Enhanced the immunity activity of HPV16 DNA vaccine by the combination CpG oligonucleotides adjuvant cloned into plasmid backbone and IL-28B gene adjuvant

doi:10.21203/rs.3.rs-4338069/v1

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Enhanced the immunity activity of HPV16 DNA vaccine by the combination CpG oligonucleotides adjuvant cloned into plasmid backbone and IL-28B gene adjuvant

https://doi.org/10.21203/rs.3.rs-4338069/v1

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Therapeutic human papillomavirus (HPV) DNA vaccine is an attractive option to control existed HPV infection and related lesions. The two early viral oncoproteins, E6 and E7, are continuously expressed in most HPV-related pre- and cancerous cells, and are ideal targets for therapeutic vaccines. We have previously developed HPV 16 DNA vaccines encoding mE7/HSP70, which generated significant antitumor effects in mice. In this study, we utilized multiple strategies including the insertion of CpG oligonucleotides (ODNs) into the backbone of vaccine vector, selection of cytokine gene adjuvants, combination of mE6/HSP70 and mE7/HSP70, vaccination with electroporation, to further enhance the potency of HPV16 DNA vaccine, We found that combination of built-in CpG adjuvant and IL-28B gene adjuvant could induce higher CD8⁺T cell response in mice. Moreover the plasmids mE6/HSP70 combined with mE7/HSP70 could synergistically enhance the specific CD8⁺T cell response. Furthermore, vaccination with CpG-modified mE7/HSP70 and mE6/HSP70, plus IL-28B gene adjuvant, generated significantly preventive and therapeutic antitumor effect against HPV E6- and E7-expressing tumors in C57BL/6 mice. Our results suggested that it may be promising to effectively control HPV infection and associated diseases by combining these multiple strategies in HPV DNA vaccine.

CpG oligonucleotides

IL-28B gene adjuvant

HSP70

HPV DNA vaccine

Carcinogenic human papillomaviruses (HPVs) infections are associated with cervical, anogenital and oropharyngeal malignancies¹. HPV16 is by far the predominant type causing invasive cervical cancer worldwide. Recently the available prophylactic HPV vaccines (e.g. Gardasil and Cervarix) have been shown to be effective in preventing HPV infections, but without any therapeutic efficacy against pre-existing HPV infections and associated lesions². Current treatment for HPV-associated high-grade precancerous lesions and early stage cancers is limited to surgical excision, while with many complications. For advanced cancers, the effect of surgery, radiotherapy and chemotherapy is poor³. Therefore, developing effective HPV therapeutic vaccine may represent an opportunity to impact the progression of cervical associated diseases.

HPV E6 and E7 are viral oncoproteins, constitutively expressed in HPV-infected, pre-malignant and malignant cells, and are ideal tumor specific antigens⁴. Recently several HPV therapeutic DNA vaccines targeting E6 and E7 delivered by electroporation could induce complete regression of high-grade cervical lesions and viral clearance in CIN2/3 patients^5,6,7. These trials indicated that the magnitude of systemic polyfunctional CD8⁺T cell response is the main contributing factor for histopathological regression and cleared HPV infection^5,7. Thus a therapeutic vaccine capable of selectively inducing robust E6/E7-specific T cell immunity is highly desirable. However, in case of HPV-associated malignant tumors, vaccination with one HPV DNA vaccine with IL-2 induced specific CD8⁺T cell response, but the clinical regression of tumors was not shown⁸. It may be due to patients’ local immune microenvironment and the immunosuppressed state which is not conducive to the establishment of powerful tumor immunity. Therefore, inducing the stronger CD8⁺T cellular immune response of DNA vaccines is one of the hotspots of HPV therapeutic vaccines.

