Findings supported that archaeosomes promotes immune responses to DNA vaccines and a long-term CTL response was generated with a low antigen dose. Combination therapy with archaeosome/L1/E6/E7 vaccines exhibited a strong cytolytic activity against tumor cells and induced prophylactic and therapeutic effect against the development of tumor in the animal model.Cancer is a leading cause of death worldwide. Cervical cancer caused by human papillomavirus (HPV) is a major health problem in women. DNA vaccines are a perfect approach to immunization, but their potency in clinical trials has been insufficient for generating effective immunity, which may be related to the degradation of the DNA via nucleases, poor delivery to antigen-presenting cells (APCs), and insufficient uptake of DNA plasmids by cells upon injection. Archaeosome is a nano-delivery systems based on liposomes with their immunological role have been developed for gene delivery. In this study, human papillomavirus type 16 genes, containing truncated L1, E6, and E7, were simultaneously used in combination therapy with archaeosome and assessed  Due to issues associated with HPV biology and its pathogenesis, progress in developing effective therapies against infection of these viruses has been slow2. Numerous therapies are accessible for the management of HPV-related diseases, including surgery, cryotherapy and trichloroacetic acid. Although these strategies are effective, but cannot necessarily eradicate HPV infection as they are applicable for removing only the visible lesions3. Since the diagnosis of HPV happens at the late stages of this disease, as little as 7.7% of women demonstrate this infection without any clinical lesion4.More than four decades have elapsed since the preliminary hypothesis concerning the possible role of human papillomavirus (HPV) as etiological agent of human cancers was suggested. Nowadays, HPV-16 and -18 accounts for approximately 76.7% of cervical cancer worldwide5. Future strategies to combat HPV may rely on therapeutic vaccines, oncolytic virotherapy, immunotherapies as well as antivirals, natural derivatives/herbs, and therapies based on RNA interference, which are being thoroughly utilized and can interfere with several stages of the viral life cycle. Several of these therapeutic agents are in advanced stages of clinical assessment5. HPV vaccine is considered as an encouraging approach to hinder HPV related infection. Although the immunologic effect is not always sufficient to prevent of cancer progression, vaccines are commonly well tolerated and offer valuable anticancer effects in some situations. Several types of vaccine may be used to treat cancers, such as adjuvant and DNA vaccines6.Due to the high frequency of infection following the current management approaches, it is crucial to explore substitute methods for an effective inhibition of the cervix and other HPV-related premalignant and malignant lesionsCurrently, there are three HPV prophylactic vaccines vaccines based on L1 virus-like particles (VLPs) (Cervarix® and Gardasil®) which are authorized for the prevention of cervical cancer with some limitations. First, HPV protection is type-restricted and more than 200 different types of HPV expressing distinct L1 major capsid proteins. Second, the existing HPV vaccines do not confer therapeutic effect. The third limitation is the relatively high cost of production that prevents their widely use in low- and middle-income countries where nearby 90% of deaths from cervical cancer happens.7. The strategy of somatic gene therapy, the use of DNA as a therapeutic candidate, is an excellent approach to open new horizon for medical applications. Many clinical trials have been conducted in this field. In this method the internal production of antigen let the antigen to be presented to the immune system by means of MHC I antigen-processing machinery while still allowing the MHC-II molecules to act. The production of intracellular antigens via exogenous gene more closely mimics live infections, so they can develop the coordinated triggering of both potent cell-mediated and antibody protective immune responses. Furthermore, DNA vaccines administration in humans has already been demonstrated to be safe and well-tolerated8.Hence, there is a vital necessity for an efficient therapeutic vaccine through which HPV related neoplasia can be eliminated without any surgical treatment9.The expression of late HPV capsid proteins (L1) occur merely in differentiated epithelial cells, whereas HPV E6 and E7 are expressed throughout the layers of epithelium and act as viral oncoproteins that not only cause carcinogenesis, but also are mainly expressed in HPV-associated pre-malignant and malignant cells. In this view, the simultaneous gene therapy via HPV viral oncoproteins, E6, E7 and L1 major capsid protein, can be a well-combined model for immunotherapeutic approaches for stimulation of immune responses to prevent current HPV-16 related malignancies10.Despite naked DNA plasmid raise measurable levels of antigen-specific immunity and being successful in some preclinical studies; their efficacy in clinical trials has been unsatisfactory for generating effective immunity. This low immunogenicity may be due to the DNA degradation via nucleases, inadequate uptake of DNA plasmids by cells, and poor delivery to antigen-presenting cells (APCs) following injection. Therefore, both viral and nonviral vectors have been utilized to develop delivery systems that offer protection to pDNA for improving the efficacy of DNA vaccination strategies. Effective nonviral delivery systems, however, remains a major problem to clinical gene therapyNano-delivery systems based on liposomes have been the first systems being elucidated to provide adjuvant function. Their immunological character has also been advanced for gene delivery. This role supports that they could boost immune responses to DNA vaccines through enhancing their uptake via APCs in the lymphoid tissues.11. The main limitation to the application of conventional liposomes is their instability, relatively short half-life and uneconomical production especially in large scales. Conventional liposomes can be quickly removed from the circulation by liver Kupffer cells and spleen macrophages, vesicle disruption at low pH in the gastrointestinal areas and lipid hydrolysis because of lipases12.Liposomes composed of synthetic esters have been explored as drug or antigen carriers, and a nano liposome-based vaccine against hepatitis A being licensed for use in humans13. Archaeal lipids are composed of typically fully saturated and branched phytanyl chains in many species which are being linked through ether linkage to the glycerol backbone at the positions of the sn-2, 3. The unique structures of archaeal lipids confer considerable stability on archaeosomes. These remarkable molecular and physicochemical characteristics of archaeal lipids confers some superiority over the custom ester phospholipid liposomes which led to the adaptation of their membrane lipid compositions to stringent environments, suitable for the highly stable archaeosomes preparation14. Archaeosomes are demonstrated extremely effectual as a self-adjuvanting vaccine strongly eliciting antigen-specific, both cell-mediated immune and humoral responses, to encapsulated antigens. Some superior characters of Archaeosomes compared with conventional liposomes and other adjuvants are the stimulation and recruitment of dendritic cells; antigen orientation to process via MHC class I which leads to the potent activation of CD8 + cytotoxic T lymphocyte responses that is pivotal to combat against intracellular pathogens and cancer; and archaeal lipid cores stability which make a profound immune memory easier16.Archaeosomes belong to a biological liposomes family which are composed of total polar lipids extracted from archaea, such as methanogens, halophiles and thermoacidophiles17. Therefore, liposomes formed by archaebacterial membranes lipids represent a promising alternative to the classical liposomes and virosoms, consequently found extensive applications in vaccine and drug delivery20.Additional significant advantages of archaeosomes include a low number of boosts required to reach a high circulating antibody titer. Archaeosomes are safe without any noticeable toxicity. They are more resistant to chemical hydrolysis, oxidation, bile salts, acidic or alkaline pH, and high temperatures with various vaccine and drug delivery applicationsWith these in mind, the current study aimed to assessment the ability of the cellular antigenic regions of the HPV 16 genes, containing truncated L1, E6, and E7, used simultaneously in combination therapy with archaeosomes as an adjuvant and nanoparticle carriers. In this study, archaeosome was prepared and characterized, then formulated with DNA vaccine (L1/E6/E7 recombinant genes). In the next step, the cervical cancer tumor model was provided in C57BL/6 mice and the ability of the vaccine in stimulating different facets of immune responses evaluated; also, the DNA fragmentation in tumor cells following administration of DNA vaccine/archaeosomes formulation in animal models was assessed.The recombinant DNA vaccine candidate (pIRES- L1/E6/E7) utilized in this study was generated in our previous study and validity of synthesized sequences was confirmed using bioinformatics software and western blot. The list of the amino acid sequences of the peptides can be found as Supplementary Table In current study, to confirm the expression of the recombinant plasmid carrying the L1/E6/E7 gene, we examined the GFP protein expression using a fluorescence microscope.The recombinant DNA vaccine candidate L1/E6/E7) was subcloned into pIRES2-EGFP vector. This vector contains the internal ribosome entry site  of the encephalomyocarditis virus (ECMV) between the Multiple Cloning Site (MCS) and the enhanced green fluorescent protein (EGFP) coding region. This permits both the gene of interest (cloned into the MCS) and the EGFP gene to be translated from a single bicistronic mRNA. Therefore, the pIRES- L1/E6/E7 plasmid was utilized for the HEK293 cell transfection assay. After 24 h of culture, the GFP protein expression was visualized using a fluorescence microscope . Secondary antibodies were goat anti- Mouse IgG H&L (HRP) (Abcam). Binding signals were visualized with TMB substrate . The result of western blot provided as a supplementary file  was assayed in the present study by the LDH release assay in triplicate in 96-well plates by culturing the effector cells with TC-1 target cells at the effector/target (E/T) ratio of 25:1. The data presented in Fig. To evaluate the frequency of T cell immune responses induced by various formulation of vaccines and investigation of the potential mechanisms responsible for the therapeutic efficacy of the vaccine candidate on TC-1 tumor, the proportion of different cytokines between experimental groups was compared.Tumor-bearing C57BL/6 mice were immunized three times at one-week intervals using each DNA vaccine alone or in combination with the archaeosome .ex vivo, and various cytokine profiles such as IFN-γ, IL-2, IL-10, and IL-4 secreted via T cells were assessed by ELISA kits. As shown in Fig. Seven days after the last immunization, freshly isolated splenocytes of immunized mice were cultured in the presence of different mitogens The pDNA alone (L1/E6/E7 recombinant gene) induced IFN-γ production compared with control groups, and the rate of this stimulation was significantly increased after being formulated with archaeosome, representing the adjuvant and protective properties of archaeosome nanoparticles. Furthermore, the number of IFN- γ- and IL-2-secreting splenocytes in mice immunized with antigen-free archaeosomes was statistically significant and approximately two-fold higher than in the rest of the mock plasmid and PBS control groups, demonstrating the intrinsic adjuvant property of the archaeosome nanoparticles.Data in Fig. To assess the possibility that the administration of vaccines candidate would affect apoptosis in the cervical cancer model, the tumor cell was analyzed following vaccination with L1/E6/E7 gene with or without the formulation of archaeosome. For this purpose, DNA fragmentation in tumor cells was examined by TUNEL assay in pDNA-surface localized archaeosomes, pDNA-encapsulated archaeosomes, L1/E6/E7 recombinant gene alone, and the results were compared with DNA fragmentation from PBS control groups. Nuclear staining was observed using a DAPI filter (magnification for g through n: 40X). All vaccine treatments displayed an elevated number of apoptotic cells by TUNEL assay when compared to control groups.The majority of these apoptotic cells were found in the tumor following pDNA-surface localized archaeosome and pDNA-encapsulated archaeosome treatment groups. The number of apoptotic cells in tumors significantly decreased following L1/E6/E7 recombinant gene vaccine treatment alone compared to that following the main treatments.Therefore, according to the results, treatment with the combination of L1/E6/E7 genes alongside archaeosome nanoparticle was associated with the greatest extent of apoptosis, ranging from 40–60%. Treatment with L1/E6/E7 recombinant gene led to less apoptosis, ranging from 15–20% for pDNA alone. Finally, treatment controls resulted in background apoptosis levels ranging from 0 to 1.5%. These results suggest a synergistic effect between L1/E6/E7 gene and archaeosome vaccines individually . Seven days after the end of administration (Day 28), the average tumor volume was 601.9 ± 125.67 mm3 and 530 ± 198.09 mm3 in the pDNA-surface localized archaeosome and pDNA-encapsulated archaeosome group, respectively; 1225.4 ± 190.68 mm3 in the L1/E6/E7 recombinant gene alone group; 2297.5 ± 284.15 mm3 in the antigen-free archaeosomes; and 2696.28 ± 240.86 and 3212 ± 314.62 mm3 in the mock plasmid and PBS control group, respectively.Based on Fig. in vivo anticancer efficacy studies demonstrated that the combination treatment with archaeosome-pDNA formulation greatly hindered the tumor progression compared to each treatment alone.Although tumor growth in the L1/E6/E7 recombinant gene alone group was much slower than the mock plasmid and PBS control groups after treatments (P < 0.0001) Fig. ; and theIn addition, a statistically significant difference was observed in tumor weight between pDNA-surface localized archaeosome and pDNA-encapsulated archaeosomes groups-treated mice and other groups. Tumors treated with PBS and mock plasmid control group reached 10.28 ± 0.5 g and 7.79 ± 1.2 g, respectively. However, tumor weight was reduced to 3.64 ± 0.7 g and 3.6 ± 1.1 g Fig.  in the pIn this study there were six animal groups, and each group consisting of eight pathogen-free female C57BL/6 mice. Five Mice of each group were euthanized one week after the 3rd immunization  and spleens and tumor were collected for the evaluation of different experiment. Three mice remained in each group were closely monitored up to 42 days and the cumulative survival rate was calculated.Figure 21. It is now estimated that 20–25% of human cancers worldwide have a known viral etiology22. Human papillomavirus plays essential roles in the initiation as well as progression of cervical cancer, and continued expression of their viral transforming activities is required for the maintenance of the transformed phenotype4. Investigating of the association between the high-risk HPV, especially HPV 16 (60–50%), and the development of cervical cancer, initiates a wide range of studies aimed at producing therapeutic or prophylactic vaccines against HPV infection23. To design a vaccine with a high potential to boost immune responses, considering of several parameters such as the dose, time, and routes of administration are essential. Also an appropriate adjuvant in vaccine formulation to increase the stimulation of the immune system are necessary. Although the infection by high-risk papillomavirus can be prevented with introducing of Gardasil and Cervarix vaccines, but the problem remained still due to the general non- vaccination of communities and the presence of non-responder infected populations24. It seems that there is no ideal vaccine with a high efficacy in clinical phases, which can act as a therapeutic and preventive. Therefore, designing these types of vaccine was the aim of the present study.Despite advances in the treatment of cancer, nearly 530,000 new instances of disease and 275,000 deaths from cancer occur annually worldwide. Viruses play a significant role in many human cancers25. DNA vaccines are a valuable form of antigen-specific immunotherapy which are transcribed in eukaryotic cells using a proprietary viral promoter, and the target gene supply is performed through the MHC-I pathway which activates CTLs and stimulates the host immune system. In addition, the induced immune response is long-term26. The cellular immune response in contrast to humoral immunity plays an important role in effectively responding against human papillomavirus. It is believed that the human papillomavirus vaccine should not only prevent the formation of primary infections, but also provide treatment approach. Antibody‐mediated immunity is only able to prevent infection, while cell-mediated immunity against the intracellular proteins of the papillomavirus can perform both functions by activating T helper cells for the proliferation and differentiation of B cells and CD8+ (cytotoxic) T cells and the production of inhibitory cytokines27.In viral diseases and cancer, the activation of CTLs to eliminate infected and cancerous cells is necessary. Thus, it is vital to use strategies for activating CTLs28. The HPV-E7 oncoprotein is an appropriate candidate for designing anti-tumor vaccines, which in addition to the production of anti-E7 IgG in mice, induces the activity of cytotoxic T cells. Consequently this vaccine has both preventive and therapeutic properties28. Liu et al. (2002) showed that the immunogenicity of the polynucleotide vaccine increases when using optimized HPV-E729 and, in the case of HPV 16 E7, reported that N-terminus and C-terminus are immunogenic. On the other hand, numerous studies have indicated that HPV16 E6-based vaccines are an effective way to prevent the E6 gene expression in the tumor.In the initial human studies, the protective effect of L1 protein against HPV infection and the therapeutic role of E6 and E7 proteins after exposure to the papillomavirus have been demonstratedet al. (2006) at Johns Hopkins University concluded that the DNA vaccine containing E6-gene has a strong ability for protein expression in the cell culture models and can more effectively induce CTL responses30. Mirshahabi et al. (2012) showed that E6 protein of type 16 human papillomavirus can induce cytotoxic T-cell responses in a cell culture model31. Since E6 and E7 proteins are continuously expressed at all stages of cervical cancer, they can be employed as an ideal target to produce therapeutic vaccines and develop immunotherapy methods against papillomavirus tumors. Blounet et al. (2009) demonstrated that specific T-CD8+ and CD4+ lymphocytes of L1 gene have therapeutic effects in patients with cervical cancer28.Cheng In the present study, we used the perfect combined construct contains the immunogenic regions of the papillomavirus L1, E6, and E7 recombinant gene in the formulation of archaeosome nanoparticles as an adjuvant and carrier system to increase and strengthen the immunogenicity of these DNA vaccine. So far, no report has been presented based on the investigation of archaeosome nanoparticles as a carrier and adjuvant in formulation with DNA vaccine for the treatment of cervical cancer in the mice model.33. Archaebacteria are a group of non-pathogenic organisms not associated with endotoxins or other toxic metabolites, and thus archaeosomes are predicted to be safe34. Early studies have demonstrated that archaeosomes can induce a long-term antibody response in their associated antigen only after two initial injections on Days 0 and 2133. In the initial stage after immunization by bovine serum albumin-archaeosomes, more than 95% of CD8 + T cells responded to the antigen15. The study by Patel et al. revealed that the archaeosomes made of lipids extracted from M. smithii are an appropriate adjuvant for a multivalent mucosal vaccine. The archaeosome containing encapsulated antigen OVA and egg lysozyme demonstrated a robust and specific antibody response to all antigens16. Krishnan et al. (2010) reported that archaeosomes made of lipids extracted from M. smithii are a strong adjuvants for