Pre-challenge Evaluation of Immune Response in Serum Cytokines (Th1 and Th2) and T-cell Markers (CD4, CD8, CD3, and CD25) Following Administration of New Formulated Leishmania Vaccine in Balb/c Mice

Copyright: © 2017 Latifynia A, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Pre-challenge Evaluation of Immune Response in Serum Cytokines (Th1 and Th2) and T-cell Markers (CD4, CD8, CD3, and CD25) Following Administration of New Formulated Leishmania Vaccine in Balb/c Mice Afshineh Latifynia1,2*, Mohamad Hosein Niknam1, Samad Farashi Bonab1, Bita Ansaripour1, Zahra Gheflati1 and Mohammad Javad Gharagozlou3 1Department of Immunology, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Islamic Republic of Iran 2Research Center for Immunodeficiencies, Childeren’s Medical Center Hospital, Tehran University of Medical Sciences, Tehran, Islamic Republic of Iran 3Department of Pathology, School of Veterinary Medicine, University of Tehran, Tehran, Islamic Republic of Iran


Introduction
There are three forms of leishmaniasis-namely cutaneous, visceral, and mucocutaneous. Leishmaniasis often occurs in areas where poverty, malnutrition, famine, and illiteracy are common. The disease is transmitted to vertebrate hosts by the bite of female phlebotomine sandflies [1]. Until now, an effective vaccine capable of controlling the leishmania infection by preventing cutaneous leishmaniasis has not been available, and it is speculated that one will be developed in the near future. Patients who have recovered from L. major infections develop high levels of immunity to the pathogen [2]. Leishmaniasis has a long history. Designs on pre-Colombian pottery and the existence of thousand-year-old skulls with evidence of leishmaniasis prove that the disease had been present in the Americas for a long time. It had also been present in Africa and India since at least the mid-18 th century [3]. Today, there are an estimated 12 million cases of leishmaniasis worldwide with an estimated number of 1.5-2 million new cases occurring annually, which include 1-1.5 million cases of cutaneous and 500,000 of visceral leishmaniasis [1,4].
In vertebrate hosts, leishmania parasites survive and multiply as nonmotile amastigotes, primarily in macrophages. The genus Leishmania comprises 30 species, of which around 20 are pathogenic for humans [5]. Sores can result in permanent scars and disfigurement. Treatment may reduce their severity. Medication can sometimes cure the disease; however, treatment is most effective when it is started before injuries enter the immune system. Visceral leishmaniasis often proves fatal within two years if it is not treated properly. For most species, humans are accidental hosts since leishmania is primarily a zoonotic disease [6]. Until now, a successful vaccination strategy against leishmaniasis has been limited to cutaneous leishmanization in which small doses of living virulent L. major promastigote are deliberately injected intradermally [7]. Since the mouse model is close to humans, it has been used for the study of both cutaneous and visceral leishmaniasis, but reflects the human cutaneous leishmaniasis rather than the visceral disease. Until now, several vaccine formulations have been prepared, but they were not found to be protective in primates [8,9]. That is why there is a need for new methods to assess the immune response of animals to vaccination that can better predict the response to virulent organism challenges in primates and humans. New methods would be useful for the detection of parasites as well as assessment by using pathological methods. The effective diagnosis of the progression of infection with my candidate vaccine will require an assessment of the genetic stability of the agent. The results obtained from the mouse model, although practical and informative, must be further confirmed in a primate model that more closely predicts pathogenesis and immunogenicity in humans [10]. The limitation of this unique alternative is that by default, the Th1/Th2 unique parameter of the mouse model is reflected on the leishmaniasis. In other words, compensating for the Th2 strategy of the parasite can be an effective alternative for the development of the vaccine against leishmaniasis instead of enhancing the Th1 response that occurs during the infection [11]. To date, two host systems have been classified for studying leishmania infection on the basis of host susceptibility and resistance. This observation extends to the murine L. major model-for example, C57BL/6 mice being uniformly resistant and BALB/c consistently susceptible, which reflect the immune potentiality toward leishmania infection [12]. Dendrite cells (DCs) are potent antigen-presenting cells and can induce T cell activation efficiently [13]. It has been also shown that DCs are the source of different cytokines such as IL-12, IL-10, and IFN-γ [14][15][16].
The incubation of leishmania promastigote with dendritic cells can induce early IL-12 production in vitro, which might have originated from the preexisting pool of IL-12 p70 that was secreted soon after the ligation of any microbial product [17], suggesting the role of DCs in the initiation of T cell immune response in leishmania infection. It is also reported that the uptake of leishmania amastigotes by skin-derived DCs induces IL-12 p70, and up-regulates co-stimulatory molecules and vaccinates against L. major infection. In marked contrast, L. major inhibits IL-12 production in macrophages [18,19]. IL-10 appears to constitute a major regulatory control in the outcome of infection, as well as the failure to produce IL-12 associated with the active form of the disease [20]. Our previous findings on the same newly formulated vaccine in two groups of mice (susceptible/Balb/c) and (resistance/ conventional) showed that it produced positive DTH [21] and led to an increase in white pulp size [22], which was statistically significant and correlated with effective immune responses, and was dependent on antigen doses, types of adjuvants, and injection groups [23]. Also, in other studies, our new formulated provisional vaccine was fortified by using BCG and the alcoholic extract of the Teucrium polium plant as an adjuvant in Balb/c mice, and after that the vaccinated subjects were challenged with live promastigote that were harvested from the culture medium. In this experiment, Types 1 and 2 cytokines, spleen changes, and rate of survival were evaluated [24][25][26]. The findings indicated the safety, efficacy, and productivity of the new leishmania vaccine.
The aim of the present study is to further elucidate the effect of the new adjuvanted vaccine on the immune responses of Balb/c mice, pre challenge and post vaccination. In the present experimental design, we evaluated Th1 cytokines (IFN-gamma and IL-12) and Th2 cytokines (IL-4 and IL-10), and the markers belonging to T cell subtypes, including CD4, CD8, CD3, and CD25 markers, which are considered to be mostly involved in immune response against leishmania parasites and occur in the vaccinated animals.