Our previous studies have found that HPV fusion DNA vaccine encoding modified E7 and human heat shock protein 70 (mE7/HSP70) inducing E7-specific CD8⁺T cell response⁹. Here, we empolyed multiple strategies to further strengthen CD8⁺T cell response induced by HPV16 DNA vaccine. We constructed mE7/HSP70 and mE6/HSP70 plasmids respectively, inserted CpG ODN into the backbone of the plasmid vector, selected cytokine genes as adjuvants, and immunized mice by electroporation. Then the antigen-specific cytotoxic T cell responses and anti-tumor effect in vivo were evaluated to explore whether the combined multiple strategies could strengthen the effect of HPV DNA vaccine.

Construction of CpG-modified DNA plasmids

The optimized CpG ODN sequence (5’TCGTCGTTTTGTCGTTTTGTCGTT3’) was synthesized by Sangon (Shanghai, China). To study the built-in adjuvant activity of CpG motif in the backbone of DNA plasmid pVAX1EI, 20 copies of tandem sequences were inserted into the backbone of pVAX1EI via PamC I restriction site resulting in CpG-modified immunization vector pmVAX1EI.

The mE7/HSP70 antigen gene was designed previously by our group. The mE7 was derived from wild type E7 modified by gene shuffling, site directed mutagenesis and codon optimization^9,10. Thus the mE7 was characterized by the increasement of T cell epitopes and elimination of transformation activity. The mE7/HSP70 fusion gene was amplified by PCR using pVR1012-mE7 /HSP70 (conserved by our group) as template with the primers: 5’-CGGGATCCGCCACCATGGATCTGCTCAT-3’, 5’-CCGCTCGACTAATCTACCTCCTCAATGGTGGG-3’, then was cloned into BamH I/Xho I-digested pmVAX1EI and pVAX1EI to generate pmVAX1EI-mE7/HSP70 and pVAX1EI-mE7/HSP70.

The wild type E6 was dissected into two segments at amino acid position 70, two HLA-I epitopes E6aa29-38 and E6aa49-67 were inserted between these two segments. The artificial gene was then codon optimized for human system and synthesized by Sangon (Shanghai, China), named as mE6. The mE6 was also characterized by the increasement of T cell epitopes and elimination of transformation activity. The mE6/HSP70 fusion gene was generated by replacing mE7 of the pVR1012-mE7/HSP70 with mE6, and then was cloned into BamH I/Xho I-digested pmVAX1 and pVAX1EI to generate pmVAX1EI-mE6/HSP70 and pVAX1EI-mE6/HSP70.

Human IL-28B and IL-15 gene were codon optimized and synthesized by Sangon (Shanghai, China), then were ligated into BamH I/Xba I-digested pmVAX1EI and pVAX1EI to obtain pmVAX1EI-IL-28B, pmVAX1EI-IL-15 and pVAX1EI-IL-28B, pVAX1EI-IL-15.

All DNA constructs were validated by restriction enzyme digestion and DNA sequencing.

Animals

Six- to eight-week-old female C57BL/6 mice were purchased from SPF (Beijing) Biotechnology Co.,Ltd.. All animals were maintained in the animal facility of the Institute of Laboratory Animal Sciences(HBNU20231222116). All animal procedures were performed according to approved protocols and in accordance with recommendations for the proper use and care of laboratory animals. The procedures with animals described in this protocol have been reviewed by the test facility’s Institutional Animal Care and Use Committee. At the end of study, all mice were sacrificed by CO₂ euthanasia.

DNA Immunization

DNA plasmids for injection were prepared with Plasmid Maxprep Kits from Vigorous Biotechnology (Beijing China), resuspended in endotoxin-free TE buffer at a final concentration of 1 µg/µl and stored at -20˚C until used for injection.

The quadriceps muscles of C57BL/6 mice were injected plasmids 2 times, 10 days apart followed by electroporated using BTX ECM830 electroporation system (America), each plasmid was used as 15 µg. Animal grouping and corresponding injected plasmids composition were shown in Table 1.