evoking a CD8 + T cell response. Their results exhibited that system could be used in the formulation of cancer vaccines13. Rethore et al. (2009) examined the archaeosome made of synthetic tetraether lipids as a new gene delivery system, and the results showed that these archaeosomes could be used for the delivery of genes in vitro35. Due to their high stability, archaeosomes can be considered as suitable carriers for proteins, peptides, and genes17.The archaeosome technology is a new self-adjuvant delivery system with the ability of antigen delivery to the MHC class I processing pathway and stimulating the intrinsic immune system to induce a strong and long-term immunity against subunits of antigen36.Nanoparticle carriers provide adjuvant activity by increasing the delivery of the antigen or activating the inherent immune response. The strength and mechanism of stimulating the immune system induced by nanocarrier vaccines depend on various factors, including chemical composition, particle size, homogeneity, load, nature, the position of the antigen within the carrier, and site of administration (123). DNA-liposome complexes enter the cell through endocytosis. In this method, the cytoplasmic membrane surrounds the liposome like a sheath and creates an intracellular carrier called endosome; liposome is decomposed inside the endosome; and the DNA is released into the cytosol and transferred to the nucleus37. The presence of cholesterol is required in stabilizing the dual-layer membrane and increasing the liposomes’ persistence in the in vivo environment. In the absence of cholesterol, liposomes are destabilize by HDL and followed by the release of the contents, and the drug is rapidly removed from blood circulation38. In the endocytosis process for entering the cells, liposome is swallowed by cells, like the phagocytosis of foreign particles by phagocytes. It has been shown that, by decreasing the size of liposomes, their surface potential is reduced. Also, increasing the particle size of liposomes to more than 1000 nm leads to rapid swallowing by macrophages and, consequently, early removal from blood circulation39. Macrophage cells which have different endocytic activities swallow almost all types of liposomes. According to studies by Allen et al., elimination of liposomes with a very small size (about 8–4 nm) does not occur, and these particles are only partially removed by glomerular filtration in the kidneys. However, due to their inherent instability, vesicles with a particle size of less than 40–50 nm are not constructed38.Choosing lipids to make liposomes as carriers for drug or DNA vaccine depends on several factors40. According to the present results, it seems that the size of the nano- archaeosomes is within an appropriate range and, therefore, one of our main goals has been achieved.In the present study, the nano-archaeosome particle size was optimized and measured by the Zetasizer Nano ZS, which approximately equaled 98.1 ± 1.3 nm before formulation with plasmid and about 429 ± 5 nm and 127 ± 2.1 nm after formulation in the pDNA-encapsulated archaeosomes and pDNA-surface localized archaeosomes, respectively. Studies have shown that immunization with small liposomes induces a Th2 response, whereas large liposomes induce a Th1 response, higher interferon gamma (IFNγ) levels, and immunoglobulin IgG2a/IgG1 ratios41.In this research, the zeta potential after formulation with plasmid DNA became more negative, reaching −29 ± 0.7 mV from −6.84 ± 0.8 mV at surface formulation and −30.8 ± 0.7 mV at encapsulation form, which can be related to the addition of DNA to archaeosome. In many studies, it has been concluded that the negative charge contributes to a better transfer of DNA into the cell and an increase in transfectionin vivo; was provided the cervical cancer tumor model in C57BL/6 mice using the subcutaneous injection of TC-1 tumor cells. In many similar studies, the amount of injected DNA vaccine was 100 μg/100 μl per dose. In this study, in order to reduce the vaccine dose and investigate the effect of archaeosome as carriers and adjuvants on increasing the efficacy of low-dose DNA vaccine, it has been shown that fewer amounts of DNA vaccine in formulation with archaeosome significantly reduce the tumor size and stimulate cellular immune responses.In order to investigate the ability of the vaccine for the induction of immune response, and the evaluation of the properties of archaeosome nanoparticles 34. In addition, the antigen-specific activation of both Th1 and Th2 cells was corroborated by splenocytes from archaeosome-antigen immunized mice relative to the antigen Alum group42. Archaeosome-antigen presentation occurs through the classical cytosolic pathway and MHC class I molecules, in which a strong adjuvant activity is sufficiently potent to bypass a requirement for CD4 + T cell help. The published results clearly demonstrated the superior adjuvant activity of archaeosome in comparison with conventional liposomes and Alum42.Given the importance of the cytokine response in antiviral and anti-tumor immunity, if the type 1 cytokine response is produced in vaccinated mice, the immune system will be able to eliminate the infected tumor cells. Therefore, in designing therapeutic vaccines, the use of a carrier or adjuvant with the goal of shifting the cytokine balance towards Th1 immune responses and cell-mediated responses should always be considered. Thus, in this research, the cytokine secretion of the splenocytes of the vaccinated mice in test groups was compared with negative control groups. One of the first problems in the use of Alum as an adjuvant has been its inability to induce strong IgG2a and cell-mediated immunity, in particular, Th1 cells. Archaeosome strongly induces both IgG1 and IgG2aTh1 cells produce cytokines such as IFN-γ and IL-2 which are involved in the differentiation of precursor mouse CD8 + T cells into cytotoxic CD8 + T cells. In this study, IFN-γ secretion in two main vaccine groups, that received the surface localized and encapsulated pDNA/archaeosome vaccines, was significantly higher than that in other groups. Moreover, the DNA vaccine alone (L1/E6/E7 recombinant gene) somewhat induced an IFN-γ-type cytokine response, suggesting that the presence of three truncated E6, E7, and L1 genes together leads to an appropriate immune response. In this research, the rate of immune response increased after the formulation of L1/E6/E7 recombinant gene and archaeosome, representing that the archaeosome provides a potent adjuvant activity and protects the pDNA from degradation and oxidation. This protective phospholipid shield undamaged until the contents of the liposome are delivered to the exact target where the contents will be utilized. In addition, since empty archaeosomes alone with a significant difference in comparison with PBS and mock plasmid groups have caused IFN-γ secretion, one can note the intrinsic adjuvant property of archaeosome, consistent with other studies.Interleukin-2 plays a determining role in controlling the survival of immature and mature T cells. In this study, groups vaccinated with DNA vaccine-archaeosome formulation have significantly improved IL-2 secretion compared to control groups. Surface-localized and encapsulated pDNA/archaeosome vaccines induced the highest IL-2 production, and these findings can indicate the adjuvant and delivery property of archaeosome, causing appropriate protectiveness and boosting the DNA vaccine efficiency. Since nanoparticles alone induced IL-2, an inherent self-adjuvanting property of archaeosome nanoparticles was well demonstrated in this study.Interleukin-4 is produced by Th2, NK, and mast cells. In this study, IL-4 cytokine measurement showed that, in groups receiving surface localized L1/E6/E7/archaeosome vaccines, they significantly induced IL-4 secretion in comparison with the control groups. In addition, encapsulated L1/E6/E7 recombinant gene in archaeosome induces IL-4 secretion, but it is not remarkable in comparison to other groups; this can be due to the lack of DNA access in the encapsulated state and the slow release of plasmid from archaeosome.Based on the results of this study, it was observed that in groups receiving the L1/E6/E7 recombinant gene DNA vaccine in the surface of archaeosome, they significantly induced IL-10 secretion compared to PBS, mock plasmid, L1/E6/E7 recombinant gene, and empty archaeosomes alone groups. As previously stated, IL-10 cytokine is a stimulant for the humoral immune system and stimulates B lymphocytes. In the present study, the surface localized L1/E6/E7/archaeosome vaccines group stimulated the secretion of IL-10 more than the encapsulated L1/E6/E7/archaeosome vaccines group. It can be said that a high-level induction of IL-10 in the form of surface formulation is expected due to the availability and faster release in comparison to the encapsulated state, while the enclosed antigens need a disturbance in vesicles to be available. IL-10 secretion level in the other groups is not significantly different.43 and consistent with the goals and hypotheses of our plan due to the higher levels of Th1 cytokine production, IFN-γ and IL-2, in comparison to Th2 cytokines, IL-10 and IL-4; considering our purpose in the selection of specific sequences of E6, E7, and L1 genes which stimulate cytotoxic T lymphocytes (CTLs); and considering the archaeosome properties for the activation of the cellular immune system.It can be concluded that the results of this research were similar to those of other published studiesFurthermore, the location of the antigen in liposome affects immunity. Both surface localized and encapsulated antigens induce T-cell response. The advantage of the first case is availability as well as fast and easier release from archaeosome, while the enclosed antigens need disruption within the vesicle in order to be available. The results demonstrated significant differences between the efficacies of archaeosome-containing DNA vaccine compared with when DNA was injected nakedly, similar to others studies. The cytokine profile represents switching the immune response toward Th1 using the archaeosome, well demonstrated in the reduction in the size of the tumor. Moreover, in this study, no significant difference between negative control groups is shown.The impacts