Culture and isolation of Leishmania parasites
Leishmania parasites and antigens from the promastigote of the L. major (WHO) strain were provided by the Tehran University of Medical Sciences, and they were grown in NNN medium (14 g bacto peptone, 6 g NaCl, 300 ml rabbit blood, and up to 1200 mL H 2 O 2 ) and in the second step were grown in RPMI 1640 culture, both FCS 5% and 10%. The harvested parasites were washed three times with normal saline solution (0.9%) or phosphate buffer saline (PBS). The parasites were counted in a Neubar chamber and then kept at 70°C till use. After parasite accumulation in one flask, it was diluted to a concentration of 1.87 × 10 10 .
For details of the procedure, please refer to the previous studies by Latifynia et al. [21][22][23][24][25][26]. In time, the harvested parasites were diluted to a concentration of 5.92 × 10 10 parasites per milliliter. Based on the previous studies, 100 μg/0.1 ml or 200 μg/0.1 ml Leishmania protein per dose of the provisional vaccine was selected for the formulation and preparation of the vaccine. The protein content of each dose was estimated by the Lowry method [27]. The vaccine was stored at 4°C until injection. BCG Vaccine (Mycobacterium bovis, Bacillus Calmette Gurine, BCG Strain Pasteur Institute of Iran, Frozen-dried BCG Vaccine Pasteur France. 1173 P2 secondary seed lot C, batch no. 179, Feb. 1995) was suspended and diluted in the SSI solution (125 mg MgSO 4 , 125 mg K 2 PO 4 , 1 mg L-aspargine, 12.5 mg iron ammonium citrate, 18.4 mg 85% glycerol, 0.5 mg citric acid, and 1 ml H 2 O for injection).The amount of BCG for each injection dose was 2 × 10 5 CFU⁄0.1 ml. To prepare the Teucrium polium adjuvant, 400 mg of alcoholic extract of Teucrium polium [28][29][30] was dissolved in 1 ml of distilled water; 2.5 mg/0.1 ml was used for each of the injection dosages of the antigen (100-200 μg⁄0.1 ml). The adjuvants were added to the leishmania antigen solutions mentioned previously, and two injection doses containing 100 μg/ml and 200 μg/ml of antigens supplemented with adjuvants were prepared.