Table 1

vaccination groups and plasmids composition
Groups	plasmids
1	saline
	pmVAX1EI-mE7/HSP70
	pmVAX1EI-mE7/HSP70 + pmVAX1EI-IL-28B
	pmVAX1EI-mE7/HSP70 + pmVAX1EI-IL-15
2	saline
	pVAX1EI-mE7/HSP70 + pVAX1EI-IL-28B
	pmVAX1EI-mE7/HSP70 + pmVAX1EI-IL-28B
3
	saline pmVAX1EI-mE7/HSP70 + pmVAX1EI-mE6/HSP70 + pmVAXEI1-IL-28B
	pmVAX1EI-mE7/HSP70 + pmVAX1EI-IL-28B
	pmVAX1EI-mE6/HSP70 + pmVAX1EI-IL-28B
	pmVAX1EI-mE7/HSP70 + pmVAX1EI-mE6/HSP70

ELISPOT

IFN-γ ELISPOT was performed to determine HPV16 E6 and E7 specific CD8⁺T cell response. The ELISPOTs were carried out according to the manufacturer’s protocols (BD biosciences) using 96-well plates. Briefly, each well was coated with 5 µg/ml rat anti-mouse IFN-γ antibody in 100 µl of PBS overnight at 4˚C. After washing, the wells were blocked with RPMI-1640 culture medium containing 10% fetal bovine serum for 2 hours at room temperature. Splenocytes from each vaccinated mouse were added to the wells along with 50 IU/ml IL-2, 1 µg/ml E7-specific MHC class I CTL epitope (H-2D^b, amino acids 49–57, RAHYNIVTF) and/or 1 µg/ml E6-specific MHC class I CTL epitope (H-2D^b, amino acids 50–57, YDFAFRDL) for 40 hours at 37˚C. After washing, the wells were incubated with 2.5 µg/ml biotinylated IFN-γ antibody in 100 µl PBS for 2 hours at room temperature. For detection, HRP-streptavidin and AEC substrate were used. Spots were counted using ImmunoSpot Analyzer.

Real time PCR

Real time PCR was used to detect the expression of granzyme B in spleen cells from immunized mice. Briefly, total RNA were extracted from splenocytes restimulated with E7-(H-2D^b, amino acids 49–57, RAHYNIVTF) and E6-specific MHC class I CTL epitope (H-2D^b, amino acids 50–57, YDFAFRDL) for 20 hours at 37˚C. 1 µg RNA was reverse transcribed into cDNA with SuperRT cDNA Synthesis Kit. The cDNA was quantified using the UltraSYBR Mixture (Low ROX) (Beijing Comwin Biotech Co.,Ltd) with granzyme B sense primer 5’-CCTCCAGGACAAAGGCAG-3’ and antisense primer 5’-CAGTCAGCACAAAGTCCTCTC-3’. The samples were quantified by the standard under the following conditions: 15 min at 95℃and 40 cycles of 15 s at 95℃ and 1 min at 60 s. The data were normalized relative to GAPDH by using the following formula: relative mRNA expression = 2-△△^Ct, Ct is the cycle threshold.

In vivo tumor protection experiment

C57BL/6 mice (six per group) were vaccinated via intramuscular injection by electroporation with 15 µg /plasmid two times, ten days apart as described above. One week later after the last vaccination, each mouse was challenged subcutaneously with 7.5×10⁴ TC-1 cells in the right flank and then was monitored twice a week for tumor growth.

In vivo tumor treatment experiment

Each mouse was challenged subcutaneously with 7.5×10⁴ TC-1 cells in the right flank. After 3 days, the mouse was immunized with the plasmids as described above. Then tumor growth was monitored twice a week.

Statistical analysis

Statistical analyses were performed with SPSS 13.0 software. The difference between immunization groups was assessed by ANOVA. Comparisons between samples with a P value less than 0.05 were considered statistically significant.

3.1 Generation of plasmid vector containing optimized CpG ODNs

CpG ODN is an agonist of TLR9 and enhance adaptive immune response by stimulating human B cells and plasmacytoid dendritic cells¹¹. In numerous studies, CpG elements were recognized to improve vaccine immune activity for the prevention/treatment of cancer, allergy, and infectious disease¹². The number and type of unmethylated CpG motifs within the plasmid backbone are shown to be critical to enhance immune response¹³. we have selected CpG motifs that showed good stimulating activity on immune cells¹² and 20 copies of tandem sequences were inserted into the backbone of pVAX1EI to obtain CpG-modified vector pmVAX1EI(Fig. 1).

