGE analysis of released G10E. As a cationic polymer, chitosan can bind strongly to cell surfaces with a predominantly negative charge, thereby increasing residence time and releasing peptides in a sustained fashion in targeted cells (Koppolu et al., 2014). In this study, we observed a slow-release effect of G10E-CS microspheres. It is worth noting that nano- or micro-particles used in vaccines are often limited by their toxicity and the difficulty with which they are manufactured (Zhang et al., 2015a). These effects have been observed with chitosan microspheres in the past. Glutaraldehyde, the traditional cross-linking agent used in the production process of CS microparticles may be built into the vaccine carrier and show cell cytotoxicity (Agnihotri et al., 2004).

FIGURE 5 | Cellular immune response induced in BALB/C mice after vaccination with G10E-CS. BALB/c mice were immunized with G10E-CS, CS, G10E, or PBS three times at 2-week intervals. Then splenocytes were harvested from three mice per group 2 weeks after the final immunization. Cellular immune responses were analyzed after stimulation with G10E peptides (10µg/ml) for 72 h. (A) The lymphocyte proliferative response in immunized mice was measured by CCK-8 assay. The absorbance was detected at 450 nm and the stimulation index (SI) was calculated. (B) The percentages of T lymphocyte subsets (a) and ratio of CD8+/CD4+ (b) in immunized mice. The G10E stimulated splenocytes were stained with anti-mouse CD3-APC, anti- mouse CD4-FITC and anti-mouse CD8-PE for 30 min and cell population was analyzed by flow cytometry. (C,D) The production of cytokines (IFN-γ, IL-2) (C) and cytokines (IL-4, IL-10) (D) in the immunized mice. The splenocytes were stimulated with G10E peptides for 72 h in the presence of Cell Stimulation Cocktail to inhibit the secretion of cytokine into the extracellular and were fixed using an Intracellular Fixation & Permeabilization Buffer Set Kit. After stained with anti-mouse CD4-FITC, anti-mouse CD8-PE, anti-mouse IL-2 (APC), anti-mouse IFN-γ (PerCP-Cyanine 5.5), anti-mouse IL-4 (APC) and anti-mouse IL-10 (PerCP-Cyanine5.5) for 30 min, the cells population was analyzed by flow cytometry. Data represent the mean ± SD and splenocytes from three mice in each group were tested individually. \**p* < 0.05, \*\**p* < 0.01.

To overcome this problem, we replaced glutaraldehyde with genipin, an aglucone of geniposide extracted from gardenia fruits and used in traditional Chinese medicine with demonstrated low cell cytotoxicity (Lin et al., 2013). Moreover, its low crosslinking speed with chitosan could avoid the occurrence of agglomeration (Jin et al., 2004). This difference led to the more uniform particle size observed herein. As expected, all concentrations of G10E-CS microspheres studied showed

tachyzoites challenge. Two weeks after the final immunization, 10 mice per group were intraperitoneally infected with 1 × 103 tachyzoites of RH strain and observed daily for mortality. (B) The cyst number in the brain of mice after sublethal PRU strain cyst challenge. 10 mice per group were challenged orally with a sublethal dose of cysts (30 cysts) of the PRU strain (type II). Cyst load was counted from whole brain homogenates of mice 45 days after challenge. Data are representative results of three independent experiments and are represented as the means ± SD. \**p* < 0.05, \*\**p* < 0.01.

almost no cytotoxicity, a direct result of the choices of molecules with studies supporting good biocompatibility and low cytotoxicity.

Generally, immune responses mediated by CD4 + Th1 cell, especially the cytotoxic T lymphocyte (CTL) response, can directly suppress the growth of dividing tachyzoites and reactivation of the cysts of T. gondii (Ching et al., 2016). Thus, activation of T cells and production of Th1 cytokines are pivotal for the control of the parasite (Wang J. L. et al., 2017). Given the effective delivery system of the chitosan (CS) microspheres, the MAP (G10E) were stably released, inducing a strong cell-mediated immune response. In this study, we observed that T lymphocyte proliferation in mice immunized with G10E-CS microspheres was significantly higher than other groups. Meanwhile, percentages of CD8+ and CD4+ T lymphocyte were both increased dramatically in this group, together with the highest ratio of CD8+/CD4+ T cells. The ability of G10E-CS microspheres to stimulate T lymphocytes proliferation and differentiation was similar to that noted in a previous study (Xu et al., 2015), which encapsulated T. gondii ROP38/18 into PLG microparticles. The CD8+ T lymphocyte subset is important in responding to T. gondii infection and the helper CD4+ T subgroup appears to cooperate in the control of chronic infection by producing IFN- γ and redundant CD8+ T cells (Caetano et al., 2006).