Experimental design
Forty young adult female and male Balb/c mice were obtained from the Pasture and Serum Research Institute, and they were randomly assigned to four standard polycarbonate boxes of four treatment groups. All the groups were fed ad lib with commercial mice kept in the polycarbonate boxes in a well-ventilated animal room located in the University of Medical Sciences of Tehran, School of Medicine, Tehran, and Islamic Republic of Iran. The experimental design consisted of six antigen-injected groups (LT 100, LT 200, LB 100, LT 200, LBT 100, and LBT 200) and a control group that received no antigen injection. Group LT received 100 or 200 μg/0.1 ml of the crude cocktail antigen preparation plus alcoholic extract of Teucrium polium as adjuvant, Group LB received 100 or 200 μg/0.1 ml of the crude cocktail antigen plus BCG as adjuvant, Group LBT received 100 or 200 μg/0.1 ml of the crude cocktail antigen preparation plus the alcoholic extract of Teucrium polium and BCG as adjuvants. Groups LT, LB, and LBT were injected subcutaneously with the antigen preparations at the base of the tail. The control group did not receive antigen injection. All the six groups received a booster a week later. All the mice were scarified 25 days after the booster injection.

ELISA method
To evaluate cytokine levels in the sera of the animals, at most 2 ml of blood sample was taken from each mouse, and its serum was separated by using the routine standard method. The levels of IL-4, IL-10, IL-12, and IFN-γ in the six injection groups and the control group of mice were determined by the sandwich ELISA method according to the recommendations of the manufacturers. Mice serum levels of IL-4, IL-10, IL-12 and IFN-γ in the subjects were measured by using an automated micro plate reader set at 450 nm. The sensitivity limit was 20 pg/ml for IL-4, IL-10, and IFN-γ.

Spleen cell isolation and flow cytometry
Splenic cells and lymphocytes were obtained from Balb/c mice by the collagenase method. Following this, 10⁷ cells were treated with 4 mg/ml proteinase-free collagenase (Sigma-Aldrich, # C6079) for 20 min at 37°C in saline solution at pH 7.5 under gentle agitation, followed by the neutralization of collagenase with an equal volume of complete RPMI media. The cells were centrifuged at 8006 g, re-suspended in saline solution containing 1% BSA, and passed through a 100 µm filter mesh before analysis. Negatively sorted CD4 T cells were obtained at higher than 90% purity according to FACS analysis by cell passage through mouse CD4 subset column kit #MCD4C according to the manufacturer's instructions (R&D Systems Minneapolis, MN). DNCD3 T-cells were isolated either by depletion of CD4 and CD8 T-cells using tandem CD4 and CD8 mouse column kits ( # MCD4C and # MCD8C 1000, R&D Systems), or by FACSAria cell sorter (BD, San Jose, CA) at 98% purity. For purification of DN, DP, and SP4 T-cell subsets, single-cell suspensions were triple-stained with CD3 Ab-FITC, CD4 Ab-PE, and CD8 Ab-PerCP conjugates (BD Phar Mingen, CA) and then FACS-sorted in three simultaneous windows in a FACS Aria instrument. In some experiments, the TCRcd/NK cell depletion of FACS-sorted DNCD3 splenocytes was carried out by the incubation of cells with 2 µg/10⁶ cells of anti-mouse TCRcd Ab-PE (clone #GL3, BD Phar Mingen, San Jose, CA) and 2 µg/10⁶ cells of anti-asialo-GM1 Ab-PE conjugates (clone #SH34, ATCC), followed by incubation with anti-PE Abs coupled with magnetic beads and passage through MACS paramagnetic columns according to the manufacturer's instructions (Miltenyi Biotech Inc., Auburn, CA) [28].

Data analysis and ethics
The data obtained from the experiments were analyzed by using SPSS (SPSS Inc., Chicago, IL, USA). The means were compared by the standard analysis of variance/simple factorial tests, and by one-and two-way student Newman Keuls methods. The correlation coefficient analysis was determined by a Pearson average two-tailed test of significance. The study was done in compliance with the Helsinki Declaration, and the protocol was approved by the research deputy of the Tehran University of Medical Sciences, Tehran, Iran.

