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Human Papillomavirus E6&E7 Oncoproteins for Cervical Cancer
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This entry is adapted from the peer-reviewed paper 10.3390/vaccines10010053

Cervical cancer is recognized as a serious public health problem since it remains one of the most common cancers with a high mortality rate among women despite existing preventative, screening, and treatment approaches. Since Human Papillomavirus (HPV) was recognized as the causative agent, the preventative HPV vaccines have made great progress over the last few years. However, people already infected with the virus require an effective treatment that would ensure long-term survival and a cure.

cervical cancer cervical intraepithelial neoplasia HPV E6 oncoprotein E7 oncoprotein therapeutic vaccine DNA vaccine DNA therapeutic vaccine

1. Clinical Efficacy

Clinical efficacy was evaluated according to the histopathological regression to CIN ≤1, which is less than one-third of the thickness of the cervical epithelium, on a colposcopy-guided biopsy 15, 20, or 36 weeks after the first injection. All six studies [1][2][3][4][5][6] report tumor size decrease to some extent (Table 1).
Table 1. Comparison of the trials’ results.
Table
Vaccine Clinical Efficacy (Histopathology, Colposcopy, Tumor Size) Viral Clearance Immunogenicity (E6 and E7 Specific CTL Activity) Adverse Events/Toxicity Additional Findings Limitations
GX-188E,
[1]		 HP regression to CIN < 1 in 33/64 patients (52%) at V7, and 35/52 (67%) at V8 (Visit 8, week 36).
Lesions that cover <50% showed better efficacy than the ones >50% after GX-188E injection, 63% vs. 41% (V7; x2 test; P 1⁄4 0.133.		 Of the patients with HP regression, 73% (24/33) exhibited HPV clearance at V7 and 77% (27/35) exhibited clearance at V8.
Of the nonregressors, 16% (5/31) exhibited HPV clearance at V7 (Visit 7, week 20) and 12% (2/17) exhibited clearance at V8.
HPV clearance and HP regression were significantly associated at the V7 [OR 1⁄4 13.867; 95% confidence interval (CI), 4.070—47.249; p < 0.001] and V8 visits (OR 1⁄4 25.313; 95% CI, 4.750–14.883; p < 0.001).		 A higher percentage of the patients (16/25) with HP regression exhibited > 3-fold increase in IFN-γ ELISpot responses compared with the group without HP regression (x2 test (P 1⁄4 0.028), but 7 of 22 nonregressed patients developed more than 3-fold increase in these responses.
Patients with HPV clearance (n 1⁄4 26) presented significant increases in IFN-γ ELISpot responses compared with those without clearance (n 1⁄4 21; fold changes were 28 and 10, respectively; t test; P 1⁄4 0.002).		 GX-188E-well- tolerated.
The AEs relating to the injection site-pain, erythema, induration, and swelling/edema in both groups; pain was the most common AE (occurring in 94.4% and 100.0% in the 1 and 4 mg GX-188E groups, respectively)
One patient was lost to follow up due to pregnancy (1 mg GX-188E group).		 HPV sequence variants: HP regression in 42% (11/26) of the CIN3 patients with HPV variants, whereas 75% (12/16) occurred in those without any of the three variants.
1 vs. 4 mg: 1 mg was found to have better efficacy at V7 and V8. (x2 test; P 1⁄4 0.006 and P 1⁄4 0.027, respectively)
HLA types: HLA- A02 was associated with HP regression at V7 (20 weeks after the first injection; P 1⁄4 0.032; OR 1⁄4 2.381; 95% CI, 1.064–5.327), but not at V8 (36 weeks after the first injection; P 1⁄4 0.404; OR 1⁄4 1.490; 95% CI, 0.582–3.811.		 Lack of control/placebo group.
The selection bias-patients recruited into the study were diagnosed with CIN 3 only.
The attrition bias-20/72 participants withdrew from the study due to various reasons.