IFN-γ plays a major role in protecting the infected host against T. gondii infection. Its secretion is directly proportional to the mortality rate of infected mice, because it can inhibit tachyzoite propagation during the early stages of infection by activating phagocytes such as macrophages, provide the appropriate cytokine environment (Innes et al., 2009; Sonaimuthu et al., 2016). IL-2 is essential for the development of cytotoxic CD8+ lymphocytes (Sa et al., 2013). As expected, we found that mice injected with G10E-CS generate the highest levels of IFN-γ and IL-2 in all immunized mice. Thus, we speculate that G10E-CS induces a stronger cell-mediated immune response than G10E due to enhanced delivery via the CS delivery system, and the response is stronger than CS alone due to the immunogenicity of the MAP, G10E. IFN-γ and IL-2 are critical for coordinating protective immunity against T. gondii (Luo et al., 2017). Many nano- and micro-particle vaccine formulations against T. gondii have observed similar results (Chuang and Yang, 2014; El Bissati et al., 2014; Dimier-Poisson et al., 2015; Xu et al., 2015).

Previous studies have also indicated a key role for vesicle size in determining the Th1/Th2 bias of the resulting immune response. Vaccination using influenza A antigen entrapped in the larger bilosome formulation (980 nm) resulted in a more biased Th1 response as measured by antigenspecific IgG2a serum levels and splenocyte IFN-γ production than vaccination using the smaller bilosomes (250 nm) (Mann et al., 2009). In this study, the size of G10E-CS microparticles was about 1,000 nm which result in Th1 bias immune response.

Th2-related IL-4 and IL-10 possess the antagonistic effect to IFN-γ (Bessieres et al., 1997), making this observation compelling. Interestingly, we also found spleen cells from mice immunized with G10E-CS produced slightly more IL-4, IL-10 than other groups. It has found that the early mortality of infected mice in the acute phase of toxoplasmosis is caused by severe inflammatory effects and lethal immunopathology provoked by IFN-γ, rather than parasitic infection itself (Ching et al., 2016). Even it has been recognized that a protective immune response against T. gondii infection requires a Th1 predominance response (Kumar et al., 1998; Meira et al., 2014). Th2-related cytokines play a vital role in downregulating this short-term deleterious inflammatory effect and reduce fatality following T. gondii infection (Neyer et al., 1997; Pinzan et al., 2015). Our findings are consistent with this conclusion. It is worth mentioning that GRA10 is found to translocate into the host cell nucleus and interact with signal transducers and promote activation of transcription factor 6 (STAT6). STAT6 plays a central role in the activation of the IL-4 response, triggering the expression of anti-apoptotic factors. In sum, mice immunized with G10E-CS have induced a combined Th1/Th2 immune response, skewed predominantly toward a Th1 response.

The critical role of antibody in immunity against T. gondii has long been recognized (Correa et al., 2007). Antigenspecific antibody can inhibit the tachyzoite directly and impair attachment to host cells (Sayles et al., 2000). In the present study, the increased production of total IgG, IgG2a and IgG1 were all observed in the sera of mice immunized with G10E-CS microspheres. As IgG1 is Th2 related and IgG2a is associated with Th1-driven immunity (Germann et al., 1995; Wang S. et al., 2017), our data supports the idea that G10E-CS microspheres generated Th1 polarize immune response with more IgG2a than IgG1.