Result
In continuing the pre challenge experiments, we compared the immune responses of the Th1 cytokine profile (IL-12, INF-γ) and Th2 (IL4, IL10) with cluster determinants (CD4+, CD8+, CD3+, and CD25+) that were induced by the new vaccine of L. major, which was injected in each of these three injection groups (LB, LT, LBT) that received 100 or 200 µg/0.1ml protein with only one booster dose. Our results were as follows:

IL-12:
The ANOVA test showed that the means square of IL-12 compared between the injection groups and the others (INF-γ, IL4, IL10, CD4+, CD8+, CD3+, and CD25+) was significant (P<0.001), and between the injection and the control groups, Th1 cytokine (IL-12) had significant differences (P=0.001) too ( Table 1). The highest amount of IL-12 was related to normal, which was not vaccinated (3896 pg/ml), but among the three injection groups and two injection doses, LB 200 µg/ ml (3688.3 pg/ml) had the highest level of IL-12 and the lowest amount belonged to the LBT injection group of dose 100 µg/ml (2353 pg/ml) ( Table 3). The amounts of Il-12 were as follows: Normal>LB 200>LBT 200>LT 200>LT100>LB 100>LBT 100 (Figures 1-3). Considering IL-12 and multiple comparisons of IL-12 with Tukey's (HSD) test washonestly significant different (HSD) test and 95% confidence interval showed that the mean difference was significant at the 0.05 level (Table 3). Pearson correlation with two-tailed test revealed that virtually none of INF-γ, IL-10, CD3+, CD8+, and CD25+ was significantly associated with IL-12, but IL-4 had more correlation. IL-12 and correlation with IL-10 and INF-γ, although low, were inversely related, and when IL-
The highest amount of IL-4 (2.84 pg/ml) was related to LBT 100 µg/0.1 ml and the lowest concentration was related to the normal group (1.29 pg/ml). The injection groups LB 100 and 200 µg/0.1 ml were equal (2.43 and 2. 46 pg/ml) and LB 200 was 1.6 pg/ml  ( Table 3; Graphs 1 and 2). Pearson correlation with a two-tailed test of IL-4 with IL-12 was 0.225 and not significant (p=0.174), but near to it. This means that both IL-12 and IL-4 can increase or decrease. In this context, there was no observed significant correlation between IL-4 and two injection doses (100, 200 µg/0.1 ml) with another measured cytokines and the other Th1 and Th2 measured cytokines and Cluster determinants (CDs) ( Table 2).The results of multiple comparisons, Tukey's HSD test, and 95% CI between the two injection doses of 100 and 200 µg/0.1 ml were not significant at the 0.05 level (P<0.05).    Figures 4 and 5). The highest level of IL-10 was related to LBT 100 µg/0.1 ml (5.7 pg/ml), and the lowest concentration was related to the control group (1.29 pg/ml). The results also showed that the mean of IL10 for the two injection doses 100, 200 µg/0.1 ml and 0.00 µg/0.1 ml (control), while considering the group injection in the serum of Balb/c mice after vaccination with the new leishmania vaccine with 95% CI, showed that the doses of 100 and 200 µg/0.1 ml had no significant differences with each other, but had significant differences with the 0.00 injection dose (see Graph 3; Figures 4 and 6).

Cluster Determinant 8 (CD8+):
The ANOVA test showed that the means square of Th2 cytokine (CD8+) between injection groups and compared to others (IL-12, INF-γ, IL4 , IL-10 , CD4+, CD3+, and CD25+) had no significant statistical differences but were very near to it (P=0.158) (  Figure 5). Among the injection groups, CD8+ did not show statistical differences (P=0.158), which was not significant ( Table 2), but very near to it. The highest amounts were related to LB 200 and the lowest was related to LBT 100 (Table 3). Pearson correlation with a two-tailed test on CD8+ with doses of 100 and 200 µg/0.1 ml (0.490) and 0.075 significance revealed relatively high results but not significant ones.