The confirmation bias—in the discussion part, authors concluded that immunologic response and HP regression had weak association. However, earlier in the results they mentioned an association between HP regression and systemic immune response.
GX-188E
[2]		 At 8 weeks post last vaccination (VF1), 6/9 patients were free of lesions—2 patients from each cohort (A01 and A03 from 1 mg cohort, A05 and A06 from 2 mg cohort, A07 and A08 from 4mg cohort).
GX-188E vaccination led to the clinically and virologically meaningful complete response rate of 78% (7/9).		 At week 12, 5/9 patients showed viral clearance.
At week 20, 6/9 patients showed viral clearance.
At week 36, 7/9 patients showed viral clearance.		 All subjects exhibited a marked increase in the vaccine- induced E6- and E7-specific IFN-g ELISPOT response compared with the background level before vaccination. Vaccine-induced cellular immune responses became progressively stronger in all patients during GX-188E vaccination.
The response against the E6 antigen was more vigorous than against E7 as determined by the magnitude of response (69–89% against E6 versus 11–31% against E7 at VF1).
GX-188E vaccination-induced E6/E7- specific memory T-cell response can be maintained for at least 24 weeks post last vaccination.
Apart from patient A04, GX-188E vaccine elicited activation of both HPV16-specific CD4 and CD8 T cells.
The amount of Th1 effector cytokines, such as IFN-γ, IL-2 and tumour necrosis factor α (TNF-α) increased after vaccination in most of the patients (median 49.9−, 13− and 22.9−fold increases for IFN-γ, IL-2, and TNF-α, respectively).		 AEs associated with GX-188E vaccination-chills, injection site pain, swelling and hypoaesthesia in 19/49 patients.
AEs-headache, rhinitis and fatigue in 7/49 of the cases could be potentially associated with the vaccine.		 6/7 responders carrying HLA-A*02 exhibited high polyfunctional CD8 T-cell responses as well as complete regression of CIN3.
Among the two non-responders, patient A04 with HLA-A*26 and -A*30 did not induce HPV-specific CD8 T-cell responses at all.		 Too small study population, which does not allow for generalization of the results and drawing conclusions.
No stratification by age, ethnicity, monoinfection/mixed infection.
Randomization or masking of the population was not introduced as well.
No control group.
All patients had CIN 3 –both severe dysplasia and carcinoma in situ (selection bias).
The confirmation bias)-no consideration of spontaneous regression.
VGX-3100
[3]		 HP regression in 53/107 patients (49.5%) in treatment group, 11/36 (30.6%) in placebo group (PPD 19·0, 95% CI 1·4–36·6; p = 0·034).
Modified intention-to-treat analyses: HP regression in 55/114 patients (48.2%) in treatment group, 12/40 (30%) in placebo group. (percentage point difference 18·2, 1·3–34·4; p = 0·034)
Post-hoc efficacy analyses: HP regression to normal in 43/107 patients (40.2%) in treatment group, 6/36 (16.7%) of placebo recipients (PPD 23·5, 95% CI 4·4–37·0; p = 0·012).		 Viral clearance occurred in 56/107 patients (52·3%) in treatment group, 9/35 patients (25.7%) in placebo group (percentage point difference 26·6, 95% CI 6·8–42·2; p = 0·006).
Among those with HP regression, viral clearance was more likely among VGX-3100 recipients (about 80%) than among placebo recipients (about 50%).		 In post-hoc immunological analyses, T-cell responses to HPV-16 and HPV-18 E6 and E7 peaked at week 14 for VGX-3100 recipients, with a 9.5 times greater median response than in placebo (p < 0·0001).
VGX-3100 elicited significantly increased frequencies of antigen-specific, activated CD8+ T cells, identified by cell surface expression of CD137, that also expressed perforin compared with placebo (p = 0·001).