Although there are some effective nano- or micro-particles vaccines against T. gondii, almost no delivery system can simultaneously act as a good adjuvant. A potent adjuvant required for T. gondii vaccines is able to elicit a Th1-type immune response (Verma and Khanna, 2013). It is exciting to find that chitosan can also serve as an attractive candidate adjuvant to induce dendritic cells (DCs) activation and, thereby, promote Th1 cellular immune responses (Riteau and Sher, 2016). In particular, internalized chitosan induces mitochondrial stress and increase reactive oxygen species (ROS) production, which act as a trigger for cGAS-STING pathway (Carroll et al., 2016). This pathway could mediate the production of IFN-γ, responsible for triggering the activation of DCs and T helper type 1 (Th1) immunity responses (Dubensky et al., 2013). DC activation is characterized by elevated surface expression of costimulatory molecules CD40 and CD86 (Reis e Sousa, 2006). The costimulatory molecules also have a key role in regulating T cell proliferation, cytokine production and generation of CTL (Chen and Flies, 2013). In our study, we found that bone-marrow-derived DCs exposed to G10E-CS microspheres in vitro showed drastically increased expression of CD40 and CD86. G10E-CS stimulated DCs could both induce efficient costimulation of T cell proliferation and substantial production of IFN-γ and IL-2. Therefore, G10E-CS microspheres could induce DC maturation and T cell activation in vitro by upregulating the expression of costimulatory molecules.

The survival rate of vaccinated mice against T. gondii challenge is considered the most direct way to assess a candidate vaccine (Zheng et al., 2017). In the present study, we found that mice vaccinated with G10E-CS microspheres have significantly prolonged survival time (21 days) against lethal challenge with the virulent RH strain of T. gondii. In contrast, mice immunized with PBS, G10E, or CS had all succumbed within 10 days after infection. Evaluation of the ability of G10E-CS to protect against chronic infection was also of interest. After challenging with a sublethal dose of cysts of the PRU strain, cyst burden in mice vaccinated with G10E-CS was greatly decreased (57.8% reduction) compared to the other groups. These results, taken together, proved that our G10E-CS microsphere vaccine was able to protect against both acute and chronic T. gondii infection.

In summary, in this study, G10E-CS microspheres have triggered a strong, mixed Th1/Th2 cellular and humoral immune response in immunized mice. At the same time, CS as a potent adjuvant, has also enhanced cellular immunity by eliciting Th1 type immune responses. Therefore, the humoral and cellular immune responses induced by G10E-CS microspheres were interrelated and synergistic in strengthening protection against T. gondii. Our results proved that chitosan microparticles loaded with MAPs could be a promising approach to vaccine development against T. gondii.

A limitation of this study was that the loading capacity (LC%) of the chitosan microsphere was low (15.3%). This may be due to liquid paraffin on the surface of the microspheres, which was hard to clean completely, potentially affecting the peptides contact with the microspheres. Interestingly, this loading capacity was slightly higher than that observed with chitosanbased microspheres loaded with other protein (Li et al., 2017). It will be necessary to increase the loading capacity to improve the protection efficiency of G10E-CS vaccine. Therefore, other optimized published methods for manufacturing protein-loaded chitosan particles can be used in future research.

# AUTHORS CONTRIBUTIONS

HC and HY designed experiments. HY, JG, TW, and XS performed experiments and analyzed data. JG and HY wrote the manuscript. HC and YL revised the manuscript. CZ, HZ, and SH provided advice for experiment. All authors read and approved the final manuscript.

# ACKNOWLEDGMENTS

This study was supported by grants from the development Project of Shandong Province (Grant No. 2016GSF201201), Shandong Natural Science Foundation Project (Grant No. ZR2017MH043 and ZR2016HM74) and the National Natural Science Foundation Project of China (Grant No. 81171604).

# SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcimb. 2018.00163/full#supplementary-material

Figure S1 | The release of G10E peptides was analyzed by PAGE (A) and SDS-PAGE (B). G10E-CS microspheres were dispersed in PBS (pH 7.4) using a shaking air bath (37◦C,100 rpm) for 15 d. After centrifuging (5,000 rpm, 20 min), the microspheres precipitation at the bottom of the tube was collected to be analyzed by non-denaturing gel electrophoresis (PAGE) and denaturing gel electrophoresis (SDS-PAGE).

Table S1 | Size and zeta potential of G10E-CS, CS and G10E. Size and zeta potential of chitosan microspheres loaded with G10E peptides (G10E-CS), chitosan microspheres (CS) and free G10E peptides.