3.2 IL-28B gene adjuvant could significantly enhance CD8⁺T cell response induced by mE7/HSP70

The mE7/HSP70 DNA vaccine could induce cellular immune response have been identified previously by our group⁹. In this study, IL-28B and IL-15 plasmids were first used as HPV DNA vaccine adjuvants to identify whether they could increase the cellular immune response of mE7/HSP70 DNA vaccine. Female C57BL/6 mice were selected and randomly divided into four groups (n = 6 per group). The plasmid mE7/HSP70 combined with either IL-28B plasmid or IL-15 plasmid, or saline were injected into the quadriceps muscle of mice 2 times, 10 days apart via electroporation as described in materials and methods. 7 days after the final vaccination, the splenocytes were isolated and stimulated with 10 µg/ml E7_{49 − 57} peptide for 40 hours. Results of the IFN-γ ELISpot assay showed that inclusion of IL-28B or IL-15 was able to significantly increase E7-specific IFN-γ release (740 SFU or 343 SFU per 5×10⁵ splenocytes) (Fig. 2A), and inclusion of IL-28B adjuvant could induce significantly higher IFN-γ release than that inclusion of IL-15 adjuvant.

Upon confirming that IL-28B and IL-15 could increase the release of IFN-γ, we next endeavored to determine whether they could affect the expression of granzyme B. Mice spleen cells were stimulated with 10 µg/ mL E7_{49 − 57} peptide for 20 h, the mRNA levels of specific granzyme B were analyzed by real-time PCR. Similarly, the inclusion of IL-28B adjuvant resulted in significantly higher mRNA level of granzyme B than inclusion of IL-15 adjuvant (Fig. 2C). Thus IL-28B plasmid adjuvant may be thought as more effective in mE7/HSP70 DNA vaccine than IL-15 plasmid.

3.3 CpG ODN embedded in the vaccine vector could enhance CD8 ⁺ T cell response induced by mE7/HSP70 vaccine

To investigate the built-in adjuvant activity of CpG ODN in the backbone of DNA plasmid. The CpG ODN modified or unmodified plasmids encoding mE7/HSP70 (pmVAX1EI-mE7/HSP70, pVAX1EI-mE7/HSP70) and IL-28B (pmVAX1EI-IL-28B, pVAX1EI-IL-28B) were constructed. Female C57BL/6 mice were selected and randomly divided into three groups (n = 6 per group). The plasmids were injected into the quadriceps muscle of mice 2 times, 10 days apart via electroporation as described in materials and methods. 7 days after the final vaccination, the splenocytes were isolated and stimulated with 10 µg/ml E7_{49 − 57} peptide for 40 hours. Results of the IFN-γ ELISpot assay showed that the CpG-modified plasmids induced significantly higher E7-specific IFN-γ release than unmodified group (Fig. 3A). Results of real-time PCR for the detection of granzyme B also showed that the CpG-modified plasmids induced significantly higher level than unmodified group (Fig. 3C). Thus CpG ODN embedded in the backbone of plasmid as intramolecular adjuvant could significantly enhance specific CD8⁺T cell response. So CpG-modified vector pmVAX1EI was used in subsequent experiments.