Discussion
In this study, we prepared the L. major antigen as a leishmania vaccine for the third time. According to this topic, we measured the safety, toxicity, and reproducibility in different conditions, and confirmed the authors' previous results with the same experiments as well as additional ones. Our present results showed that without considering the injection doses, IL-12 had significant differences (P=0.001) and CD4+ had nearly significant differences (P=0.097) among all the groups. INF-γ (P=0.110), CD3+ (P=0.117), and CD8+ (P=0.158) had no significant differences, but were approximately near to it. IL-4 (P=0.506) and IL-10 (P=587) showed no significant differences among all the groups (Table 1). In our previous post challenge experiments, IL-12 was found to be significant among all the groups (P=0.008), and IL-4 and Il-10 had no significant differences (P=0.323 and P=0.22) (26). The present Th1 and Th2 cytokine results confirmed our previous findings (21, 23, 24, and 26). This new vaccine increases DTH pre-challenge and leads to an increase of IL-12 in the post-challenge phase. As per the post-challenge results, we did not know whether the challenge with live leishmania promastigote increased IL-12 or if the increase was due to our preliminary vaccine. But we were satisfied to see that the new vaccine induce to production significant immune response which was significant and resistant-in mice against live promastigote with high survival rates have had with high and significantly IL-12 between all of the groups. An important finding for us was that the injections of the new vaccine pre-challenge directly increased IL-12 with different adjuvants, and the two injection doses of 100 and 200 µg/ml showed significant differences between all the injection groups (LB, LT, and LBT) ( Table 1). This is an important point for the new vaccine, because not only did Il-12 and IFN-γ increase, but also IL-4 and 10 decreased compared to normal, which suggests that this harmless new vaccine provides an immune defense against intracellular pathogens in Balb/c mice. Now, if our study was reversed, interferon gamma and interleukin 12 would decrease and IL-4 and IL-10 would increase; we had seen this as it was a breach of the vaccine, which had shifted antibodies after exposure to intracellular pathogens (L.) and ultimately fatal visceral leishmaniasis that led to failure of the vaccine. The reason for this is that in this case, the animal model has produced humoral immunity and antibody. But fortunately, this new vaccine has no significant difference in IL-4 production, and 10 injections were observed for any of the two injection doses and three injection groups (  (Figures 7 and 8). In the first study, we measured the delayed type hypersensitivity (DTH), resistant, and susceptible mice before the challenge. We came up with different results for different injection groups, and observed that the new vaccine could not only stimulate the delayed immune system, but it could also be sensitized 48 h after injecting the new antigen, and it turned from negative to positive and the expansion of spleen white pulp size was seen in resistant and  susceptible mice [31][32][33]. For the second time, the different injection doses and injection groups showed significant differences again for the Th1 and Th2 cytokine profiles, and the expansion of spleen white pulp size in susceptible mice (Balb/c) against the same new vaccine post challenge with live promastigote [34][35][36].With regard to these results, this new vaccine was found to have the ability to stimulate a delayed immune system as well as produce positive DTH 48 h after vaccination [31,33] ,and the post-challenge increases in IL-12 and IL-10 were significant in some groups [34,[36][37][38][39][40][41]. We had also observed in one of our previous studies that pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6), in non-healing, active leishmaniasis patients, and antioxidant levels were higher than in the other three patient and control groups [42]. Lipophosphoglycan leishmania could suppress TNF-α, IL-1β, and NO production by lipopolysaccharide-stimulated or PMA-stimulated macrophages. TNF-α induces mononuclear phagocytes and neutrophils to produce reactive oxygen intermediates (ROIs) [42], which led us to guess that increased amounts of Th1 cytokines such as IL-12 and IFN-γ cause a high DTH response. In contrast, Th2 cytokine (e.g. IL-4 and IL-10) levels enhanced a low DTH response in cutaneous leishmaniasis [21].
IL-4 and IL-10 act together in the presence of leishmania antigens [43]. When L. major causes a single cutaneous lesion or undergoes a spontaneous cure, the subject is resistant. In this case, probably, the infection was inhibited in the macrophage via innate immunity and production of IFN-γ and IL-12 by a Th1 response that led to the elimination of the parasite. In a future challenge, the subject would most likely be immune [23]. Scott et al. have suggested that low antigen doses may preferably promote a CD4+ Th2 response in vivo, whereas high doses may favor the development of Th1 cells [44]. The results of our present study also confirmed that CD4+ cells had significant differences depending on the two-dose injections and the three-group injections in