Immunization with Hpv16e7d Vaccine to
Tumor Bearing Mice: Changes of Cytokines
Patters in the Tumor Microenvironment
Pariya Mahin Samadi1, Mohammad Hossein Yazdi2,3*, Setareh Haghighat1 and Mehdi Mahdavi3,4*
1Department of Microbiology, Pharmaceutical Sciences Branch, Islamic Azad University, Tehran, Iran
2Biotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
3Immunotherapy Group, The Institute of Pharmaceutical Sciences (TIPS), Tehran University of Medical Sciences, Tehran, Iran
4Recombinant Vaccine Research Center, Tehran University of Medical Sciences, Tehran, Iran
Submission: January 28, 2019; Published: March 07, 2019
*Corresponding author: Mohammad Hossein Yazdi, Biotechnology Research Center and Recombinant Vaccine Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran and Evidence-Based Medicine Group, Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran, Iran
Mehdi Mahdavi, Recombinant Vaccine Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
How to cite this article:Pariya M S, Mohammad H Y, Setareh H, Mehdi M. Immunization with Hpv16e7d Vaccine to Tumor Bearing Mice: Changes of
Cytokines Patters in the Tumor Microenvironment. J Tumor Med Prev. 2019; 3(5): 555621. DOI: 10.19080/
Immune response patter in the tumor environment determines the outcome of tumor prognosis. Here, tumor-bearing mice were immunized with HPV17E7d vaccine at a distant area of the tumor and balance of cytokines were assessed in the tumor microenvironment. Tumor was established in C57BL/6 mice and mice were vaccinated subcutaneously, three times with two weeks intervals with 20 μg of E7d protein. Two weeks after final immunization, tumor homogenate was prepared and the quantity of IL-2, IL-12, IL-17 and TNF-α cytokines were evaluated with commercial ELISA kits. In addition, lymphocyte proliferation of spleen cells was determined with BRDU method. Results show that immunization with vaccine candidates increased IL-2, IL-12, IL-17 and TNF-α cytokines and lymphocyte proliferation versus un-vaccinated PBS control group. As regard to results of cytokines and lymphocyte proliferation could conclude that tumor microenvironment has been modified by changing cytokines patterns to defeat tumor cell using tumor vaccination even at a distant area of the tumor.
Cervical cancer is the second most common cancer in the worldwide . Over these last 25 years, proved the direction relation between cervical cancer and the persistent infection by specific genotypes of the HPV is the most important discovers in the etiologic researches . In developing countries, statistics show that more than half of women who suffered by Human Papilloma Viruses (HPVs) lose their lives . Papilloma virus is a DNA virus, which has three main protein regions containing early, late and non-coding areas . Actually, E6 and E7 are two oncoproteins of HPVs that are related to early region. Among the tasks of these two proteins can be mentioned to disturb tumor prognosis and cell cycle of infection . These small proteins have powerful connection to the regulatory proteins of host cells . Protein E6 could attach to p53 and E7 protein by connecting to pRb can inactivate these proteins and thereby the cells transformed and divided without controlling mechanisms [4-8]. Many candidate
vaccines such as DNA vaccine, peptide or protein vaccines, whole
cell vaccine, viral and bacterial vectors are some of wide variety procedure of producing vaccine versus HPV; however, they are not indicate enough efficacy and potency for therapeutic purpose. Because protein vaccines are a series of candidate vaccines and at the same time, they are not sufficient immunogen, therefor needed to change their formulations. Adjuvants increase vaccine immunogenicity and efficacy via various mechanisms. Some mechanisms of adjuvants act at systematic levels and some mechanisms act at local levels of a host. For example adjuvants are able to increase antibody responses versus an organism in serum as systematic level and also at an specific organ such as lung as a local and thereby increases the clearance of the invader in this organ [9,10]. In recent years, adjuvants due to the effect of raising capacity and efficacy of the vaccination are under consideration. Various adjuvants are able to modify immune responses versus of the organisms in different immunologic sites. Because of the importance of immune responses at the tumor microenvironment site, and its effect on the tumor prognosis, here we mainly focused
on the cytokines patterns in the tumor microenvironment .
Herein, we hypothesized that maybe vaccination at a distant
area of the tumor, changes the immunologic profiles of immune
responses in the tumor microenvironment. By this viewpoint, we
focused on the cytokines patterns of tumor milieu after immunization
course of experimental mice and observed that immunization
of mice with vaccines far away from the tumor site changed the
cytokines patterns in the tumor microenvironment.