of each vaccine in the induction of immune responses has been studied by measuring the tumor size. There was a significant difference between the tumor volumes of test groups compared to control groups. Finally, with regard to the profile of cytokine production and their results in inhibiting tumor growth, the synergistic effect of the L1/E6/E7 recombinant gene and archaeosome can be concluded. The findings clearly indicated the positive effect of some of the structures on the reduction of the HPV-E6/E7-expressing tumors size. By monitoring the tumor size in the remaining mice, it was well-established that archaeosome-formulated pDNAs are well able to stop tumor growth in the animal. In general, the results of spleen cell cytokine secretion and measuring tumor volume in this study confirm that the candidate DNA vaccine in formulations with archaeosome nanoparticles in both surface and encapsulation formulation has created the best anti-tumor response in comparison with other vaccinated groups.In order to further study the possible effect of L1/E6/E7-archaeosome vaccine on cervical cancer, we detected apoptosis signals in tumor sections with the TUNEL method. TUNEL staining data indicated that apoptosis occurred in tumor cells from mice immunized with L1/E6/E7 DNA vaccine. It is interesting that apoptosis in the tumor cells of immunized mice clearly increased after the administration of both plasmid DNA immunogens adsorbed onto or encapsulated inside archaeosome nanoparticles. However, no apoptosis occurred in mice immunized with the PBS control group. The result suggested that there was some relation between the combination treatment of L1/E6/E7 recombinant gene/archaeosome and apoptosis.Humans are constantly exposed to archaeal-like lipids, so archaeosome as a natural adjuvant can be safe and suitable for human body. One of the main obstacles to many new adjuvant technologies, especially those that are able to augment potent immune response, is their toxicity, particularly at high doses. Archaeosomes lack toxicity and can be activate the immune system.Simultaneous utilization of HPV 16 genes, L1, E6, and E7 in combination therapy with archaeosomes can be an excellent strategy for stimulate immune responses against existing HPV-16-associated malignancies, holding great promise for the therapeutic vaccine development.This therapeutic approach offers a novel prospect for the improvement and rational design of potent and safe vaccines capable of eliciting T cell immunity against cancers and intracellular infections. Although there is a long way to reach the final result in the use of archaeosome, it seems that the application of this technology has its undeniable benefits. Archaeosomes have been introduced as highly valuable carrier for vaccines which, besides appropriate presentation of antigen to immunocompetent cells and improving antigen stability, they have the ability to overcome biological obstacles, such as skin and mucosa. Moreover, they provide controlled and slow release of antigens. In addition to the ability to induce a potent immune response by adjuvant property, archaeosome-based vaccines have features that are essential for the improvement of modern vaccine formulations. It is predictable that this therapeutic and prophylactic strategy will be increasingly employ in the near future successfully and can lead to major advances in vaccine development.44. To generate the recombinant construct (L1/E6/E7) encoding CTL-epitopes restricted by both human and mouse, the cellular antigenic regions of the human papillomavirus type 16 genes, containing L1, E6 and E7 selected and linked by a spacer of glutamic acid, alanine, alanine, alanine, lysine. The list of the amino acid sequences of the peptides can be found as Supplementary Table The recombinant DNA vaccine candidate (L1/E6/E7) utilized in this study was generated in our previous study45. For the large-scale preparation of the plasmids, pIRES-L1/E6/E7 and pIRES were performed by ion exchange chromatography with a Plasmid Mega kit  according to the manufacturer’s instructions.In current study, the pIRES-L1/E6/E7 and pIRES (mock plasmid) was used to construct archaeosome-plasmid DNA (pDNA) formulations. The competent cells of Escherichia coli DH5α strain were transformed via recombinant pDNA in the Luria-Bertani medium in the presence of kanamycin. Amplification and purification of plasmids were performed with a GeneAll® ExprepTM Plasmid SV mini (Korea). The quality and quantity of the pDNA were spectrophotometrically detected with a Thermo Scientific NanoDrop™ 2000/2000c, and electrophoresis was performed on a 1% agarose gelIn current study, we examined the GFP protein expression using a fluorescence microscope. The recombinant DNA vaccine candidate (L1/E6/E7) utilized in this study was subcloned into pIRES2-EGFP vector. This vector contains the internal ribosome entry site  of the encephalomyocarditis virus (ECMV) between the Multiple Cloning Site (MCS) and the enhanced green fluorescent protein (EGFP) coding region. This permits both the gene of interest (cloned into the MCS) and the EGFP gene to be translated from a single bicistronic mRNA.4 cells per well seeded into 24-well culture plates, and incubated under standard culture conditions for 24 hour. Two hours before transfection the culture medium was renewed. Sub-confluent HEK293 cells were transfected with pIRES-L1/E6/E7 using Lipofectamine 2000  according to the manufacturer’s protocol and incubated at 37 °C with 5% CO2 for 24 h. Empty pDNA was used as the control in these experiments. The medium was replaced 6 h after the transfection to remove plasmids not internalized by cells. Twenty-four hours post-transfection, the transfected cells were analyzed by immunofluorescence microscopy (IF).The HEK293 (Human Embryonic Kidney 293) cells were cultivated in DMEM  cell-culture medium without any antibiotics, supplemented with 10% fetal bovine serum , 1 mM sodium pyruvate, 2 mM glutamine, and 2 mM nonessential amino acids, and incubated at 37 °C with 5% CO2 and 95% humidity. The cells were passaged at 80% confluency and 5 × 10Halobacterium salinarum  was provided by Pasteur Institute . The cells were grown aerobically at 40 °C in the modified growth medium for the cultures described by Choquet et al., containing (g/l): (NH4)2SO4 (3), K2HPO4 (0.5), MgSO4⋅7H2O (0.5), Ca(NO3)2⋅4H2O (0.01), and KCl (0.1). Sulfur (10 g/l) and 0.1% yeast extract were added to the basal medium and adjusted to pH 7.2 using sulfuric acid. Cultures were incubated in rotary shakers for 10 days at 40 °C, monitored by absorbance at 660 nm, harvested in the late stationary phase, and stored at −20 °C as cell pastes prior to lipid extraction.H. salinarum by the modified method of Bligh and Dyer and the total polar lipid fraction collected by precipitation by cold acetone. Cell paste was thawed by water and solvents were added to obtain a volume of methanol/chloroform/water at the ratio of 2:1:0.8 v/v and the mixture was stirred for 24 h at room temperature. The suspension was centrifuged at 5000 g for 20 min in glass centrifuge bottles. The supernatants containing the lipids were combined and transferred into a separatory funnel. Subsequently, chloroform (CHCl3) was added to produce a biphasic system. The lower organic phase (chloroform layer) included the lipids, and the top methanol-water layer generally contained the non-lipid components. The chloroform layer was withdrawn, the volume of CHCl3 was depleted to dryness by rotary evaporation at 60 °C, and the lipid films thus obtained were dried under high vacuum. The lipid residue was dissolved into CHCl3 adding the minimum volume of methanol required, and the polar lipids were precipitated by adding ice-cold acetone . After storage at −20 °C for 24 h, the white precipitate of total polar lipids was collected by centrifugation and dissolved in CHCl3. Lipid extracts dissolved in CHCl3 were stable indefinitely but were placed at 4 °C to minimize solvent evaporation over long storage periods and the resultant precipitation of lipids from concentrated extracts. The chloroform phase was dried with lyophilization, and the TPLs were stored at 4 °C until required.Total polar lipids were prepared from frozen and thawed pastes of The formation and morphological characteristics of uncomplexed archaeosomes were analyzed with TEM. Archaeosome samples were examined by a negative staining technique using adsorption on a carbon-film–covered copper grid, stained with 2% (w/v) uranyl acetate, and investigated with an electron microscope  at an accelerating voltage of 60 kV.H. salinarum. For the preparation of encapsulated plasmid DNA-archaeosome formulation, dried total polar lipids were hydrated at 37 °C in phosphate-buffered saline (PBS) supplemented with either plasmid DNA. The solutions were vortexed and hydration was allowed to proceed at 37 °C with shaking for 24 h. Empty archaeosome was formed following the same procedure. For the latter, manufacturing of the surface localization of plasmid on lipid vesicles, plasmid DNA was surface-bound by incubating empty archaeosomes at 37 °C with shaking overnight. To improve complex formation between pDNA and archaeosomes, CaCl2 as a helper molecule was also added into the medium20. In the end, the suspensions were sonicated at room temperature for three times 5 min with intervals of 3 min using a sonication bath . Any plasmid not associated with the vesicles was removed by centrifugation (25000 × g for 1 h), followed by washing the vesicle pellet twice in PBS. The formulations were stored at 4 °C until use.Archaeosomes were prepared aseptically by hydrating the lyophilized TPL from The average size and zeta potential of the archaeosomal formulations were measured using a Zetasizer Nano ZS . Sample size were detected by dynamic light backscattering. All measurements were conducted at 27 °C and in triplicate.Inbred female C57BL/6 mice five to six weeks of age were purchased from the Pasteur Institute  and maintained in the animal facility of the Tarbiat Modares University . All animal procedures were performed according to the approved protocols and in accordance with recommendations for the proper use and care of the Animal Ethics Committee of the Faculty of Medical Sciences, Tarbiat Modares University. The approval No. IR.TMU.REC.1394.193.2 