3.4 The CpG-modified plasmids mE7/HSP70 combined with mE6/HSP70 could synergistically enhance the specific CD8 ⁺ T cell response

At present, HPV16 therapeutic vaccines that focused on E7 or E6 oncoprotein have been reported, while there were relatively few DNA vaccines that targeted both E6 and E7 oncoproteins. In this study, we identified the combined effect of the mE7/HSP70 and mE6/HSP70 in the presence of IL-28B gene adjuvant. Female C57BL/6 mice were selected and randomly divided into five groups (n = 6 per group). The plasmids were injected into the quadriceps muscle of mice 2 times, 10 days apart via electroporation as described in materials and methods. 7 days after the final vaccination, the splenocytes were isolated and stimulated with 10 µg/ml E7_{49 − 57} and 10 µg/ml E6_{50 − 57} peptides for 40 hours. Results of the IFN-γ ELISpot assay showed that vaccination with mE7/HSP70 combined with mE6/HSP70 induced significantly higher specific IFN-γ-secreting CD8⁺T cells than vaccination with either mE7/HSP70 or mE6/HSP70 alone (780 vs 103 vs 102 SFU per 2.5×10⁵ splenocytes) (Fig. 4A), the number of specific IFN-γ-secreting CD8⁺T cells was 3.8 times the sum of mE6/HSP70 alone and mE7/HSP70 alone. Results of real-time PCR for the detection of granzyme B showed that mE7/HSP70 combined with mE6/HSP70 induced significantly higher mRNA levels than single mE7/HSP70 or single mE6/HSP70 (Fig. 4C). The results also showed that the inclusion of IL-28B adjuvant could significantly enhance CD8⁺T cell response induced by mE7/HSP70 combined with mE6/HSP70.

From the above results, vaccination with the CpG-modified plasmids mE7/HSP70 combined with mE6/HSP70 in the presence of IL-28B gene adjuvant could induce the most effective cellular immune response.

3.5 Vaccination with CpG-modified plasmids mE7/HSP70 combined with mE6/HSP70 in the presence of IL-28B gene adjuvant could effectively prevent TC-1 transplanted tumor growth

C57BL/6 mice were immunized with mE6/HSP70 combined with mE7/HSP70 in the presence or absence of IL-28B gene adjuvant by electroporation. Vaccination with single IL-28B gene adjuvant was used as control. As shown in Fig. 5, on day 60, all mice immunized with mE6/HSP70 combined with mE7/HSP70 in the presence or absence of IL-28B gene adjuvant were tumor free. In contrast, the mice immunized with IL-28B gene alone exhibited tumor beginning 16 days after TC-1 injection, and the average size of tumors was about 387 ± 18 mm² by day 60, then the mice were sacrificed. The results suggested that vaccination with mE6/HSP70 combined with mE7/HSP70 or synergistic with IL-28B gene adjuvant was capable of generating stronger antitumor effects to completely prevent growth of tumors in C57BL/6 mice after prophylactic challenge.

3.6 Vaccination with CpG-modified plasmids mE6/HSP70 combined with mE7/HSP70 in the presence of IL-28B gene adjuvant could effectively suppress progression of established TC-1 transplanted tumors

The therapeutic effects of mE6/HSP70 combined with mE7/HSP70 in the presence of IL-28B gene adjuvant were shown in Fig. 6. On day 14 after TC-1 challenge, 50% mice receiving mE6/HSP70 combined with mE7/HSP70 in the presence or absence of IL-28B gene adjuvant were tumor free rate. On day 32, in the presence of IL-28B gene adjuvant, the tumor free rate of mice inoculated with mE6/HSP70 and mE7/HSP70 was up to 100%, while in the absence of IL-28B gene adjuvant was 67%. 100% mice receiving IL-28B plasmid alone formed tumors and the average size of tumors was about 433 ± 27 mm² by day 60. The above results suggested that mE6/HSP70 combined mE7/HSP70 in the presence of IL-28B gene adjuvant showed the strongest immune clearance activity against the transplanted tumor.

In the past, DNA vaccines have proved effective in animal models, while DNA vaccines did not afford sufficient immunogenicity in human clinical studies¹⁴. Therefore, it is essential to modulate and augment the efficacy of DNA vaccines. Now a number of strategies have been investigated to increase the immunogenicity of DNA vaccines including adjuvants (such as cytokines^15,16, CpG motif or CpG oligonucleotide^17,18,19,20, liposomes^21,22, lipopolysaccharide,^23,24etc), alterations in the codon bias^25,26 electroporation^27,28,29, heterologous prime/boost immunization^27,30,31and so on.