VGX-3100 recipients with HP regression and viral clearance developed antibody responses to both HPV-16 and HPV-18 E7 that were significantly higher than for non-regressors, at the time of peak response (post-dose 3) but also as early as post-dose 2 and as late as week 24.		 Injection site erythema—98/125: 78.4% in treatment group, 57.1% in placebo group.
4 patients discontinued due to AEs—2 injection site pain, 1 maculopapular reaction, 1 allergic reaction.
No serious AEs reported.		 None Skewing of the population towards more severe disease and older age.
92.8% of the participants had genotype of HPV 16 + at the entry.
The attritition bias-18 patients in treatment group and 6 patients in control group were excluded from the study due to different reasons.
HPV genotyping, which was based on the cervical swabs, included the possibility of only HPV16 or/and HPV18. Therefore, mixed infection study group could be underestimated.
The confirmation bias-Vaccination induced HP regression and viral clearance in about 40% of women with CIN2/3 positive for HPV-16 or HPV-18, whereas surgical excision would have eliminated the dysplastic tissue in 85–90% of women.
pNGVL4a-CRT/E7(detox)
[4]		 HP regression in 8/32 patients (30%).
Remaining 70% of patients had persistent CIN 2/3.
  • 1 patient had regression to CIN1.
  • 7 patients had no residual CIN.
 No differences between pre- and post-vaccination viral loads in any of the treatment cohorts. Immune response to E7 was minimal and was not significantly different than response to E6.
Intraepithelial CD8+ T cell infiltrates increased after vaccination in intralesional administration cohort (p = 0.0313).		 Total vaccine specific AEs in 22/32 patients (69%).
55% of IM vaccination patients, 80% of PMED patients, and 73% of intralesional vaccination patients experienced AEs.
Most common–constitutional and injection site grade 1 or less AEs.
No grade 3 or 4 AEs.
No vaccine-related serious AEs.
1 bleeding after LEEP, 3 pregnancies unrelated to vaccine.		 None This was a small phase I trial designed to primarily evaluate the feasibility and safety of pNGVL4a-CRT/E7(detox).
Only HPV 16 positive CIN patients were included in the study. The majority of these patients were Caucasians.
Patients were required to have a hemoglobin of 9 g/dL or greater. The selection bias-anemia is considered strong prognostic factor.
The ND10 PMED has a reduced number of components to ease large-scale manufacturability, compared to previously used ND5.5. This could potentially lead to discrepancies in results due to device error.
pNGVL4a-Sig/E7(detox)/HSP70 [5] No HP progression was observed.
3/9 patients (33%) had complete histologic regression of disease at week 15 in the highest dose cohort.		 NA E7 specific T cell response was identified in 3/15 patients:
  • 1 patient–response increased subsequent to vaccination at week 15
  • 1 patient–stable response
  • 1 patient–declined response.
E6 specific T cell response: 5/15 patients. Overall, responses to E6 were not of greater absolute magnitude in regressors compared with non- regressors at either of the two time points. (p = 0.4228 and p = 0.4964, respectively).
At 6 months response to E7 was detected in 5/9 patients (55.6%) in highest dose cohort.		 Transient local reactogenicity was reported in 5/15 (33%). Systemic symptoms (malaise, myalgia, headache) after vaccination were also reported by 5/15 subjects.
No dose-limiting toxicities were observed.		 None This was a small phase I study–15 patients only. No masking.
Follow up period was 19 weeks, whereas the average follow-up period in selected studies was 36 weeks.
Vaccine targets specifically HPV16 E7 oncoprotein, without HPV 18, or E6 oncoprotein.
Local and systemic AEs were assessed by patients, which may result in self-reporting bias such as social desirability or recall bias.
MEDI0457
[6]		 All cohort 1 patients remain alive with no evidence of disease clinically or by PET/CT.
