Introduction
Cervical cancer is an infection-related cancer that is caused by persistent human papillomavirus (HPV) infection [
1]. High-risk HPV type 16 or 18 can inhibit the antiviral immune response by expressing oncoproteins to inhibit interferon (IFN) secretion by keratinocytes and reducing the expression level of toll-like receptors (TLR9) after infecting the basal layer of the cervical epithelium so that the virus can exist in vivo for a long time. During persistent infection, the virus expresses E6 and E7 proteins to induce host cells carcinogenesis, resulting in tumorigenesis [
2‐
4]. In general, the time span from persistent HPV infection to the development of cervical cancer is long, averaging 15 years [
5].
HPV prophylactic vaccines provide effective immune protection against high-risk HPV infection. The memory B cells induced by HPV prophylactic vaccines can persistently produce high-affinity neutralizing antibodies, thus directly preventing the virus from infecting host cells [
6]. Gardasil 9, in particular, provides protection against approximately 90% of cervical cancer cases caused by HPV infection and significantly reduces the incidence of other HPV-related diseases [
7]. Studies have shown that the protection provided by HPV prophylactic vaccines decreases with the increased age of vaccination [
8], and HPV prophylactic vaccines are not recommended for those who are not in the appropriate age range for vaccination. Moreover, because neutralizing antibodies cannot enter cells, the vaccines cannot provide protection for people are already infected with HPV or have a therapeutic effect on existing HPV-related diseases. The risk of HPV infection is lifelong. Although HPV can be actively cleared by the immune system in most cases, it is still capable of causing latent infection by inducing a defective immune response. Therefore, current HPV prophylactic vaccines still have protective limitations. It is worth considering establishing memory T-cell immunity to provide long-term immune surveillance and generate a rapid response against lesional cells to prevent HPV infection-related diseases such as cervical cancer.
Tumor vaccines are a promising immunotherapy method. They can effectively deliver tumor-associated antigens (TAAs) to the immune system by vaccine vectors and induce specific T-cell responses, especially CD8
+ T-cell mediated CTL responses against TAAs, to clear cancerous cells. Memory T cells (Tm cells) are formed by the differentiation of effector T cells during the initial immune response, and the intracellular gene expression profile in Tm cells is rearranged. Tm cells have a shorter delay time and faster rate of cell division and can express different effector molecules simultaneously [
9,
10]. Tm cells are not only present in immune organs but also distributed in peripheral organs for rapid responses. In addition, Tm cells can maintain long-term immune memory without repeat antigen stimulation, and the stable presence of Tm cells provides long-term immune surveillance [
11‐
13]. Clinical studies have found that the proportion of CD45RO
+CD8
+ Tm cells infiltrated in tumor tissue was an independent factor in the prognosis in patients with squamous non-small cell lung cancer [
14]. In a study by Li T et al., 40 d after treatment with nanovaccine in tumor-bearing mice, the increase in the proportion of CD8
+ Tcm cells in peripheral blood was detected, and the levels of tumor necrosis factor-α (TNF-α) and IFN-γ were significantly upregulated after reinoculation of tumor cells, suggesting that CD8
+ Tcm cells might recognize the reinvading tumor cells and thus elicit an antitumor immune response [
15]. These results suggest that tumor vaccines have the potential to induce the formation of TAAs-specific Tm cells which are involved in antitumor immunity. The application of cervical cancer tumor vaccines for tumor prevention has been reported [
16‐
18], but these studies did not evaluate the immune response induced by preimmunization with tumor vaccines, especially the Tm cells responses or explore the specific antitumor mechanism of tumor vaccines. Therefore, there are still gaps in the research on the immune regulation mechanism of cervical cancer tumor vaccines in tumor prevention. We have successfully constructed two
Listeria-vector cervical cancer vaccine candidate strains, LM∆E6E7 and LI∆E6E7, and confirmed that combined immunotherapy with these two vaccine candidate strains could effectively inhibit tumor progression [
19]. Therefore, in this study we aimed to investigate the role of TAAs-specific Tm-cell responses induced by preimmunizing with LM∆E6E7 and LI∆E6E7 in the tumor prevention and the potential antitumor mechanism.