# REFERENCES

and the parasite genome, binding assays, studies of human T cell responses, and immunization of Hla-A∗ 1101 transgenic mice including novel adjuvants provide a foundation For Hla-A03 Restricted Cd8+T cell epitope based, adjuvanted vaccine protective against Toxoplasma gondii. Immunome Res. 6:12. doi: 10.1186/1745-7580-6-12

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Guo, Sun, Yin, Wang, Li, Zhou, Zhou, He and Cong. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

digital media

of impactful research

article's readership


**2. Literature Review**

Recently, Data Envelopment Analysis (DEA) has become one of the main tools for environmental assessment. This methodology was initially proposed by Charnes et al. [7], and since then it has become an important tool for measuring relative efficiency in different fields [8]. This tool can serve as a guideline for firms and policy makers. Since Faere et al. [9] introduced the concept of the undesirable output, the use of DEA has widely spread in environmental assessment, becoming the most popular application area within the DEA methodology [10]. This section presents a complete review of this methodology and its application in the power generation sector, and its different variations in terms of environmental models and dynamic models.


**4. Discussion**

Overall, studies on the application of the broad strategies used in SMT show that it is most of the time effective and for an extensive category of difficulties. For instance, it sometimes has a physical impact on medical conditions and is clearly effective to help patients cope with associated psychological reactions. Most studies, but not all, support the effectiveness of SMT. When it comes to dealing with chronic physical illness, strategies having an enduring impact on patient's life and coping style might also be more effective than brief interventions.

The same can be said when looking at mental disorders. Our literature review showed that SIT and AMT are effective tools for PTSD and, to some extent, for other disorders such as generalized anxiety disorder. The broad set of SMT intervention are powerful enough to treat mental disorders, as long as they are structured an include ingredients at the core of SIT and AMT like exposure, cognitive restructuring and homework assignments. Softer techniques often included in SMT, like relaxation and basic coping skills, can be learned and mastered effectively by people who suffer from significant life impairment like schizophrenia or alcoholism in order to deal with stress-related issues instead of treating the disorder itself.

As for the control of non-clinical anxiety symptoms, a large number of studies successfully used SMT to train people to control their stress. These studies represent a secondary prevention approach, where people are already dealing with stress that has not yet reached a clinical level of significance. Broadly defined SMT interventions are effective to deal with stress in the workplace, with academic stressors and for healthcare professionals. Structured approaches like SIT and AMT, as well as biofeedback, appear to be superior. Adapting the coping strategies and how they are presented to the trainees may be inevitable when it comes to applying them to specific contexts such as school and police work. Along those lines, some authors have noted the need to also adapt SMT programs to attitudes of trainees towards stress and emotion regulation.

On another application of SMT, the studies analyzed almost invariably mention that people attending debriefing programs appreciated the experience and were under the subjective impression that it had been beneficial to them. However, empirical data, especially when collected in rigorously designed studies, do not support its efficacy. The selection criteria for the articles were not designed to target debriefing, therefore our analysis may be incomplete. Nevertheless, the limited efficacy of debriefing to reduce the incident of PTSD has been confirmed several times and in other more comprehensive reviews [73, 86] and the consensus is that debriefing trauma victims is not an effective approach, at least when the goal is to reduce the incidence of mental disorders.

Finally, a few interesting studies could be used to appraise the usefulness of SMT in the training of military personnel to cope with acute stressors. Their results suggest that broadly defined SMT strategies could be effective in preparing individuals to cope with a highly specific upcoming stressor. Studies with military personnel and other people facing lifethreatening stressors are scarce, and the breadth of stressors they are likely to experience might be too great to provide effective training options for some of the training programs (e.g., SIT). However, the existing research does suggest that some SMT strategies could be effective, even for life-threatening situations. Their efficacy might be increased if the SMT strategies are structured, sufficiently long to be well learned, and practiced until they are well mastered in stressful situations. Attitudes towards using such coping skills may be a

Overall, studies on the application of the broad strategies used in SMT show that it is most of the time effective and for an extensive category of difficulties. For instance, it sometimes has a physical impact on medical conditions and is clearly effective to help patients cope with associated psychological reactions. Most studies, but not all, support the effectiveness of SMT. When it comes to dealing with chronic physical illness, strategies having an enduring impact on patient's life and coping style might also be more effective than brief

The same can be said when looking at mental disorders. Our literature review showed that SIT and AMT are effective tools for PTSD and, to some extent, for other disorders such as generalized anxiety disorder. The broad set of SMT intervention are powerful enough to treat mental disorders, as long as they are structured an include ingredients at the core of SIT and AMT like exposure, cognitive restructuring and homework assignments. Softer techniques often included in SMT, like relaxation and basic coping skills, can be learned and mastered effectively by people who suffer from significant life impairment like schizophrenia or alcoholism in order to deal with stress-related issues instead of treating the

