Aloe Vera Extracted Polysaccharides Shift the Immune Responses of Tumor Bearing Mice Toward Th1 Pattern: Animal Study

Experimental Procedure: Sixty C57BL/6 mice were divided into six groups as follow: Group 1, E7d-ALUM-GEL; Group 2, E7d-ALUM; Group 3, E7d-MONTANIDE ISA 206GEL; Group 4, E7d-MONTANIDE ISA 206; Group 5 and Group 6, GEL ALONE and PBS . The oral feeding of Aloe Vera gel with 0.05% polysaccharide was done for a period of 30 days. Mice were immunized with HPV16 E7d vaccine three times on day 0, 14 and 28th. Then tumor was implanted subcoutaneosly and 2 weeks later, the serum samples of the mice were collected to evaluate IL-4 and IFN-γ cytokines and specific total IgG, IgG1 and IgG2a antibodies levels by ELISA.


Introduction
Cervical cancer is a public health problem and is well-known as the fourth most common cancer and the fourth major cause of cancer-associated death in women in the developing countries [1]. Human Papilloma Virus (HPV) may cause an infection with no symptom and would be self-alleviated but infection persistansy of some HPV species may cause warts and also precancerous lesions [2,3]. This virus, especially its "high-risk" genotypes such as HPV16 and 18 are shown to have a critical role as the most important etiological factor in the induction of cervical cancer [4][5][6]. The proteins E6 and E7 are two oncogenes that are necessary for malignant conversion and are expressed in oncogenic HPVs (e.g, types 16 and 18) and have transforming properties. To date, vaccination has been the most effective method to improve and control infectious diseases in terms of effectiveness and economy [7]. Prophylactic vaccines such as Gardasil and Cervarix which are approved by America's Food and Drug Agency (FDA), stimulate humoral immune response by induction of antibodies secreation against non-infectious Virus Like Particles (VLPs) which are part of Major capsid protein of HPV (L1 of HPV types 6, 11, 16, and 18 for Gardasil, and types 16, and 18 for Ceravix) [8,9]. HPV16E7 is a small protein (11KDa) known as Tumor Specific Antigen (TSA) has been expressed in eukaryotic and prokaryotic cells and agitated immune cells against, TC-1 tumor growth (TC-1 is the mouse tumor cell line, which expresses the E6 and E7 oncoprotein of HPV-16).
Recombinant E7 protein is a good candidate as a therapeutic HPV vaccine, but it is not enough immunogen for stimulating the cytotoxic T lymphocyte (CTL) responses [10][11][12][13][14]. Aluminum hydroxide (Alum) is the most common used adjuvant for human vaccines. Alum although is able to stimulate antibody secretion and shifts immune responses toward Th2, it has low potency for stimulating the cellular (Th1) immune responses which beside Th2 and humoral immunity is so important for protection antigens HPV infection. Montanide is another adjuvant that raises the TH1 response and improves IgG2a production [15][16][17] [18][19][20]. It is recommended that the macrophage mannose receptor as a carbohydrate-binding endocytic receptor, can adjoin to Acemannan mannose residues [21,22]. Glocomannan has a great declination to coupling with macrophage that express mannose receptors on their surface abundantly. Meanwhile, Glucomannan is an inhibitor of the tumor-associated macrophages (TAMs) activity.
TAMs play the main role in tumor angiogenesis and metastasis [23]. Glucomannan and Acemannan will trigger the mannose receptors on the surface of macrophages and thereby increase the expression of pro-inflammatory cytokines, nitric oxide and agitate phagocytosis [14,24]. In the present work, we hypothesized that the oral feeding of Aloe Vera gel containing natural polysaccharides to mice which are immunized with HPV16E7d by stimulating the immune responses may be result in improvement of E7d vaccine efficacy or treatment outcome.

Experimental Animals
Seven-weeks-old male C57BL/6 mice (n=65, 19.4-22.9gm) (Supp Figure) were purchased from the Pasteur Institute of Iran (Karaj, Iran) and were manipulated and maintained under standard

TC-1 Cell Culture
The TC-1 cell line was prepared from Department of Cell Bank in Pasteur Institute of Iran and propagated in complete RPMI-1640 to a high density.

Injection of TC-1 Cell Line to Prepare Stock Tumor Mice
The mice that were injected with TC-1 cells line are called stock mice which were used as a source of tumor tissue for induction of tumor through surgery in pre-vaccinated mice. Cultivated TC-1 cell line were prepared in PBS buffer and brought the suspension to density of 1×107 cells/ml and 100μl and injected into the right-side of five mice flank; and waiting for tumor growth for two or three weeks.

Tumor Implantation
Initially, Mice flank were shaved by depilatory, then mice were
Mice from group I, II, III and IV were vaccinated three times with 10 days intervals.

Oral Administration of Aloe Vera Gel During Vaccination
The oral feeding of mice from group I, II and VI accomplished

Serum Collection
Two weeks after implantation of tumor in the experimental group, blood samples were collected from the inner corner of the eyes and blood serum was collected with centrifuge and saved at -200C until use.

Tumor Growth Evaluation
In this study, the largest length and width of tumor were measured by Digital caliper. Also, the experimental mice tumor volume (mm 3 )were measured by formula of ab 2 /2. Relative Tumor Volume (RTV) increase can be calculated by dividing the last day tumor size to the first day of transplanted tissue and this index was used in the statistical calculations.