Discussion
T cells infiltrated in the tumor microenvironment (TME) cannot respond to antigen stimulation and kill tumor cells, but Tm cells isolated from the TME can be activated by tumor antigens and restart antitumor capability [
22]. An increasing number of studies have shown a correlation between the Tm cells response and tumor disease progression. In tumor infiltrating lymphocytes (TILs) of patients with esophageal squamous cell carcinoma, the level of CD45RO
+ Tm cells correlated with prognosis and tumor metastasis [
23]. Enhancing the proportion of Tm cells at tumor sites in patients with glioblastomas might promote the therapeutic efficacy of dendritic cells vectored tumor vaccines [
24]. Esquerre M et al. studied the protective effect of a therapeutic vaccine (GTL001) against HPV-16 and HPV-18 and found that tumor-bearing mice successfully cured by GTL001 produced a Tm-cell response against the reinoculated tumor cells to prevent tumor recurrence [
25]. These findings suggest that Tm cells are directly involved in the antitumor immune responses and play important role in inhibiting tumor metastasis and maintaining long-term protective responses.
As the TAAs of HPV infection-associated cancers, E6 and E7 proteins are ideal target antigens for cervical cancer vaccines. In this study, the tumor antigens carried by LM∆E6E7 and LI∆E6E7 are formed by the cross-fusion of the E6 and E7 proteins of the HPV-16 type. Our study has shown that both strains have good immunogenicity and safety in mice [
19]. In addition, we confirmed that the cross-immunization strategy, that is LM∆E6E7 as the first dose, LI∆E6E7 as the second dose and LM∆E6E7 as the third dose, could avoid the anti-carrier effect to obtain better tumor treatment effect than the single-strain immunization strategy (multiple immunizations with the same vaccine) [
26]. In this study, healthy mice were cross-preimmunized with LM∆E6E7 and LI∆E6E7, and then we observed whether mice could resist the tumorigenesis induced by TC-1 cells. In reported studies on the tumor preventive effect of cervical cancer tumor vaccines [
16‐
18], the time intervals between the completion of immunization and tumor cells inoculation in mice were varied, but none exceeded half a month. Study has found that after 40 days of immunization, CD8
+ Tm cells in mice possess more stable and more definite immune function than those from earlier timepoints [
27]. Due to the time interval is too short to form stable Tm-cell responses in mice, the observed results cannot fully represent the protective immune responses. Therefore, according to the time schedule rule of establishing immune memory in the body and to let the mice establish a stable immune memory response in vivo, we inoculated tumor cells at 40 d after completion of vaccine immunization, which can better simulate the process of vaccination against tumorigenesis in humans. This is the biggest difference between our study and the existing reports in terms of the study protocol of the tumor prevention effect of cervical cancer vaccines.
The mice in the PBS and LM∆ & LI∆ groups died successively due to tumor burden at one month approximately after tumor cells inoculation. However, 60% of mice that were preimmunized with LM∆E6E7 and LI∆E6E7 had no tumor burden until the endpoint (Fig.
2A). This indicates that direct immunization with cervical cancer tumor vaccines is able to induce protective immunity against tumors. After inoculation with tumor cells, the proportions of CD4
+ Tcm, CD4
+ Tem, CD8
+ Tcm, and CD8
+ Tem cells in the spleen and lymph nodes in all groups mice increased with different degrees (Fig.
3), and the proportions of CD8
+ T cells and NK cells in the spleen were also increased. However, the proportion of IFN-γ
+CD8
+ T cells was significantly increased only in the E6E7 group after splenocytes were stimulated by tumor antigen epitopes (Fig.
4G, L, Q). IFN-γ is the basis of adaptive immunity, which plays an important antitumor role in the body. A study showed that tumor-bearing mice treated with tumor vaccines could clear the reimplanted tumor cells, and the Tem cells response has also been detected in vivo [
28]. It suggested that only the mice that preimmunized with LM∆E6E7 and LI∆E6E7 were able to establish tumor antigen-specific T-cell immune memory which played an important role in inhibiting the tumor formation at early stage. But we lack further detection of immune response molecules in TME, such as perforin and granzyme B, which limiting the elaboration of tumor prevention mechanisms. Follow-up studies will focus on the immune response at this stage.
In general, tumor development induces the generation of high levels of regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSCs) and thus forms a suppressive immune response that is favorable for tumor development [
29,
30]. However, we did not find a significant increase in the proportions of Treg and MDSCs in the spleen of mice at 5 d after inoculation with tumor cells (Fig.