and 95% humidity. The cells were harvested at 80% confluency, and 7 × 105 cell suspensions in 100 μl of PBS were inoculated subcutaneously in the right flank of C57BL/6 mice.TC-1 cells were purchased from Pasteur Institute of Iran . The cells were propagated in DMEM  cell-culture medium supplemented with 10% fetal bovine serum  and 100 IU/ml of penicillin, 100 µg/ml of streptomycin, 2 mM glutamine, 1 mM sodium pyruvate, and 2 mM nonessential amino acids, and incubated at 37 °C with 5% COSix animal groups, each consisting of eight pathogen-free female C57BL/6 mice, were handled according to animal care ethics committee of the Faculty of Medical Sciences, Tarbiat Modares University,  and immunized according to the schemes presented in Table 4) as target cells were co-cultured with effector cells in a volume of 100 μl in phenol red-free RPMI containing 3% FBS at effector/target ratios of 25:1. Generation of effector CD8 + T cells (CTLs) was assayed by the LDH release assay in triplicate in 96-well plates according to the manufacturer’s instructions . Optical density was read at the wavelength of 492 nm (A492), and cytotoxicity was calculated according to the formula:In order to assess cell cytotoxicity, lactate dehydrogenase (LDH) level was determined in splenocytes. LDH is an intracellular enzyme found in the cells of many body tissues. one week after the last immunization, a single-cell suspension of splenocytes was prepared and used as effector cells. A precise number of TC-1 cells  or 10 µg/ml of Escherichia coli LPS ; and then incubated at 37 °C with 5% CO2. Culture supernatants were collected after 72 h for the measurement of IFN-γ, IL-2, IL-4, and IL-10 cytokine secretion using commercial DuoSet enzyme-linked immunosorbent (ELISA) cytokine assay kits  according to the manufacturer’s instructions. The lower limit for the detection of the cytokine was 32 pg/ml. Values were presented as pg cytokine/ml .To assay proliferation, single-cell suspensions of splenocytes from vaccinated and control mice were obtained one week after the last treatment by gentle homogenization. Selective lysis of erythrocytes was performed with Tris-buffered ammonium chloride ,  for 5 min at room temperature, and the pelted splenocytes were resuspended in RPMI supplemented with 20% FBS, 2mM L-glutamine, 100 U/ml of penicillin and 100 µg/ml of streptomycin. The cells at the concentration of 2 × 10in vivo with the In Situ Cell Death Detection kit  according to the manufacturer’s instructions. Briefly, seven days following the final administration, the mice were euthanized and histological studies were conducted. Autopsied tumors from animals were fixed in 10% phosphate-buffered formalin. The formalin-fixed paraffin-embedded tumor tissue was sectioned in three different regions from the central part of the tumor, and deparaffinized. Each section was rehydrated through a graded series of ethanol and double distilled water, washed in PBS, incubated with PBS containing 20 μg/ml of proteinase K for 20 min, and permeabilized with 0.1% Triton x-100 and 0.1% sodium citrate. The sections were then incubated in TUNEL solution at 37 °C for 1 h. Finally, the samples were washed with PBS and observed under a fluorescent microscope (×40). Apoptotic cells were counted on a basement membrane of seminiferous tubules in each group.Tumor cell apoptosis was determined Data were expressed as mean ± SD. One-way ANOVA and two-way ANOVA with Tukey’s test were performed for data comparisons. The GrapPad Prism software version 7.04 was used to plot charts. Asterisks indicated groups are significantly different from each other .The animal experiments followed the guidelines of the Laboratory Animal Ethical Commission of the Faculty of Medical Sciences, Tarbiat Modares University. The approval No. IR.TMU.REC.1394.193.Supplementary Figure S1.Supplementary Table S1.Supplementary Table S2.


Head and neck cancers (NHCs) are of multifaceted origins, and tobacco and alcohol are the primary risk factors. Currently, other factors associated with the genesis of these tumours are being considered, among these viral infections, especially human papillomavirus (HPV) infection.The objective was to evaluate HPV infection, HPV-16 E6 load and its physical status in patients with squamous cell carcinoma in the head and neck and evaluate its effects in the survival of these patients.A total of 80 fresh biopsies of HNC were evaluated. The genetic material was extracted using the commercial kit QIAGEN. The detection and classification of HPV were carried out using INNO-LiPA, whereas the quantification and analysis of integration of the viral genome into the host cell were carried out using real-time PCR.p < 0.05).The average age of the patients included was 60.34 ± 14.48 years, with a predominance of the male gender. The most frequent HPV infection was genotype 16 (52.8%), with an average of 10 copies of the HPV-16 E6/β-globin gene. Furthermore, an integration of the viral genome in the host cell was observed in 86% of cases with a statistically significant relationship between the location of the tumour and the viral load (HPV-16 is the most common infection, and its physical status in the host cell is the determining factor in establishing response to treatment. However, more studies are needed to demonstrate the role of HPV infection in carcinogenesis. Head and neck tumours of any oncological pathology are of multifactorial origin, and tobacco and alcohol are the primary risk factors. It is estimated that between 85% and 90% of cases of head and neck squamous cell carcinomas (HNSCC) are explained by exposure to these factors; the risk is being proportional to the intensity of the exposure . In receCurrently, there is epidemiological and molecular evidence that human papillomavirus (HPV) is related to squamous cell carcinomas, such as in the case of cervical cancer  and oropDriven by everything set out previously, the objective of this investigation is to evaluate HPV infection, HPV-16 E6 load and its physical status in patients with squamous cell carcinoma in the head and neck and evaluate its effects in the survival of these patients.A total of 80 fresh biopsies from HNSCC tumours were evaluated. These were gathered by Dr Juan F. Liuzzi of the Head and Neck Service in the Oncology Hospital ‘Padre Machado’ in Caracas.Each patient included in this study had to give their approval by signing an informed consent as well as responding to a survey, which can be found as part of the FONACIT projects G-2005000408, 2013000413 and Misión Ciencia (Science Mission) LPL-20074001088.The extraction of the nucleic acids was carried out using the commercial DNA Mini Kit QIAGen (250) following the firm’s specifications.From each biopsy, a piece of tissue approximately 25 mg was cut, and this, in turn, was cut into small pieces and incubated in ATL buffer (180 μL) and 20 μL of proteinase K overnight with agitation at 56ºC. Afterwards, 200 μL of buffer AL was added, vortex mixed for 15 sec and incubated at 70ºC for 10 min.200 μl of absolute ethanol was added, and then, it was transferred to a tube and centrifuged at 6,000 xg for 1 min; Nearly 500 μL of the buffer of washed AW1 was added and centrifuged at 6,000 xg for 1 min; 500 μL of the buffer of AW2 was added and centrifuged at 20,000 xg for 3 min. Then, 200 μL of AE buffer was added, and it was incubated at room temperature for 1 min and centrifuged at 6,000 xg for 1 min. The genetic material gathered was stored at −80ºC until its subsequent use.HPV detection was performed using the INNO-LiPA HPV Genotyping Extra Kit (Innogenetics), following the commercial specifications. The kit allows specific detection of 25 HPV genotypes , and it is based on reverse hybridisation. The test included a PCR amplification of a 65 bp fragment within the L1 region of the HPV genome using the broad-spectrum SPF10 biotinylated primers. Biotinylated amplicons are subsequently hybridised with HPV type-specific oligonucleotide probes which are immobilised as parallel lines on membrane strips. After hybridisation and stringent washing, streptavidin-conjugated alkaline phosphatase is added and bound to any biotinylated hybrid formed. Incubation with BCIP/NBT chromogen yields a purple precipitate, and the results can be visually interpreted.The quantification of HPV-16 E6 as well as evaluation of the genome’s integration was carried out using real-time PCR according to the specifications described by Koskinen et al. .μL made up of 1X SSoFast Evagreen (BIORAD), 900 nM of each of the initiators and approximately 50 ng of the DNA. The amplification process consisted of 1 cycle at 95°C for 10 min, 44 denaturing cycles at 95°C for 15 sec and the binding and extension phase at 60°C for 1 min, followed by the dissociation curve.Real-time PCR was performed with the 7500 Real-Time PCR System (Applied Biosystem) in the Molecular Biology Laboratory at the Immunology Institute of the Central University of Venezuela. The sequences for the initiators are shown in All the samples that tested positive for HPV genotype 16 were evaluated to check whether gene E2 had broken off, as well as quantifying genes E2 and E6 of HPV genotype 16.