Of the cohort 2 patients:
  • 1 died
  • 1 had persistent disease
1 remains free of disease.
The estimated PFS at 12 months was 88.9% overall, 100% in cohort 1, and 50% in cohort 2.
7/8 patients achieved a complete response (6/7 in cohort 1 and 1/3 in cohort 2), and 1 (cohort 1) achieved partial response (decreased or stable hypermetabolic activity after CRT+MEDI0457) after completion of the immunization series.		 All patients cleared detectable HPV DNA at week 16 after immunizations.
5/6 patients cleared HPV RNA by in situ hybridization at the completion of immunization.		 8 patients had detectable cellular or humoral immune responses after chemoradiation and MEDI0457.
6 patients showed increased IFN-γ responses over baseline against HPV16 E6 and E7.
5 patients showed increased IFN-γ responses against HPV18 E6 and E7.
Anti-HPV responses were numerically greater in cohort 1 (23.3 SFU/106 PBMC to 369 SFU/106 PBMC) compared with cohort 2 (6.7 SFU/106 PBMC to 63.3 SFU/106 PBMC.
6/10 patients exhibited de novo sero-responses to HPV16 antigens, and 6/10 patients exhibited de novo sero-responses to HPV18 antigens.		 Vaccine related AEs in 8 patients–grade 1 injection site bruising (n = 2), injection site pain (n = 2).
Treatment related AEs occurred in 8 patients, mainly grades 1 or 2.
Grade >3 AEs in 4 patients–abdominal pain and pneumonia in cohort 1;
pathologic fracture, anemia, intestinal perforation (grade 5).
were followed after chemoradiation and 3 doses of INO3112.		 Expression of PD-L1 on panCK+ tumor cells, CD68+ macrophages, and CD8+ T cells in serial biopsy specimens:
  • post-CRT and post-CRT+MEDI0457 showed decreased epithelial cells, consistent with tumor regression.
  • PD-L1 was detectable on panCK+ tumor cells and CD68+ cells at pre-CRT and post-CRT biopsies.
  • PD-L1 was detectable on CD8+ T cells.
Compared with pre-CRT and post-CRT time points, post-CRT þ MEDI0457 biopsies were associated with decreased PD-L1+CD8+, PD-1+CD8+, and PD- L1+CD68+ subpopulations		 Too small study (n = 10) population, which does not allow for generalization of the results and drawing conclusions.
Study included several histologic diagnoses–squamous cell carcinoma, adenocarcinoma, adenosquamous cell carcinoma of the cervix with various prognosis.
The confirmation bias-patients received a vaccine 2 to 4 weeks after chemoradiation, which could impact the vaccine effect on organism. It is unclear whether longer period of recovery would result in better outcome.
Dosing and timing regimen of MEDI0457 was based on studies of preinvasive cancer, thus the applicability of the regimen for invasive cancer types is questionable [4].
There was no control group of “chemoradiation only” in order to assess the sole effect of vaccination.
Abbreviations:—histopathological—HP; percentage point difference—PPD; progression-free survival—PFS; positron emission tomography—PET; computer tomography—CT; adverse events—AEs; particle-mediated epidermal delivery—PMED; human leukocyte antigens—HLA; chemoradiotherapy—CRT;—intramuscularly—IM; electroporation—EP; cervical biopsy—CB; maximum tolerated dose—MTD; cervical intralesional—IL; calreticulin—C-RT; heat shock proteins—HSPs; squamocolumnar junction—SCJ; viral load—VL.
GX-188 E in phase I trial by Kim et al. [2] showed a 78% success rate of complete response both histologically and virologically. The same vaccine in the phase II trial by Choi et al. (2019) resulted in histopathological regression to CIN < 1 in 52% of patients 20 weeks and 67% of patients 36 weeks after the first dose [1]. MEDI0457 by Hasan et al. showed 87.5% of complete response to the vaccine and 1 patient had a partial response to the treatment [6]. pNGVL4a-CRT/E7(detox) [4] and pNGVL4a-Sig/E7(detox)/HSP70 [5] vaccines both showed a similar response rate of around 30%.