As for the control of non-clinical anxiety symptoms, a large number of studies successfully used SMT to train people to control their stress. These studies represent a secondary prevention approach, where people are already dealing with stress that has not yet reached a clinical level of significance. Broadly defined SMT interventions are effective to deal with stress in the workplace, with academic stressors and for healthcare professionals. Structured approaches like SIT and AMT, as well as biofeedback, appear to be superior. Adapting the coping strategies and how they are presented to the trainees may be inevitable when it comes to applying them to specific contexts such as school and police work. Along those lines, some authors have noted the need to also adapt SMT programs to attitudes of trainees

On another application of SMT, the studies analyzed almost invariably mention that people attending debriefing programs appreciated the experience and were under the subjective impression that it had been beneficial to them. However, empirical data, especially when collected in rigorously designed studies, do not support its efficacy. The selection criteria for the articles were not designed to target debriefing, therefore our analysis may be incomplete. Nevertheless, the limited efficacy of debriefing to reduce the incident of PTSD has been confirmed several times and in other more comprehensive reviews [73, 86] and the consensus is that debriefing trauma victims is not an effective approach, at least when the

Finally, a few interesting studies could be used to appraise the usefulness of SMT in the training of military personnel to cope with acute stressors. Their results suggest that broadly defined SMT strategies could be effective in preparing individuals to cope with a highly specific upcoming stressor. Studies with military personnel and other people facing lifethreatening stressors are scarce, and the breadth of stressors they are likely to experience might be too great to provide effective training options for some of the training programs (e.g., SIT). However, the existing research does suggest that some SMT strategies could be effective, even for life-threatening situations. Their efficacy might be increased if the SMT strategies are structured, sufficiently long to be well learned, and practiced until they are well mastered in stressful situations. Attitudes towards using such coping skills may be a

**4. Discussion**

interventions.

disorder itself.

towards stress and emotion regulation.

goal is to reduce the incidence of mental disorders.

factor to take into account when designing the training protocols. In the case of Hytten et al.'s [79] work, it is possible that the use of SIT may not have been optimal. SIT involves strategies that should be learned over many sessions, accompanied with extensive practices and includes several techniques that may be more appropriate for dysfunctional primary and secondary appraisal than dealing with the adequate appraisal of an objective lifethreatening stressors. In any case, Hytten et al.'s [79] paper suggests that a brief, one hour, SMT training is probably not sufficient to learn how to cope effectively with objective lifethreatening stressors. Longer programs, with extensive practice, may be required.

Overall, our goal was to assess whether or not SMT could be used to help military personnel develop effective coping skills while in the theatre of operation. Studies reported in the present chapter point towards a positive answer. Many specific strategies have been shown to be useful, from tactical breathing [87] to cognitive restructuring [10] and exposure [11].

However, a challenge may reside in the low motivation of soldiers in using and practicing psychological tools that are viewed as making a person weak or unmanly. It would therefore be strategic to find ways to help military personnel apply SMT without the negative perceptions. One way of accomplishing that could be to combine SMT with virtual reality [88,89,90]. Although virtual reality is sometime viewed as requiring considerable technological equipment, studies are being conducted to assess the capability of video games and a television screen in helping soldiers control their anxiety. With this technology, it is believed that the negative perception would be reduced and soldiers could then benefit from stress management training in a way that would prove beneficial for their health and mission without appearing weak.


*2.3. Tissue-Mimicking Phantoms*

The performance of the three different illumination methods was evaluated with regards to the PA imaging depth using a phantom made of polyvinyl chloride (PVC) (M-F Manufacturing Super Soft, Fort Worth, TX, USA), with 0.2% fine ground silica (US Silica MIN-U-SIL5, Stow, OH, USA) used as an optical diffuser. To create a PVC phantom [43], the plastisol was first mixed with the ground silica and then heated in a microwave to 170 ◦C. In a mold, three graphite rods with a 2 mm diameter were placed horizontally, and the PVC was poured to create the three layers as shown in Figure 3. After cooling, the phantom was removed from the mold and used for the described experiments. Graphite was chosen as an absorber due to its broadband absorption characteristics and ease of placement inside the phantom.