4X, Y). We think the reason may be that the detection timepoint (5 d post-inoculation in tumor cells) is too early to induce immunosuppression in vivo. In our other research, we continuously monitored the immune status of cervical cancer tumor-bearing mice and found that the elevation of the suppressive immune level occurred one week after tumor cells inoculation (data not published). Other studies have also reported that there is no difference in the proportions of Treg and MDSCs in the spleen at one week after tumor cells inoculation, but a significant increase could be detected two weeks later [
31,
32]. Our previous study found that combined immunotherapy with LM∆E6E7 and LI∆E6E7 could significantly reduce the proportion of Treg at the tumor site [
19], suggesting that preimmunization with LM∆E6E7 and LI∆E6E7 may also prevent tumor formation and delay tumor progression by weakening tumor-induced immunosuppression. In fact, the E6E7 group mice were not tumor-free for the entire process. Rice-sized masses formed after subcutaneous inoculation of TC-1 cells, but subcutaneous masses were completely cleared within two weeks in 60% of the mice. We speculate that at the early stage of tumor formation, an antitumor immune response was generated but was not sufficient to completely clear the tumor cells. With the persistence of the antitumor immune response and the weakness of the suppressive immune response later, the tumor cells were gradually cleared, and the mice remained tumor-free until the endpoint. In subsequent studies, the influence on the immune response caused by vaccines should be more fully explored by increasing the monitoring timepoints. It reminds us that there is no significant suppressive immune response in healthy women compared to cervical cancer patients. Tumor vaccines can induce a higher level of T cell response in healthy bodies than in cervical cancer patients, to generate stronger resistance against tumor antigens. This is also one of the key factors for women preimmunize with tumor vaccines in advance so as to prevent cancer.
Tm cells respond rapidly after stimulation by antigens. Tcm cells proliferate and differentiate into Tem cells. Tem cells exert immune effects and can localize to tumor sites to mediate protective responses. Immunohistochemical staining analysis showed that the cell proliferation rate in the tumor tissues of mice in the E6E7 group was significantly lower than that in the PBS group (Fig.
5A, B). CD44, L-selectin and CCR7 are expressed on the cell surface as signaling molecules that can drive the recruitment of T cells to inflammatory sites, activate leukocytes and promote the immune response [
33‐
36]. In the TME, higher mRNA expression levels of
CD44,
L-selectin and
Ccr7 were detected in the E6E7 group mice than in the PBS group or the LM∆ & LI∆ group, and the proportion of infiltrated T cells was increased in the LM∆ & LI∆ and E6E7 groups (Fig.
5A, C, E-G). Angiogenesis in tumors is correlated with tumor growth and metastasis. Immunohistochemical analysis revealed a slight decrease in angiogenic capacity in the tumor tissues of mice in the E6E7 group, as well as a decrease in the expression level of
Vegf (Fig.
5A, D, K). Activation of the transcription factor EOMES contributes to enhancing the clonal diversity of the memory pool [
36], and a slightly higher expression level of
Eomes was detected in the tumor tissues of mice in the E6E7 group than in the other two groups (Fig.
5H). These results indicate that the protective immunity induced by the preimmunizing cervical cancer vaccines LM∆E6E7 and LI∆E6E7 may inhibit tumor progression by promoting the enrichment of T cells at tumor sites to exert a specific immune response and by inhibiting intratumoral angiogenesis and cell proliferation. The specific effector molecules and mode remain to be clarified.
Our study suggests that preimmunization with cervical cancer vaccines can provide protection against tumor in healthy individuals. The prevalence of HPV infection among women without cervical abnormalities is 11.7% globally [
37]. The implementation of effective preventive measures for cervical cancer, such as HPV vaccination and HPV screening, is severely limited in less developed countries and regions due to many factors, such as economic level, cultural ideology, and policy diffusion [
38,
39], which also delays the early detection and treatment of cervical cancer. Pay attention to cervical cancer intervention has great significance to improve women’s health benefits. It is worth mentioning that Yvonne Paterson et al. studied on the vaccine LM-LLO-E7 suggests that the sustained expression of E6 and E7 within HPV carriers may induce a low affinity anti-tumor immune response to antigens through a tolerance mechanism. This also means that the protective effect of cervical cancer vaccines may be influenced by the expression levels of viral antigens in HPV infected individuals [
40,
41]. Therefore, the protective effect of cervical cancer vaccines in people who are infected with HPV is still to be investigated.
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