2. Viral Load Clearance

Viral load was measured by means of PCR amplification to assess the clearance of HPV DNA from the cervical biopsy after vaccination. Choi et al. established that HPV clearance was associated with the histopathologic regression as 77% of regressors had no trace of HPV DNA, while only 12% of non-regressors had no viral load in the tissue biopsy [1]. Kim et al. results show that GX-188E takes time to clear off the virus [2]. MEDI0457 [6] and VGX-3100 [3] report the association between viral clearance and tumor size reduction, whilst pNGVL4a-CRT/E7(detox) [4] did not result in any difference between pre- and post-treatment viral load.

3. Immunogenicity

Immunogenicity is one of the key features of the therapeutic vaccines as it represents the potential of the vaccine to induce virus-specific T cell response, in particular HPV E6 and E7 specific CD8+ T cell immune response. IFN-γ response was measured by means of ex vivo ELISpot assay with cryopreserved and thawed peripheral blood mononuclear cells (PBMCs) at pre- and post-treatment stages. The vaccine response is considered positive when the increase in T-cell frequency was at least three times greater compared to the study entry measurement. GX-188E both in phase I [2] and phase II [1] studies showed a significant increase in IFN-γ response, which was correlated with the histopathologic regression and viral clearance. Moreover, an E6 specific response was more pronounced than E7 specific [1][2]. VGX-3100 induced 9.5 times greater IFN-γ response in the treatment group compared to the placebo, which lasted as long as 24 weeks post-vaccination [3]. On the contrary, MEDI0457 induced a greater response to E7, particularly in newly diagnosed cohort 1 that persisted up to 48 weeks [6]. In cohort 1, 4 of 7 patients exhibited IFNγ-producing spots exceeding 100 SFU/106 PBMC, whereas no patients produced similar responses in cohort 2 [6]. pNGVL4a-CRT/E7(detox) and pNGVL4a Sig/E7(detox)/HSP70 showed minimal dose-dependent immune response, which was remarkable from the unvaccinated group [4][5].

3.1. Toxicity/Adverse Events

Overall, all vaccines were well-tolerated without vaccine-related serious adverse events. The most common adverse events were injection site pain and erythema, as well as constitutional symptoms (malaise, myalgia, and headache) [1][2][3][4][5][6]. No serious adverse events (Grade 3/4) related to the vaccination were reported. No dose-limiting toxicities were observed.

4. Discussion

This entry summarizes the findings of phase I and phase II clinical trials investigating the treatment of patients with histopathologically diagnosed CIN associated with HPV 16 or/and HPV 18 with DNA therapeutic vaccines. Six studies have demonstrated immunologic response in the form of lesion size regression, viral clearance, and increased T cell response of five different DNA vaccines–GX-188E (phase I and phase II), VGX-3100, pNGVL4a-CRT/E7 (detox), pNGVL4a-Sig/E7 (detox)/HSP70, MEDI0457. Vaccines were plasmid DNA encoding for either non-oncogenic E6/E7 or both, and chaperonin proteins such as HSP 70 and Calreticulin for the enhancement of the uptake by antigen-presenting cells, and MHC class I processing and presentation. MEDI0457 [6] had the same plasmid formulation as VGX-3100 [3] combined with plasmid encoding IL-12. All vaccines were well tolerated by patients, leading to only grade 1 or less systemic and local side effects.
Previous reviews have studied various existing therapeutic vaccines including live vectors, plant-based, protein, whole cell, and combinatorial vaccines [7]. This is the first systematic review of DNA therapeutic vaccines against cervical cancer expressing HPV16 and HPV18 E6 and E7 oncogenes. The feasibility of production, storage, and transportation, cost-effectiveness, the capability of multiple immunizations, and targeting different co-stimulatory genes provided the rationale for the study of DNA therapeutic vaccines [7]. However, comparatively weak immunogenicity and the risk of integration into the host genome are the main concerns, which could be addressed by modification of E6 and E7 to abolish its transformative capacity [7]. There are approaches of boosting the potency of DNA vaccines, such as increasing the number of antigen-expressing dendritic cells (DCs) by using a gene gun delivery method, enhancing antigen processing and presentation in dendritic cells via codon optimization, and improving the DCs and T-cell interaction [7]. These strategies were used in our selected studies, which led to increased antigen-specific, activated CD8+ T cell response in all of them. Patients with CIN2/3 were more likely to induce E6 and E7 specific CD8+ immune response, according to the IFNgamma ELISPOT results, compared to the invasive cervical cancer [6]. According to Hasan et al., diminished immune response in more advanced disease stages is associated with immune exhaustion, the effect of chemoradiation and selection of patients with diminished immunity against HPV [6]. The strongest evidence of the immunogenicity of DNA therapeutic vaccine VGX-3100 was observed by the increased intensity of CD8+ infiltrates in histopathologically regressed patients compared to the placebo group with regressed lesions [3].
DNA therapeutic vaccines were also assessed based on their clinical efficacy, i.e., the ability to induce cervical lesion regression. The regression to ≤CIN1 among study participants was observed in all studies with significantly varying degrees. The study of VGX-3100 vaccine with both treatment and placebo groups showed a response rate of 49.5% vs. 30.6%, respectively [3]. Meanwhile, GX-188E vaccine has resulted in histopathological regression in 67% of patients in both phase I study and phase II studies [1][2]. Choi et al. [1] have observed an enhanced response to GX-188E over time up to 83% among those with cervical lesions <50%, probably due to the enhanced memory T cell-driven therapeutic effect. The difference in clinical benefit between VGX-3100 and GX-188E could be explained with the recruitment of CIN3 HPV-positive patients only, the lack of placebo group, and the small number of participants in the latter. pNGVL4a-CRT/E7 (detox) and pNGVL4a-Sig/E7 (detox)/HSP70 had the lowest clinical efficacy of approximately 30% response rate among all [4][5]. However, the effect of these two vaccines on the lesion regression is questionable, as this rate is similar to spontaneous remission rate over a 15-week period [4].
It was established that women, after excision of the cervical lesion, are more likely to have a relapse; therefore, viral clearance is a key factor of vaccine efficacy [7]. VGX-3100, GX-188E, and MEDI0457 effectively cleared detectable HPV DNA, which was significantly associated with histopathological regression [1][2][3][6]. In contrast, pNGVL4a-CRT/E7 (detox) has not resulted in viral load reduction [4].

