A dose-escalation, randomized, double-blind, placebo-controlled phase I clinical trial was performed in healthy Iranian volunteer women to assess the safety, tolerability, and immunogenicity of NZ8123-HPV16-optiE7 vaccine involving recombinant Lactococcus lactis expressing the codon-optimized human papillomavirus (HPV)-16 E7 oncogene. Fifty-five eligible subjects were divided into 6 cohorts based on the dosages (1 × 109, 5 × 109, and 1 × 1010 CFU/mL) of either vaccine or placebo, which were administrated orally a total of 4 times at weeks 1, 2, 4, and 8. Then, adverse events, specific serum IgG and vaginal IgA, and E7-specific IFNγ-secreting CD8+ CTL responses were evaluated. The vaccination was well tolerated by 40 subjects who completed the immunization schedule, and no serious adverse effects were reported. The IgG and IgA levels peaked at day 60, and the levels for the 5 × 109 CFU/mL and 1 × 1010 CFU/mL dose groups were higher than those for the 1 × 109 CFU/mL dose group. Time-to-peak stimulation in E7-specific IFNγ-secreting CD8+ CTL responses was seen in cervical lymphocytes 1 month after the last vaccination. Again, no significant increase was seen in the peripheral blood mononuclear cells (PBMC) of the same volunteers. CTL responses in cervical lymphocytes and PBMCs at day 90 were markedly higher in the 5 × 109 and 1 × 1010 CFU/mL groups than in the 1 × 109 CFU/mL group, demonstrating the dose dependency of NZ8123-HPV16-optiE7 vaccine following oral administration. The 6-month follow-up revealed that antibody levels decreased up to day 240; nevertheless, long-term E7-specific IFNγ-secreting CD8+ CTL responses were recorded during follow-up. Overall, the safety and immunogenicity profile achieved in this study encourages further phase II trials with the 5 × 109 CFU/mL dose vaccine.

Human papillomavirus (HPV) infection is common, with a lifetime risk exceeding 50% for sexually active males and females. Because HPV is the major cause of cervical cancer, the high prevalence of genital HPV infection is considered a serious worldwide health issue. HPVs are heterogeneous group viruses that infect epithelial tissues (1, 2). HPV types 16 and 18 are often found in premalignant lesions of the cervix, which can progress to cervical carcinoma (3, 4). The oncogenic potential of HPV is associated with the expression of 2 viral early genes, E6 and E7. Because E6 and E7 are the only HPV proteins expressed in precursor lesions, they represent reliable antigenic targets for immunotherapy (5, 6). Widespread infection and serious disease, as well as inadequate modes of therapy and prevention, have led to interest in the development of HPV vaccines. Prophylactic viral vaccines have a long record as a cost-effective approach to preventing infection; however, none of these vaccines can treat an existing HPV infection. Therefore, a therapeutic approach to treating existing HPV cervical cancer is needed (7). Many viral infections are controlled by virus-specific CD8+ cytotoxic T lymphocytes (CTL). Hence, vaccination with oncoproteins of HPV-16 including E6 and/or E7, with the subsequent stimulation of antigen-specific CTL, has been a frequent immunotherapeutic approach for HPV-associated disease (8, 9).

In particular, the cellular arm of the immune system has been shown to constitute a major defense against cervical carcinoma and has utilized an extensive array of potential vaccine delivery systems (10).

It has been suggested that probiotic bacteria confer a range of health benefits in both children and adults (11). Many studies have highlighted the oral administration of specific probiotics strains (such as Lactococcus lactis) as enhancing both local and systemic immune responses and possibly improving immunization (10, 12). Therefore, probiotics are expected to enhance the immunogenicity of the vaccine. L. lactis has garnered the most attention and has been investigated intensively, as this bacterial genus is among the few that confer many positive rewards to test subjects (13, 14). L. lactis also has a number of advantages as a vaccine delivery vector; the bacterium grows very efficiently in vitro, it lacks lipopolysaccharide (LPS), they can be readily eliminated with antibiotics, and their genetic material does not integrate into the host genome (15, 16).

Using mouse models, our previous studies on immunization with Lactococcus-based vaccines have demonstrated an induction of systemic E7 cell–mediated immune responses and the regression of HPV-16 E7-positive tumors. We observed an induction of mucosal E7 cell–mediated immune responses within intestinal mucosa after oral administration of L. lactis expressing HPV-16 E7 in mice (12).