A total of 100 mice (C57BL/6, Inbred female) with age of Six
to eight weeks-old were purchased from Pasteur Institute of Iran
(Karaj, Iran) and kept in standard condition (temperature, 20-22
°C, 12/12 light/dark) with free access to food and water. After
one-week adaptation, the experiments began and carried out with
professional technician. All experiments on mice were carried out
in agreement with the Animal Care and Use Protocol of Pasteur
Institute of Iran.
TC1 cell line that predominantly express E6 and E7 proteins
of HPV was purchased from Cell Bank of Pasteur Institute of Iran
(Tehran, Iran) and cultured in RPMI-1640 medium containing
10% FBS with 1 mM non- essential amino acids, 1 mM sodium pyruvate,
and 2 mM L-glutamine. After culture of cells and enough
growth, cells were harvested with trypsin enzyme and used for
A suspension of TC1 cell line (1×107 cells per milliliter) in PBS
buffer were prepared and 100 μl of cell suspension were injected
into the flank of C57BL/6 mice to develop stock tumor mice for
further use. After 14-21 days, tumor in the flank of the mice was
appeared and these mice were used as stock for tumor modeling
in naïve mice via transplantation method.
Because of low tumor size variation in tumor transplantation
method, this method is used for tumor modeling in C57BL/6 mice.
Stock tumor bearing mice were dislocated and tumor were harvested
and putted in cold PBS containing penicillin and streptomycin.
Then tumor was cutted to the pieces between 3-5 mm in
length and used for transplantation. Mice were anesthetized with
injection of 100 mg/kg of Ketamine and 20 mg/kg of Xylosine via
peritoneal injection and tumor piece was placed at flank of anesthetized
mice. After surgery, the local was disinfected with povidone
iodine and three days after surgery mice were used for immunization
After tumor transplantation the mice were divided into five
groups (n=20). The first and second groups of mice were immunized
with HPV-16-E7d candidate vaccine formulated in Montanide
ISA 206 and alum adjuvants respectively. The third, fourth and
fifth as the control groups, alum, Montanide ISA 206 and PBS were
injected. These vaccines were subcutaneously injected three times
(20μg) with an interval of two weeks. In order to evaluate the immunologic
parameters, two weeks after the last immunization,
mice were dislocated, and tumor samples and spleen cells were
used for immunoassay.
In other to assay the lymphocyte proliferation, two weeks after
the last shot, spleen cell suspension prepared in sterile condition
and used for lymphocyte proliferation assay by BrdU method.
Single cell suspension was adjusted to 3×106 splenocytes per milliliters
in RPMI-1640 (Gibco) containing 10% FBS, 4 mM L glutamine,
1 mM non-essential amino acid, 1 mM sodium pyruvate, 100
μg/ml of streptomycin and 100 IU/ml of penicillin. Then, 100 μl of
cell suspension were dispensed into 96-well plates and stimulated
with 10μg/ml of antigen. Un-stimulated wells were used as negative
controls and PHA (5 μg/ml) was used as a positive control.
The final volume of wells was reached to 200 μl and incubation
at 37 °C for three days. Then 20 μl of BrdU labeling solution was
added to each well and the plates were incubated for 18 hrs again.
The plates were centrifuged at 300g for 10 minutes, supernatant
were discarded, and the plates kept at 60 °C for 30 minutes to be
completely dry. Then, 200μl of fixation/denaturation buffer was
added to each well for 30 min and then 100 μl of anti-BrdU conjugate
were added and incubated again for 90 minutes. The next
step, the plated washed 4 times with PBS, 100μl of TMB substrate
was added, and reaction was stopped by H2SO4. Eventually, OD450
was read using ELISA plate reader.
Two weeks after the last immunization, experimental mice
were dislocated and tumor sample were removed, dissected, and
mechanically homogenized in PBS containing 1 mM of PMSF. Then
the samples centrifuged at 14000 RPM for 10 minutes and the
concentration of protein determined with Bradford method and
frozen at - 70 °C for further assay.
ELISA test by using commercial kits allocated to measurement
of IL-2, IL-17, IL-12 and TNF-α cytokines (Mabtech, Sweden) that
was performed according to the manufacture instruction. Based
on the cytokines standards, the results were reported quantitatively
After data collection, using Non-parametric methods and
Mann Whitney U test, statistical analysis was performed, and finally,
the figures and tables based on the SD ± Mean, were plotted
by using software Prism Version 6.01.