Survival of Mice
After induction of tumor, mice were kept in cages for 50 days and were daily recorded for the mortality trends due to tumor progression.

Specific Total IgG and Antibody Assay
Specific total IgG antibody was evaluated in the serum sample which was collected two weeks after transplantation of tumor tissue to assay specific humoral immune responses with an optimized indirect ELISA method.

Cytokines Assay
Cytokines (IFN-γ and IL-4) assay was done on serum samples which was collected two weeks after the immunization by ELISA Kit (Mabtech, Sweden) according to the manufacturer's protocol.

Statistical Analysis
The data presented as means ± Standard Error of Mean (SEM) of each experiment. All statistical analysis except for survival rate were accomplished by t-Test the SPSS (Statistical Packages for Social Science) version 22 software and p-value less than 0.05 was considered that there is statistically significant differences. The results of survival rate were analysed and presented with Kaplan Meier test.

Results and Discussion
Aloe Vera gel is effective in boosting both cellular and humoral immune response through attachment on mannose receptor on macrophage. Acemannan stimulate macrophage to produce inflammatory cytocins which demonstrates tumor suppressor effect (as it have been seen in our study). Wang et al. [25] evaluated the antitumor activity of aloe polysaccharides (AP) and they observed recduction in the tumor weight and found out that AP also could help to decrease chemotherapy side-effects and also improve the antitumor effects of CTX, ADM and 5FU. It is hypothesize that aloe vera polysaccharides can increased the level of IL-2 and TNF-α in the serum of mice and enhance immune responses to suppress tumor cell and subsequently resulted in higher survival rate and may probably has the potential to be used as a safe and nontoxic supplement in cancer patient in combination of conventional therapy [25].

IFN-γ and IL-4 Cytokines Assay
Linna Zhang et al. study in 1996 on mouse macrophage cell line that situated in Raw 264.7 cells, indicated that Acemannan lead to stimulate cytokines production by macrophage, nitric oxide release, surface molecule expression and cell morphological changes [19].
Consequence of long-term Aloe Vera gel oral administration in this project, not only adjusts some immune system indicators, but also enhances vaccine efficacy in protection debate and improves vaccine response in some parameters. Although, this subject is much more observed in our control GEL group [26]. Kemp   It seems that reinforcing activities of Acemannan is occured through activation of macrophages to produce inflammatory cytokines such as IL-1, IL-6, TNF-α and IFN-γ. It is proposed that this happens by binding of Acemannan to the mannose receptors on macrophages [15,31]. In the current study, there was an increase in IFN-γ level of GEL ALONE and E7d-ALUM-GEL group in comparison to other groups but the increase was much more considerable in GEL ALONE group than E7d-ALUM-GEL group.
However the increased level of IFN-γ cytokine in these two groups were not statistically significant ( p>0.05).
The enhancement of IFN-γ in E7-ALUM-GEL group versus E7-ALUM group, has showed the stimulation of immune response toward Th1 pattern. Despite our hypothesis, GEL ALONE group showed maximum amount of IFN-γ secretion [32]. IFN-γ level in test and control groups ( p>0.05) (Figure 2). Also, an increase in IFNγ/IL-4 ratio has been detected in E7d-ALUM-GEL and GEL ALONE group in comparison to other experimental groups that support the shift of immune system in favor of cellular immunity and Th1 responses by oral administration of the Aloe Vera gel to mice that suffer from cancer, and the increase was much more significant in GEL ALONE group, although the increase was not statistically significant ( p>0.05).

Specific Total IgG Antibodies
Monitoring of total IgG antibody concentration demonstrates that the most increase in total antibody level belonged to E7d-MONT-GEL group in comparison to all of the groups. Also, a remarkable meaningful increasement of total IgG in E7d -ALUM-GEL group in camparison to GEL ALONE and PBS groups (p = 0.03) has been detected (Figure 3a).

IgG Isotypes
IgG1 and IgG2a are isotype of antibody that represent declination to Th2 and Th1 respectively. In this study the results show that IgG and IL-4 (as IL-4 switch antibodis toward IgG1, it may interprete that IL-4 level were not high enough to stimulate IgG1 secreation).
As it is known, IFN-γ swich immune system toward Th1 and also result in the production IgG2a, but the results of present study do not show this pattern, and the increasment of IgG2a in E7d-MONT-GEL and E7d-GEL group was mostly realted to the properties of Montanide as adjuvant. On the other hand, the significant increases in property of IgG2a level in E7d-MONT-GEL in comparison to E7d-MONT group and also the significant increase of total IgG in E7d-MONT-GEL group in comparison to E7d-MONT and other groups, can be concluded that aloe vera gel has this potential to be used as coadjuvant in combination with Montanide in vaccice therapy to enhance cellular and humoral immune system protection. As respected, no increase was observed in IgG1 and IgG2a (Figure 3c) level in GEL ALONE and PBS group.

Survival Rate
Based on the results of this study, highest survival rates was belonged to E7d-ALUM-GEL and GEL ALONE groups ( Figure   5)   in the body and also improves the innate immune response activity [25]. Aloe Vera extracted polysaccharides could improve cellular and humoral immune responses and can reinforced vaccine immunization although this effect was not observed in separated administration model in our work and it would be better to study the effect of Aloe Vera extracted gel polysaccharides in the formulation of vaccine instead of seprated administration. It is likely that this natural polysaccharide has impact on the stimulation of the immune responses and may be considered as a beneficial supplement in combination with chemotherapy drugs during cancer therapy.