μm of each initiator was used, 1X SsoFast Eva Green in a final volume of 20 μL. For the amplification and quantification of the β-globin gene, 1 μM of the initiators PC04 y GH20, 2X SsoFast Evagreen were used in a final volume of 50 μl. The amplification process used was 95°C followed by 40 cycles at 95°C, hybridisation at 46°C for 1 min and extension at 72°C over 30 sec. Once the amplification processes had been performed both for the standard curve and the viral load of the E6 and E2 genes, electrophoresis was carried out in a 2% agarose gel dyed with ethidium bromide (0.2 μL of a solution of 1%) and exposed to UV light on an imaging camera ChemiDoc™ XRS+ (Bio-Rad) as a photographic record, corroborating that the process had been carried out successfully.The calibration curve was performed using DNA from HeLa cells, the initiators GP5+/GP6+ were used, which allow for the detection of the HPV genome, and the initiators PC04 and GH20 were used to amplify the β-globin human gene shown in . The extX2), and the statistical significance was considered for values of p < 0.05.The relationship between the viral infection and the development of tumours was evaluated using the Chi-square test . Regarding the associated risk factors, 73.5% (p = 0.000) of the patients were smokers and 70% consumed alcohol (p = 0.000). The most frequent location of the tumour was the oral cavity (33.8%), followed by the larynx (30%) and the oropharynx (22.5%). In addition, the paranasal sinuses (5%), nasopharynx (3.8%), hypopharynx (2.5%) and nasal cavities (2.5%) are also presented as the locations of the primary tumour (The average age of the patients was 60.34 ± 14.48 years (range: 19–89). The most frequent gender was males with 68.75%  and moderately differentiated (43.75%), in the advanced states of the illness (EIII and EIV).p = 0.004), and HPV-16 was the most frequent form of single infection (17%). It could be noted that the infection for this viral genotype was present in the form of a coinfection or mixed infection in a total of 52.8% of those testing positive for HPV-16  and EIV (55%) . Of thesp = 0.00) (p = 0.016).The average viral load was 10 copies of DNA of HPV for each copy of β-globin (range between 2 and 20 copies of DNA of HPV), and the oropharynx was the anatomical location with the largest number of copies. At this site, with an elevated copies number of E6-HPV-16, the viral genome was found in an episomal or mixed form. The oral cavity and the larynx, on the contrary, presented low copies numbers of E6-HPV-16, and in these, the viral DNA was found in an integrated form ( = 0.00) . The avep > 0.05)  and they were diagnosed in advanced stages. In addition, it could be noted that the physical status of the viral genome in the host cell played an important role; in those cases, when the viral DNA presented in an episomal form, they had a more constant survival than those patients whose viral DNA presented in an integrated form although without a statistically significant relationship ( > 0.05)  and 2.HNSCC presents primarily in males, and the age of appearance is being an important factor , 11. In , 60% according to Yang et al. (2019) [et al [et al [Sixty-six percent of the samples evaluated were positive for the presence of the HPV genome, this value is higher than that reported by the other works which report the viral detection averages of 39%–40% , 14, 60%. 2019)  and even. (2019) , 17. Regl [et al , who rep  and eveThe oral cavity, larynx and oropharynx were, in this order, the anatomical locations most frequently infected with HPV. This is contrary to publications which describe the oropharynx as the area of the head and neck with the greatest infection rate . However-4 up to values as high as 739 copies/reference gene [et al [On the contrary, the result of the average viral load investigation agrees with the previous works which explain that it can vary in these types of tumours and can be anywhere from 3.4 × 10nce gene . If the nce gene . In thise [et al  reportede [et al , 29.et al [et al [et al [p = 0.016).As can be seen, viral load is not constant and presents notable differences between anatomical locations. It has been of great use evaluating the integration of the viral genome to the host cell genome since the previous studies reveal that the state of the viral genome is an important prognostic factor that, together with epigenetic factors, they are related with the cancer progression . In thiset al ; Otlthofl [et al  and Melll [et al , who allThe investigations attribute these differences to the fact that the patients with high viral load possibly induce a better immune response against cancer although this hypothesis has not been confirmed and requires more study to support it. It is important to mention that although there are notable variations in the viral load between different anatomical locations, the average percentage of integration is similar to that described in the investigation carried out in the cervix by Groves and Coleman . It is aAfter following the survival of the patients related to each one of the variables, for a period of 120 months, it could be noted that there was a downward trend in the survival of the male patients, something that was also noted in those patients who engage in risk activities such as consumption of alcohol or tobacco as well as those who presented with a diagnosis in an advanced stage (EIII and EIV). Equally, it was not possible to demonstrate that the presence of HPV infection would have a direct effect on survival. However, it would appear that an integration of the viral genome could be the determining factor since in those cases, where the HPV-16 genome was integrated, the survival was lower than in those cases, where it was in an episomal form. Although there was a statistically significant evidence to support this, this could be attributed to the low number of samples included  and 2.The oral cavity is the most common anatomical location for tumour growth, which could be due to it being in direct contact with aetiological agents. On the other hand, the presence of HPV-16 is established as the most common viral infection, considering the physical status of the viral genome as the determining factor for being able to establish the response to the employed therapies. It is suggested that HPV-positive patients, especially with oropharyngeal cancer, have a better prognosis than those who develop this type of cancer in other anatomical locations in the upper aerodigestive tract; these differences in prognosis could be due to the physical status of the viral genome in the host cell. However, the need to carry out more studies should be highlighted, to be able to evidence the role of HPV infection in carcinogenesis.The authors have no conflicts of interest to declare.


The aim of the study was to describe the course of IgG/IgA immune response in women immunized with bivalent vaccine and in women non-vaccinated with HPV infection, as well as evaluating the cross-protection against non-vaccine HPV types. Serum and cervical mucus samples were collected from infected and vaccinated women for HPV detection/genotyping and for detection of IgG/IgA anti-HPV/VLP (Virus-like Particles) by ELISA. The median absorbance detected in serum samples for anti-HPV-IgG antibodies was higher in vaccinated women when compared to HPV infected women (p <0.01), however, the median absorbance in cervical mucus samples for anti-HPV-IgA was higher in infected women when compared to vaccinated women (p<0.01). Additionally, our analyses also provided additional evidence for cross-protective efficacy of the HPV-16/18 vaccine against HPV-82, -6, -11, -13, -61, -72 and -74. The IgG antibodies were significantly more detected in the serum of vaccinated women, while the IgA was found in greater quantities in cervical samples from those infected by the virus. In addition, there is evidence that the bivalent vaccine provides cross-protection against other non-oncogenic viral subtypes. Human papillomavirus (HPV) is the most common sexually transmitted infection of the female reproductive tract which can easily be spread through direct sexual contact and is associated with a variety of clinical conditions that range from innocuous lesions to cancer . Different HPV types have been identified and classified as high-risk HPV (hrHPV) or low-risk HPV (lrHPV) based on their oncogenic potential . HrHPV types 16 and 18 are associated with 71% of all cervical cancer cases, hrHPV types 31, 33, 35, 45, 52, and 58 with 21%, while lrHPV types, like 6 and 11 cause approximately 90% of anogenital warts .®, GSK) or HPV6/11/16/18 ; more recently, a vaccine covering HPV 6/11/16/18/31/33/45/52/58 has been made available  . All available vaccines are based on non-infectious recombinant type specific L1 capsid proteins assembled into VLPs, acting as immunogens. These present an exterior surface closely mimicking HPV virions, and it is this multiplicity of L1 domains that stimulate a humoral immune response by exposing the system to VLPs, generating high neutralizing antibody titers 100 times higher than those occurring in natural infections .The first generation of vaccines targeted HPV16/18 . They coincide with the induction of high affinity polyclonal anti-L1 IgG antibody response to the HPV types included in the vaccine, and 100% seroconversion in all targeted HPV types . The immunogenicity of vaccines differs from the immune response observed after natural infection. In this case, seroconversion is found in only a proportion of individuals following incident infection. Studies point out that many vaccines can elicit cross-reactive immune responses that may have an important additional clinical impact  on disease caused by non-vaccine strains or variants of the same pathogen. Cross-protection is likely to have beneficial effects on carriage and circulation, thereby providing some form of indirect protection . The effectiveness of cross-protection against some non-vaccine types has been demonstrated for HPV-31/33/45 and, to a lesser extent, HPV 35, and for HPV 58 it was sustained and remained stable after 11 years post-vaccination . Bivalent vaccine appears to confer greater cross-protection than other HPV vaccines and this difference is reflected in antibody levels against these non-vaccine types . It has been shown that the bivalent induces the production of neutralizing immunoglobulin G/A (IgG/IgA) antibodies, which in turn play an important role in protecting against the HPV. Although HPV infects and propagates in the cervical mucosal epithelium and has almost no viremic phase, the ensuing humoral responses are most frequently detected in sera. However, immune responses in cervicovaginal secretions (CVS) are usually not investigated . Knowledge about the cross-protection is important to understand the potential clinical impact of the bivalent HPV vaccination program.®).Here, we provide direct information about the course of IgG/IgA antibody responses in CVS and serum of women with cervical intraepithelial neoplasia (CIN) and infected by HPV  versus healthy women  and 49 HPV DNA-HPV positive/with HPV-induced intraepithelial lesion confirmed by cervical biopsy. Written informed consent was obtained from all participants. All procedures were carried out in compliance with the Declaration of Helsinki. All women were informed about the methods and objectives of the research and signed an informed consent form. The study was approved by the institutional Ethical Committee for Research (1.255.691/2015 CEP-HUOL).