The results of proliferation assay in experimental groups
demonstrate that immunization of HPV16E7d vaccine candidate
with Montanide/alum as adjuvants have led to increase of lymphocyte
proliferation in HPV-alum group versus control groups
(P<0.0392). Immunization with HPV-alum does not show significant
differences versus HPV-Montanide group (P>.0.05) (Figure
The results of TNF-α cytokine assay show that immunization
with HPV-16 E7d vaccine formulated in Montanide ISA 206 cause
to significant increase of TNF-α cytokine versus CTR-MON and
PBS control groups (P< 0.0461) (Figure 2).
The results of IL-2 cytokine assay show that immunization
of HPV-16 E7d vaccine formulated in Montanide/alum as adjuvants
cause to significant increase of IL-2 versus Montanide and
PBS control groups (P<0.0347). However, there were not any significant
differences among E7d vaccinated experimental groups
(P>.0.05) (Figure 3).
The results of IL-12 cytokine assay show that injection of
HPV-16 E7d vaccine formulated in Montanide ISA 206 shows an
increase versus other experimental groups but shows significant
differences versus PBS control group (P=0.0246). No noticeable
differences were observed among vaccinated experimental groups
(P> 0.05) (Figure 4).
The results of IL-17 cytokine assay show that injection with
HPV-16 E7d-Alum shows an increase versus HPV-16 E7d-Monta nide group (P= 0.0876) (Figure 5). No other differences were observed
among experimental groups (P>0.05).
The nature of tumor microenvironment correlates with its
local immune responses and tumor prognosis . Therefore,
modulation of tumor microenvironment by immunological methods
can result in tumor controlling and suppression. Vaccine as an
approach can induce specific effector T cells against tumor antigen
and thereby can modulate tumor microenvironment by production
of cytokines and immunologic bioactive molecules that
change outcome of tumor.
Vaccine as an effective immunologic method can modulate immune
response versus tumors and due to ability to move of effector
and memory T cells, this modulatory effect can be translocated
too far away than immunization site. Herein, we hypothesized that
may be subcutaneous immunization of tumor bearing mice with
a vaccine change the nature of immune responses at the local of
Here, we used tumor bearing mice model that induced by
TC1 cell line and also HPV16 E7d vaccine that target this antigen
at the surface of this tumor [13-16]. Herein, our results showed
that administration with HPV16E7d vaccine candidates could increase
lymphocyte proliferation and also IL-2, IL-12, IL-17 and
TNF-α cytokines in the tumor microenvironment in comparison
to un-vaccinated PBS group. The increase in lymphocyte proliferation
meaning improvement of cellular immune response that is
at the first line of adaptive immune response against tumor in the
controlling of tumor as reported by other researchers [17-19]. Assessment
of cytokines pattern in the tumor homogenate show that
vaccination at a distant area from the tumor changed immunologic
cytokines at the tumor microenvironment. In fact, the level of IL-2,
IL-12, IL-17 and TNF-α cytokines in vaccinated groups increased
versus un-vaccinated PBS group. These finding show that immunization
at a local far away from tumor environment can modulate
immune responses in the tumor microenvironment. It is clear that
the microenvironment of tumor, determines the outcome of tumor
in immunotherapy toward tumor suppression or progression
. According to our finding it is possible that by immunization
at a local far away from a tumor, modulate tumor microenvironment
to combat with tumor. Studies show that microenvironment
of tumor has critical in tumor immunotherapy and modulation
of this milieu can change the prognosis of tumor . In overall,
our findings indicate that administration with HPV16 E7d vaccine
candidate can modify tumor microenvironment via increase of
IL-2, IL-12, IL-17 and TNF-α cytokines. Consequently, as regard to
results of cytokine and lymphocyte proliferation could conclude
that tumor microenvironment have been modified by changing
cytokine pattern to defeat tumor cell in cancer network. However,
immunization and also effector and memory T cell formation constitute
far away from local of tumor.
Our findings indicate that administration with HPV16 E7d vaccine
candidates can modify tumor microenvironment via increase
of IL-2, IL-12, IL-17 and TNF-α cytokines as vital cytokines to control
tumor growth and lymphocyte proliferation in comparison
to un-vaccinated PBS control group. Consequently, as regard to
results of cytokines and lymphocyte proliferation could conclude
that tumor microenvironment have been modified by changing cytokines
patterns to defeat tumor cell using tumor vaccination even
at a distant area of the tumor.
W.G.o.t.E.o.C.R.t (2010) Humans, IARC monographs on the evaluation of carcinogenic risks to humans. Ingested nitrate and nitrite, and cyanobacterial peptide toxins, IARC monographs on the evaluation of carcinogenic risks to humans/World Health Organization. International Agency for Research on Cancer 94.