**Figure 3.** (**a**) Graph illustrates the polyvinyl chloride (PVC) phantom and graphite inclusions and their dimensions. (**b**) A photograph of the experimental setup including the ring US transducer. A motorized translational stage was used to adjust the position of the phantom to acquired images at multiple cross-sections.

#### *2.4. UST and PAT Image Reconstruction*

In all instance, the waveform method was used to reconstruct the UST images [44], while filtered back-projection was utilized to reconstruct the PAT images [45]. For PAT back-projection reconstruction, the RF (radio frequency) values for each cross-section were averaged 10 times to increase the overall SNR. In UST mode, a 20 dB linear gain TGC was used for acquiring the US images, while in PAT mode, as previously described, a linear 10 dB TGC was used for data acquisition. For US imaging, the used TGC was optimized to minimize the transducer saturation, which resulted in cleaner images. This was done empirically. For PAT imaging, the value chosen was designed to reduce the signal emanating from other cross-sections from appearing in the cross-section of interest.

#### **3. Results and Discussion**

#### *3.1. A Comparison of the Three Di*ff*erent Illumination Methods*

The results discussed in this section focus on analyzing the PAT images and PA signal amplitudes from progressively deeper phantom cross-sections using the discussed illumination methodologies. Figure 4 shows the UST and PAT images for the first and third cross-sections, which are separated by 60 mm. The PAT images are masked based on the region of interest (ROI) as determined by the UST image. For each illumination method, the PA amplitude is normalized to the highest value for the method across all cross-sections. For example, for the full-ring illumination method, images were normalized to the highest amplitude of PA detected within the three slices, which are separated by 30 mm each. This allows for visualization of the effect of depth on the PA signal amplitudes for each illumination method. As can be seen in the PAT images, the graphite absorber is visible in the first and third layers (i.e., larger vertical depth) of the full-ring illumination method, which is not the case for the diffuse and point-source methods.

To further quantify the results shown in Figure 4, the PA signal amplitude across the graphite absorber for each illumination method, and for each cross-section, is plotted in Figure 5. For the full-ring illumination method, the PA values are nearly constant across the three cross-sections (Figure 5a). However, the peak amplitude value of the PA signal decreases by 25 times for point-source illumination and 15 times for the diffused-beam illumination between the first and third cross-sections. As seen by the uniformity in the amplitudes between three cross-sections for the full-ring illumination method, this imaging technique can provide a consistent image regardless of vertical depth. On the other hand, the diffused and point illumination methodologies show variance in PA amplitude signals. This finding demonstrates that the full-ring illumination is capable of providing sufficient fluences at lower vertical depths, which results in detectable and reliable PAT images across cross-sections.

**Figure 5.** PA amplitude across the graphite absorber for three different cross-sections for: (**a**) full-ring, (**b**) diffused-beam, and (**c**) point illumination.

To further compare the performance of the three illumination strategies, the SNR and contrastto-noise ratio (CNR) of the PAT images were measured. In the SNR, the value is calculated by:

$$\text{SNR} = 20 \times \log 10 \left(\frac{M\_S}{M\_B}\right)^2$$

where *MS* refers to the mean of the PA signal, as marked by the US image region of interest, while *MB* refers to the mean of the phantom background. The CNR is determined by:

$$\text{CNR} = 20 \times \log 10 \left( \frac{M\_S - M\_B}{\sigma\_B} \right)^2$$

where *MS* refers to the mean of the PA signal, *MB* refers to the mean of the phantom background, and σ*B* is the standard deviation of the phantom background. For the full-ring illumination method, the used PA amplitude values are from irradiating the target cross-section 15 mm below the cross-section of interest. As can be seen in Figure 6a,b, the SNR and CNR are nearly constant for the full-ring illumination across the three cross-sections, which is not the case for the diffuse and point illuminations. Figure 6c also plots the amplitudes across the graphite object at the third cross-section for all illumination methods. Based on the laser beam diameter of 8 mm; optical losses in the system; and 100 mJ input energy, the diffuse illumination method has a fluence of 9.3 mJ/cm2, compared to 175 mJ/cm2 for point illumination, and 7 mJ/cm2 for the full ring illumination. Full-ring calculations use a beam height of 4 mm circumferentially on a 9 cm diameter phantom, and diffused beam calculations use a beam diameter of 30 mm at the phantom. Even though point illumination has about 25 times the fluence of the full-ring illumination method, its amplitude is much smaller at this cross-sectional depth.