References

  1. Choi, Y.J.; Hur, S.Y.; Kim, T.J.; Hong, S.R.; Lee, J.K.; Cho, C.H.; Park, K.S.; Woo, J.W.; Sung, Y.C.; Suh, Y.S.; et al. A phase II, Prospective, Randomized, Multicenter, open-label study of GX-188E, an HPV DNA vaccine, in patients with Cervical Intraepithelial Neoplasia 3. Clin. Cancer Res. 2019, 26, 1616–1623.
  2. Kim, T.J.; Jin, H.T.; Hur, S.Y.; Yang, H.G.; Seo, Y.B.; Hong, S.R.; Lee, C.W. Clearance of persistent HPV infection and cervical lesion by therapeutic DNA vaccine in CIN3 patients. Nat. Commun. 2014, 5, 5317.
  3. Trimble, C.L.; Morrow, M.P.; Kraynyak, K.A.; Shen, X.; Dallas, M.; Yan, J.; Edwards, L.; Parker, R.L.; Denny, L.; Giffear, M.; et al. Safety, efficacy, and immunogenicity of VGX-3100, a therapeutic synthetic DNA vaccine targeting human papillomavirus 16 and 18 E6 and E7 proteins for cervical intraepithelial neoplasia 2/3: A randomised, double-blind, placebo-controlled phase 2b trial. Lancet 2015, 386, 2078–2088.
  4. Alvarez, R.D.; Huh, W.K.; Bae, S.; Lamb, L.S., Jr.; Conner, M.G.; Boyer, J.; Wang, C.; Hung, C.F.; Sauter, E.; Paradis, M.; et al. A pilot study of pNGVL4a-CRT/E7(detox) for the treatment of patients with HPV16+ cervical intraepithelial neoplasia 2/3 (CIN2/3). Gynecol. Oncol. 2016, 140, 245–252.
  5. Trimble, C.L.; Peng, S.; Kos, F.; Gravitt, P.; Viscidi, R.; Sugar, E.; Pardoll, D.; Wu, T.C. A phase I trial of a human papillomavirus DNA vaccine for HPV16+ cervical intraepithelial neoplasia 2/3. Clin. Cancer Res. 2009, 15, 361–367.
  6. Hasan, Y.; Furtado, L.; Tergas, A.; Lee, N.; Brooks, R.; McCall, A.; Golden, D.; Jolly, S.; Fleming, G.; Morrow, M.; et al. A Phase 1 Trial Assessing the Safety and Tolerability of a Therapeutic DNA Vaccination Against HPV16 and HPV18 E6/E7 Oncogenes After Chemoradiation for Cervical Cancer. Int. J. Radiat Oncol. Biol. Phys. 2020, 107, 487–498.
  7. Hung, C.-F.; Ma, B.; Monie, A.; Tsen, S.-W.; Wu, T.-C. Therapeutic human papillomavirus vaccines: Current clinical trials and future directions. Expert Opin. Biol. Ther. 2008, 8, 421–439.
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