®, GSK) and women infected with HPV detected by PCR and not vaccinated. The exclusion criteria for both groups were: (1) any immunodeficiency, chronic illness, or treatment that could interfere with the immune response against HPV virus; (2) known systemic hypersensitivity to any components of the trial vaccine; (3) receipt of any other vaccine within four weeks; (4) menopausal status; (5) pregnant or breastfeeding; (6) use of hormonal contraceptives and (7) administration of immunoglobulins or blood products within three months before blood sampling. Inclusion criteria were women vaccinated with 3 doses of the bivalent vaccine  according to the manufacturer’s instructions. HPV polymerase chain reaction (PCR) amplification was carried out using primers MY09 (5′-CGTCCMAARGGAWACTGATC-3′) and MY11 (5′-GCMCAGGGWCATAAYAATGG-3′) primers as described elsewhere . The PCR product was electrophoresed on a 1.5% agarose gel, stained with 1 μg/mL ethidium bromide, and photo-documented under UV light (approximately 450 bp). The samples that gave a positive PCR result were further analyzed by HPV genotyping. Two types of controls were also included in each reaction series: ‘no-DNA’ (negative control) and ‘HPV-positive DNA’ (positive control). HPV-positive samples were genotyped using a RFLP (Restriction Fragment Length Polymorphism) as described previously . 10μg/mL of each PCR sample was digested with the restriction enzyme HpyCH4V  according to the manufacturer’s instructions. The genotyping was resolved on 8% polyacrylamide gels and was determined by analyzing each band with Labimage 1D software . Comparison of the molecular weights determined the genotypes following carcinogenic potential: HR-HPV, UR-HPV (undetermined-risk-HPV) and LR-HPV. IgA and IgG anti-HPV-VLP detection by ELISA The initial antigen preparation has been described previously . A plate of 96 wells was sensitized with 50μL of antigen (HPV-16/18 vaccine) diluted in carbonate-bicarbonate buffer (Sigma-Aldrich) at a concentration of 10μg/mL and incubated overnight at 4ºC. The plate was then washed with PBS-Tween 0.05% and blocked with 100μL of PBS with 10% of fetal bovine serum (FBS-Gibco) (PBS-FBS). The next step was incubation for 2h at room temperature and washing three times with PBS-Tween 0.05%. Cervical mucus and serum samples were diluted 1:100, 1:1,000, 1:10,000, 1:100,000, 1:1,000,000 and 1:10, 1:100, 1:1,000, 1:10,000, and 1:100,000, respectively in PBS-FBS, and 50μL of this dilution was added to each well. Following this, the samples were incubated for 2h at 37°C, and was washed with 0.05% PBS-Tween. Secondary antibody  was diluted 1:10,000 in FBS 10% and 50μL was added to the wells and incubated for 1h at 37ºC. After this, the plate was washed with PBS-Tween 0.05%, and then 50μL of substrate TMB  was added to the wells before incubating for 30 min at room temperature. The reaction was stopped with 50μL of 1N sulfuric acid, and the absorbance  of each well was read using an ELISA reader at 450nm with a reference filter of 630nm. The cutoff values were established from the ROC curve , and for each antibody/sample the readings were as follows: IgG/serum, 0.616; IgG/mucus, 0.611; IgA/serum, 0.173; and IgA/mucus, 0.294.Statistical AnalysisStatistical analysis was performed with Stata 11 . The different HPV genotype characteristics (high and low risk) in the women’s groups were described in relative and absolute frequency. To analyze the effect of vaccination on immunoglobulin concentration at different dilutions, linear regression of random effects was used. Serum and mucus immunoglobulin concentration was considered the dependent variable, dilution as a fixed effect the independent variable, and intra-individual variability as the random component of the model (p <0.05). In serum and mucus samples, mean absorbance values of IgG and IgA between HPV genotypes 16/18, 82, 6/11, and 13/61/72/74 were compared by analysis of variance followed by the Tukey’s post-test (p <0.05).We enrolled a total of 84 patients divided into two groups, vaccinated (negative HPV-DNA) and unvaccinated (positive HPV-DNA) women. HPV infection was observed in 58.3% (49/84) of patients. The ELISA assay for HPV-VLP (HPV-VLP-ELISA) was performed. It was observed that 100% of serum and mucus samples from both groups were positive for IgG/IgA antibodies when antigen was present. Positivity, however, decreases according to dilutions .The median absorbance detected in serum samples for anti-HPV-IgG antibodies was significantly higher in vaccinated women compared to unvaccinated women at 1:100, 1:1,000, 1:10,000, 1:100,000 and 1:1,000,000 dilutions, presenting statistical significance (P <0.01). In relation to anti-HPV-IgA antibodies in serum, there was no significant correlation between the groups (P = 0.38) .In the detection of anti-HPV-IgA, the median absorbance in cervical mucus samples was statistically higher in unvaccinated women compared to vaccinated women at 1:100, 1:1,000, 1:10,000, 1:100,000 and 1:1,000,000 dilutions (P<0.01), while the absorbance detected for anti-HPV-IgG under the same dilution conditions was not statistically significant (P = 0.96) .Of the 84 women studied for HPV DNA, 58.3% (49/84), the unvaccinated group, were positive for an HPV genotype and 41.6% (35/84). The vaccinated group were negative for HPV DNA. All HPV-positive samples were typed by RFLP. A total of 49 HPV DNA were detected, with 9 different genotypes. The overall frequency of HR and LR HPV types is shown in Naturally acquired anti-HPV IgG / IgA specific antibodies against a single HPV type were analyzed, although without statistical significance. A total of 41% (13/32) showed high concentrations of serum IgG antibodies for HPV16, 66% (2/3) for HPV18, 83% (5/6) for HPV6 and the other types such as HPV82/6/11/61/72/74 had positivity above 50% . The perIn this study, cross-protection for HPV 6, 11, 13, 61, 72 and 74 (non-oncogenic virus) by the bivalent vaccine was present in the group of infected women. The IgG antibody was detected in greater quantities among vaccinated women, while IgA was significantly higher among those infected with non-immunized HPV. The high levels of IgG prove the efficacy of the bivalent vaccine by protecting against infection.The presence of IgG antibodies is strongly associated with protection against cervical HPV infections . As was seen in previous studies , the immune response detected after immunization indicated that the antibodies exhibited strong neutralizing activity . Regarding the infected group, although  demonstrated that cervical HPV-specific antibodies from women with active HPV16 infections have some virus-neutralizing capabilities, the local HPV-specific IgA and IgG presence did not correlate with viral clearance and was not effective in inducing the regression of established lesions.Concerning the IgA detection, this could be due to a possible transudation of the systemic circulation to the cervical mucosa, contributing to a protective environment in the cervix . However, low antibody levels in cervical mucus may be due to the shorter half-life of IgA when compared with IgG at this site, as well as the longer time required for antibody formation , suggesting that antibody development following a natural infection can be a slow process and does not necessarily occur in all women . Probably, the IgA acts with mucosal immunity neutralizing intracellular viruses  and preventing viral particles from infecting the cervical basal cell layer in the transformation zone  and, as reported by Monroy et al., after natural infection, anti-HPV-IgA antibodies are detectable in the cervix, especially in persistent HPV infections . Natural immunity to HPV is not as protective as vaccination . Although the minimum antibody titer required for protection is not defined, recent studies  have shown that anti-HPV antibodies acquired by natural infection provide protection if naturally acquired titers are high. Vaccination is not effective in patients with prevalent HPV infection, but the possible therapeutic benefit of the licensed HPV vaccines in reducing recurrent lesions in previously infected persons has been studied . The antibodies evoked by HPV vaccination during the follow-up post treatment for HPV-linked disease is recommended to reduce the risk of reactivation/re-infection or a new HPV infection, without adverse events or adverse reactions .The effector mechanism of the bivalent vaccine was mainly associated with a humoral response, as well as providing greater cross-protection over other HPV types . The cross-protection of the HPV-16/18 vaccine was observed against persistent HPV-33, -31, -45, -51 and -52 associated infection in two studies, as well as highlighting a possible prophylactic effect against new infections and lesions . Although our study addresses other types of HPV , the presence of antibodies can be explained by the fact that the effectiveness of cross-protection may differ across populations.As far as we know, our study is the first to point out that the bivalent vaccine presents cross-protection against non-oncogenic viruses, in addition to comparing the immune response between unvaccinated and vaccinated women. However, some limitations should be addressed. First, our study presented a relatively small number of subjects, and the uneven distribution of CIN among these subjects may obscure the statistical significance. Secondly, there is a lack of a follow-up for some years to verify the maintenance of the immune response.The IgG antibodies were significantly more detected in the serum of vaccinated women, while the IgA was found in greater quantities in cervical samples from those infected by the virus. In addition, there is evidence that the bivalent vaccine provides cross-protection against other non-oncogenic viral subtypes. Overall, we anticipate that there may exist substantial protection against cervical cancer and low-grade intraepithelial lesion over and above that achieved by HPV-16/18. Although further studies on the subject are needed, an expanded program on immunization is necessary to increase antibody titers and to reduce the risk of HPV infection and thus it can be argued that these immune mechanisms may provide protection against HPV.