**Figure 6.** The signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) of the PA amplitudes at three different cross-sections are plotted in (**a**,**b**), respectively. For the full-ring illumination, the values were determined based on the illumination at 15 mm below the cross-section of interest. (**c**) Plots the PA amplitude for the top cross-section (cross-section 3) for full-ring, diffuse, and point illumination.

When compared to the diffused-beam and point illuminations, full-ring illumination has a higher PA amplitude at Cross-section 3. It has a near constant SNR, CNR, and PA signal amplitude across all cross-sections, making it an effective illumination method for breast imaging. Given that imaging breast regions close to the chest wall (i.e., large vertical depth) are clinically important, the full-ring illumination shows promise in accessing these regions and thus provides a means for reliable whole breast PAT imaging with a ring US transducer.

#### *3.2. The PA Amplitude of the Targeted Cross-Section as a Function of Illumination Position*

The optimum position for the full-ring beam was investigated by evaluating the effect of the distance between the ring-shaped beam and the targeted cross-section. The distance between the targeted cross-section and the full-ring beam was changed within a range of 0–20 mm. Zero millimeters represents the case where the ring beam is illuminating the targeted cross-section at the graphite rod, while the 20 mm case is when the ring beam is 20 mm below the graphite rod (Figure 7a). In this study, a selected cross-section was imaged while changing the illumination location from 0 to 20 mm. The five different positions chosen to illuminate the targeted cross-section of the graphite rod were 0, 5, 10, 15, and 20 mm. A 532 nm laser with 100 mJ per pulse energy was used for this experiment. The location of the full-ring illumination was adjusted by translating the phantom in steps of 5 mm in the vertical direction.

**Figure 7.** (**a**) The image shows the different positions of the ring beam based on the targeted cross-section (Cross-section 1). The targeted cross-section is located in the central field of view of the US elements. (**b**) PA amplitude at Cross-section 1 plotted as a function of illumination depth below the cross-section.

In Figure 7b, the PA amplitude was measured by drawing a line across the targeted graphite for all five positions. The overall shape of the PA amplitude is constant while the maximum increases as one moves further below the desired imaging cross-section. This increased visibility could be due to the fact that the incidence angle of the beam illuminates the central part of the object more directly as it moves below the cross-section. A possible reason for the stronger PA signal, when illuminating 20 mm below the targeted cross-section, could the incident angle of the light diffusion within the tissue. In the current prototype, the ring mirror is illuminating the object cross-section at an angle of 39 degrees with respect to the object surface. The optimum illumination will occur if the ring beam falls normal to the surface. The 0 and 5 mm cross-sections use the US image as a mask to crop out the large PA peaks generated at the surface of the phantom. The graphite absorber was embedded within the PVC background with a margin from the surface. Here, we only evaluated the signal arising from the absorber. In cases where the illumination was coincident with the center of the transducer, a strong PA signal from the surface was observed (not shown in these masked images). Hence, imaging below the transducer can help to better visualize more central parts of the object due to elimination of a large PA signal arising from the light-entering surface.

The large PA signal at the surface is primarily due to the large fluence at the surface, which can affect the visualization of deeper regions due to a limited dynamic range in PA acquisition. This is not the case where the illumination was adjusted to 10 to 20 mm with respect to the center of the US detectors (i.e., illuminating below the imaged plane). The averaged PA amplitude from all five illumination methods (shown in red in Figure 7b) is similar to the 10 mm illumination results. It is worth mentioning that these results are not necessarily general for all ring illumination systems and are dependent on the incident angle of the beam with respect to the object (39 degrees in our case). The results might vary in other ring illumination systems if the incident angle is changed.

These findings are important because they help to define the illumination scenario based on the characteristics and size of the given object. For example, for a large diameter phantom one might need to acquire PAT images using more than one position of full-ring illumination to cover the regions closer to the outer surface and deeper regions (with illumination offset from the imaging plane). In addition, illuminating below the targeted cross-section (below the central line of the US elements) could help to limit the high PA signals that are generated from the outer surface of the scanned object. The used illumination method has a significant effect on the imaging's vertical depth, which is significant when visualizing anatomy such as the breast.