In the present study, we have explored the efficacy of combined multiple strategies on HPV DNA vaccine. We found that the insertion of CpG motif into the backbone of plasmid vector, mE7/HSP70 plasmid mixed with mE6/HSP70 plasmid, combined IL-28B gene adjuvant, vaccinated with electroporation could significantly enhance specific CD8⁺T cell response and to eradicate the established graft tumors in C57BL/6 mice. We did not detect specific CD4⁺T cell response and humoral immune response, just because our group and other studies previously showed that HPV DNA vaccine fused with HSP70 could significantly enhance specific CD8⁺T cell response, not significantly improve specific CD4⁺T cell response and antibody response^9,32,33,34, this may due to HSP70 mainly delivers antigenic peptides to MHC-I molecules and plays cross-delivery activity^35,36. Previous clinical researches also showed that CD8⁺T cells is necessary for therapeutic vaccine antitumor effect, while specific CD4⁺T cell response and antibody response are not the most critical factors for the antitumor effect^5,37,38,39. So we only detected specific CD8⁺T cell response.

We also found that simultaneous vaccination of C57BL/6 mice with both mE6/HSP70 and mE7/HSP70 plasmids generate enhanced synergistic effect on the response of CD8⁺T cells rather than a simple cumulative effect of two vaccines. The reason may be that the mixed application of mE6/HSP70 and mE7/HSP70 plasmids in mice stimulated the activation and secretion of cytokines of DC cells, then further stimulated the activation of E6 and E7-specific CD8⁺T and CD4⁺T cells secreting cytokines, which constituted local immunity microenvironment. These cytokines again promoted the reactivation of E6 and E7 specific CD8⁺T cells, thus enlarging the immune effect.

In this study, HPV16 E6 wild protein genes were modified to insert two HLA-A2 restriction epitopes between amino acids 70–71 in the zinc finger binding region. The E7 wild protein gene was also modified, the strategies included the mutation of Rb binding site, gene cleavage and rearrangement, the addition of one HLA-A2 restriction epitope to N and C terminus respectively. Thus, the modified E6 and E7 genes contained more human CD8⁺T cell epitopes, which laid a foundation for inducing higher CD8⁺T cell response and future clinical application.

To further enhance the immune activity of this HPV DNA vaccine, cytokine gene adjuvant was also employed. Several previous studies have demonstrated the adjuvant activity of cytokines such as IL-12, IL-15, IL-28B,IFN-γ and GM-CSF. However, the adjuvant activity of the same cytokine is often unpredictable used in different vaccines or in different ways. IL-15 gene adjuvant could significantly boost specific CD8⁺T cell response of HIV DNA vaccine and Trypanosoma cruzi DNA vaccine^40,41,42. Although IL-28B gene adjuvant could significantly augment the function of CD8⁺T cell and protective immune response of HIV DNA vaccine^43,44,45, HSV DNA vaccine⁴⁶ and Flu vaccine⁴⁷, it had no effect on the immune activity and protective immunity of tuberculosis fusion protein vaccine⁴⁸. GM-CSF gene adjuvant significantly enhanced the immune activity of SARS-CoV-2 DNA vaccine⁴⁹, anti-Lewis lung carcinoma DNA vaccine⁵⁰ and HPV16 E6/E7 DNA vaccine⁵¹, but significantly inhibited the antibody level and protective effect induced by Japanese encephalitis virus DNA vaccine⁵². For the first time, we used the IL-28B plasmid as an adjuvant for HPV DNA vaccine compared with IL-15 plasmid because IL-15 has been proved to enhance CD8⁺T cell response by our research group. Analysis of this comparison showed that IL-28B may be more effective than IL-15 in enhancing CD8⁺T cell response induced by HPV DNA vaccine.