Anal canal cancer is one of the human papilloma virus (HPV)-associated diseases with increasing incidence. High-risk sexual behaviour and the resurgence of human immunodeficiency virus (HIV) infection, associated with low HPV vaccine coverage, are risk factors for the increased incidence of this cancer. In this paper, the authors point out pertinent questions regarding the greater exposure of the population to some risk factors and discuss the latest epidemiological data of these factors, particularly those of concern to emerging countries like Brazil. The authors also discuss policies adopted that have not been successful to combat the HIV and HPV rise and that have direct consequences on the incidence of anal canal cancer. Squamous cell carcinoma of the anal canal, often broadly termed anal canal cancer, is a rare disease but its frequency is increasing, especially in high-risk groups . WorldwiKnown risk factors, except for age and immunosuppressive drug use, are behavioural . SmokingAmong the more than 240 types described, HPV 16 and 18 genotypes are considered of high risk because, by integrating their DNA into the host cell genome, they encode oncoproteins with stimulating properties, enabling the progression of preneoplastic lesions to invasive carcinoma . The losCurrently, nearly 90% of anal cancer tumour samples are HPV positive, with different rates according to geographic location. HPV 16 infection is the most common, present in 86% of cases. In some cases, coinfection with multiple HPV types has been found .n = 2,669) of the collected samples have been tested for the presence of the virus and genotyped for subtype definition. Overall, the estimated prevalence of HPV was 54.6%, and high-risk HPV was present in 38.4% of participants. The study also showed that risky sexual behaviour was observed in 83.4% of respondents, with the average of sexual partners in the last year being 2.2 (95% CI 1.7–2.7), and the average of sexual partners in the last 5 years being 7.5 (95% CI 4.5–10.6) [An ongoing Brazilian study to determine the prevalence of HPV in sexually active women and men aged 16 to 25 years and to investigate regional differences in prevalence and virus types showed that for 5.812 women and 1.774 men analysed with median age of 20.6 years (95% CI 20.5–20.7), 17.7% of the study participants reported the presence of a previous STD or tested positive for the rapid HIV or syphilis tests. Regarding HPV, 35.2% (.5–10.6) .Because it is a strongly HPV-related disease, most (approximately 85%) of anal canal cancers are of squamous cell origin . In the et al [p = 0.014) and better tolerance profile [The modern treatment of this disease has been perfected over the years by many studies, such as that of Nigro et al  and is bet al . In the  profile , 16. One profile , 18, whi profile .Next, we will discuss some pertinent and worrying aspects on the increasing incidence of anal cancer in Brazil which may apply to other emerging countries.Between 1989 and 2013, the percentage of adult smokers in Brazil dropped significantly due to the numerous actions taken by the National Tobacco Control Policy, granted in 1988 . ConsideUnfortunately, HIV/acquired immunodeficiency syndrome (AIDS) policies do not seem to achieve the same success. According to the HIV / AIDS epidemiological report of 2018, over the past 10 years, the number of people infected with HIV has increased exponentially , with anHowever, the report also shows a decrease in the number of deaths associated with HIV , which cAnother policy that has faced difficulties is immunisation against HPV. Two HPV vaccines  are licensed by the Food and Drug Administration (FDA) and available in Brazil. The quadrivalent HPV vaccine has been shown to prevent HPV 16 and HPV 18-associated vaginal, vulvar and anal precancer lesions and HPV 6 and HPV 11-associated anogenital warts , 18. TheLike cervical cancer, anal cancer is preceded by high-grade intraepithelial neoplasia . StudiesThe American Advisory Committee on Immunisation Practices then recommends that girls and boys be routinely vaccinated at the age of 11 or 12 and, for those who were not vaccinated when they were younger, young women up to 26-year old and young men up to 21 years of age should be vaccinated. The committee also recommends that men who have sex with men be vaccinated by age 26 .In Brazil, the Unified Health System included, in March 2014, the quadrivalent HPV vaccine in the National Immunisation Programme (NIP), currently available for girls aged 9 to 14 and boys aged 11 to 14, in two doses at 6 months intervals. For people living with HIV, transplanted and cancer patients undergoing chemotherapy and radiotherapy and who are 9 to 26 years, the quadrivalent HPV vaccine is available in three doses [Low vaccination coverage, however, is not unique in emerging countries. In the United States, underuse of vaccines has been a permanent concern. A 2012 CDC report estimated that adolescent immunisation coverage was 54% for 1 dose and only 33% for the 3 dose recommendation.  Reasons Brazilian studies to ascertain the reasons for the low vaccination coverage showed that parents’ acceptance of the HPV vaccine for daughters and children under 18 was high  . ParentsHowever, it is known that 92.1% of vaccine-related adverse events are classified as non-serious. Among non-serious adverse events, the most commonly reported generalised symptoms were syncope, dizziness, nausea, headache, fever and hives; the most commonly reported local symptoms were injection site pain, redness and oedema. Among the 600,588 doses of quadrivalent vaccine given to women aged 9 to 26 years, there was no significant increased risk of any of the pre-specified adverse events following vaccination, including Guillain-Barré syndrome, seizures, syncope, appendicitis, stroke or venous thromboembolism, anaphylaxis and other allergic reactions .Increased use of HPV vaccines by young adults may be the most cost-effective mechanism to prevent anal canal cancer morbidity and mortality in the coming decades. The major public health challenge is to develop accurate forms of communication about HPV for people to understand the importance of prevention and problems associated with the virus.As we witness the exponential increase in the incidence of HIV infection, a new study shows that most young people exhibit high-risk sexual behaviour and a high prevalence of high-risk HPV . People Another previous Brazilian study showed that almost all women with HIV are infected with HPV, with high frequency of multiple HPV genotypes. Of the 208 HIV-infected women, 98% were found to be HPV DNA positive. Overall, reverse hybridisation genotyping revealed unique genotypes in 43 (21.1%) and multiple genotypes were detected in 161 (78.9%) of 204 HPV DNA positive patients, with an average of 3.1 genotypes per patient (ranging from 1 to 10 genotypes). HPV 6 was the most prevalent genotype and was observed in 39.2% of patients, followed by genotypes 51 (31.9%), 11 (26.0%), 18 (24.0%) and 16 (22.5%). Of the detected genotypes, 40.9% were low risk, 21.2% of the study population contained only high-risk genotypes, while 64.3% had high- and low-risk genotypes. The study also evaluated cervical oncotic cytology and found that at least one high-risk genotype was present in 89% of class I samples, 84% of class II samples and 82% of class III samples. Of the 28 patients with squamous intraepithelial lesions, 18% had exclusively low-risk genotypes, 46% had low- and high-risk genotypes and 36% had exclusively high-risk genotypes .It seems logical, then, that the HIV-infected population should be prioritised in HPV immunisation campaigns. HIV, however, often reduces vaccine responsiveness and effectiveness. Even individuals who have been treated and who have suppressed viral loads for more than 5 years have specific defects in memory helper T-cell function, leading to reduced B-cell responses . A randop < 0.05). There was a higher incidence of genital warts and abnormal cervical oncotic cytology in those infected with HIV [A Canadian cohort evaluated 432 HIV-positive patients aged 9–65 years  who received three doses of quadrivalent vaccine. Of the women who met the inclusion criteria for the 2-year follow-up efficacy analyses (95.3% of those receiving 3 doses), the intention-to-treat population was 279 and the incidence rates were 2.3/100/year for persistent infection of genotypes 6, 11, 16 and 18 (most were due to HPV 18) and 2.3/100/year for genital warts. Persistent cases of HPV showed lower CD4 counts (median 333 cells/μL) . A thirdThere are several studies examining the immunogenicity of HPV vaccines in people with HIV, and in general, there is some reduction in HPV antibody levels compared to HIV negative individuals . HoweverDespite the decline in smoking, emerging countries like Brazil have witnessed a relentless upsurge in HIV infection, undoubtedly associated with a change in the now-at-risk population’s pattern of sexual behaviour. These important behavioural risk factors should cause the epidemiology of HPV infection, either by higher incidence or by co-infection with multiple subtypes and HIV, to change in the coming years causing an increased incidence of HPV-related tumours, among them anal canal carcinoma. There is still no solid evidence to screen anal cancer and HPV prophylaxis measures have faced serious difficulties with misinformation by the population, low coverage of immunisation programmes and likely lower efficacy of vaccines in immunosuppressed people. It is imperative and urgent to disseminate knowledge clearly and objectively about the risks related to high-risk sexual practices and to effectively guide the population’s protection measures, encouraging technical debate and ensuring access to health, insisting on improving immunisation coverage and HIV control programmes. Otherwise, in addition to witnessing an increase in other HPV-associated diseases, we will be outraged to see not only the rising incidence of anal canal cancer but also the cost it entails soar, whether related to its complex treatment or to quality of life and, of course, to life itself.Since the increase in the use of HPV vaccines by young adults may be the most economical mechanism to prevent morbidity and mortality from cancer of the anal canal in the coming decades, a strategy that could increase vaccination coverage is the mandatory immunisation of all adolescents in schools. In addition to this, the expansion of public funds aimed at combating HIV / AIDS aimed at its prevention, that is, expanding the supply of condoms and carrying out wide-ranging advertisements emphasising the seriousness, costs and consequences of AIDS for the population and that an increase in this disease is closely linked to high-risk sexual behaviours, so it also suggests greater care in relation to these behaviours through educational materials in schools as well. High-risk individuals, such as MSM and HIV infected patients, should be routinely screened for anal cancer through clinical examination; the detection of early stage tumour improves the cure rates. The successful policy of combating tobacco must continue and count on the renewal of funds and the policies themselves. Finally, with the projected increase in cases of anal canal cancer, oncology services must be expanded and prepared with specialised professionals and infrastructure capable of meeting this demand.The authors declare that they have no conflicts of interest.This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors.The opinions expressed in the report presented are those of the authors and do not necessarily represent the official position of the institution to which they belong.Both authors contributed equally for the manuscript.None to declare.