The previous studies also have demonstrated that the immunogenicity of plasmid DNA may be modulated and augmented by the presence of CpG motif or ODN in the backbone. In humans, four classes of synthetic CpG ODN have been identified that differ in structure and immune activity, they are A-, B-, C- and P-type⁵³. One study suggested that insertion of C-type motifs in anthrax DNA vaccine enhance higher cell-mediated and protective immune response than A- and B-type motifs¹³. Another study demonstrated mice vaccinated with C-type CpG-modified vaccines resulted in enhanced Th1-biasd T-cell response⁵⁴. Morever, several clinical studies indicated that B-type CpG such as CpG7909 or CpG1018 was used as adjuvants in vaccines targeting infectious diseases and cancers, could enhance Th1-skewed immune response of these vaccines^55,56. These studies suggested CpG motif might be appropriate for using as “bulit-in” adjuvants in DNA vaccine. Furthermore, the result by comparing the efficacy of vaccines modified with 5, 20 and 40 copies of CpG motifs showed that with the addition of more copies of CpG motifs produced stronger adjuvant effects¹³. In this study, we synthesized one CpG motifs including B type showed good stimulating activity on human immune cells¹², then inserted 20 copies of CpG motif into the backbone of plasmid. Our data showed that CpG-modified HPV DNA vaccine induced significantly higher CD8 + T cell response than non-modified HPV DNA vaccine.

In general, our findings might provide one efficient strategy to optimize DNA vaccines by inserting CpG ODNs into plasmid vector, mixing mE6/HSP70 and mE7/HSP70 plasmids and combining with IL-28B gene adjuvant, vaccination with electroporation.

Our findings firstly found that IL-28B gene adjuvant can enhance the CD8⁺T cell immune response induced by HPV16 DNA vaccine. Previous studies suggested that IL-28B gene adjuvant also significantly down-regulated the number of Treg cells in the spleen of immunized mice^40,44. Thus further analysis is needed to study the effect of IL-28B on Treg cells in HPV16 DNA vaccine.

In this study, there was no obvious difference in anti-tumor activity of HPV16 DNA vaccine with or without IL-28B in vivo. The possible reason might be the low tumor burden in mice, so the future study should increase the number of TC-1 tumor cell inoculated or prolong the interval between tumor cell inoculation and the first immunization.

Author contributions

Y.Z. wrote the main manuscript text; T.Z. prepared figures1-6. ZR.W. did the data analysis；X.X. revised and edited manuscripts. All authors reviewed the manuscript.

Funding

This work was supported by the basic scientific research business of Hebei North University (JYT2022005).

Competing Interests Statement

The author(s) declare no competing interests.

Data availability

The data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval

This study was approved by the Ethical Committee of Hebei North University (Approval No.HBNU20231222116).

Consent for publication

All authors approved the publication of this manuscript

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No competing interests reported.

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You are reading this latest preprint version

Enhanced the immunity activity of HPV16 DNA vaccine by the combination CpG oligonucleotides adjuvant cloned into plasmid backbone and IL-28B gene adjuvant

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

Construction of CpG-modified DNA plasmids

Animals

DNA Immunization

ELISPOT

Real time PCR

In vivo tumor protection experiment

In vivo tumor treatment experiment

Statistical analysis

Results

3.1 Generation of plasmid vector containing optimized CpG ODNs

3.2 IL-28B gene adjuvant could significantly enhance CD8⁺T cell response induced by mE7/HSP70

Discussion

Deficiencies and Prospects

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

Enhanced the immunity activity of HPV16 DNA vaccine by the combination CpG oligonucleotides adjuvant cloned into plasmid backbone and IL-28B gene adjuvant

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

Construction of CpG-modified DNA plasmids

Animals

DNA Immunization

ELISPOT

Real time PCR

In vivo tumor protection experiment

In vivo tumor treatment experiment

Statistical analysis

Results

3.1 Generation of plasmid vector containing optimized CpG ODNs

3.2 IL-28B gene adjuvant could significantly enhance CD8+T cell response induced by mE7/HSP70

Discussion

Deficiencies and Prospects

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

3.2 IL-28B gene adjuvant could significantly enhance CD8⁺T cell response induced by mE7/HSP70