The current study evaluated the safety and immunogenicity in healthy women of 3 dose levels (1 × 109, 5 × 109, and 1 × 1010 CFU/mL) of the HPV-16 E7 vaccine (NZ8123-HPV16-optiE7) given in aqueous solution. The results demonstrate that the immunization of healthy women with HPV-16 E7 oncogene induced both serum and genital tract antibodies and vaginal cytokines.

Study population and parameters

One hundred six healthy Iranian non-pregnant volunteer women ages 18 to 59 years were vaccinated at the Keyvan Virology Specialty Laboratory (KVSL) from June 2018 to August 2018. Upon enrollment, written consent was obtained from each participant or her legal guardian. The absence of HPV contamination was verified for all subjects. Residual ThinPrep cervical cytologic specimens were centrifuged, and the concentrated cell pellet was selected for DNA extraction using a High Pure Viral Nucleic Acid Kit (Roche Diagnostics GmbH). Genomic DNA of the specimens was used in PCR with MY09 and MY11 degenerate consensus primers. DNA quality and absence of PCR inhibitors in the samples were confirmed using the beta-globin PCR assay (17). Screening procedures were conducted within 28 days of the baseline visit and included a clinical history, physical examination including genital and pelvic examination, electrocardiogram, and provision of a blood and urine sample for routine laboratory tests including HB level, WBC count, PLT count, normal UA results, AST, ALT, ALP, CA, phosphorus, total protein, ESR, glucose, BUN, bilirubin, albumin, and creatinine values within normal ranges.

Volunteers were excluded from participation if their medical history (history or clinical manifestation of genitourinary disease, history of immunodeficiency in the previous 30 days, history of cancer or chronic hepatitis), physical examination (anogenital warts within the previous year), or laboratory tests (positive urine pregnancy test or abnormal Pap smear, abnormal serum immunoglobulin level, positive serum antibodies to hepatitis C, B, and HIV-1) showed clinically significant abnormalities. Other criteria for exclusion from the study included allergies to any vaccine component, receipt of any blood product or component in the previous 6 months, presence of any known immune or coagulation disorder, and receipt of any other vaccination or immunosuppressive medications. Some volunteers were excluded from the study, because they were lost to follow-up. The current study was approved by the Iran University of Medical Sciences (IUMS). This trial is registered with the Iranian Registry of Clinical Trials (IRCT; https://www.irct.ir/trial/39227) under registration number IRCT20190504043464N1.

Vaccine and placebo

Details of the recombinant L. lactis strain NZ9000 vaccine producing the codon-optimized full-length E7 oncoprotein of HPV-16 (NZ8123-HPV16-optiE7) have been previously reported (4). The NZ8123-HPV16-optiE7 was cultivated in GM17 medium containing chloramphenicol antibiotic (10 μg/mL), tested for microbial contaminations based on the recommendation of the United States Pharmacopoeia 41 (USP 41 NF-36 (chapter <61>, <62>, and <1111>), and designated the master seed stock (18). Working seed stocks of NZ8123-HPV16-optiE7 were also produced. Clinical lots of NZ8123-HPV16-optiE7 vaccines were produced in accordance with GMP conditions in a fermenter under carefully controlled conditions within a clean room offered by Prof. Keyvani. In brief, the resultant construct was diluted in PBS (pH 6.2) to the appropriate concentrations of 1 × 109, 5 × 109, and 1 × 1010 CFU/mL based on OD600 readings and colonies counting from serial dilutions in duplicate, vialed, and stored at 2°C to 8°C. The placebo utilized contained PBS having L. lactis containing pNZ8123 (named NZ8123) to those in the vaccine, in total concentrations of 1 × 109, 5 × 109, and 1 × 1010 CFU/mL. Vaccine and placebo were not visually discernible.

Vaccination schedule

This study was a double-blind, randomized, placebo-controlled dose-escalation, phase I safety and immunogenicity trial. Volunteers who met eligibility criteria were randomized into 6 clinical groups in a 2:1 correlation using Research Randomizer software (version 3.0) to receive 4 rounds of oral vaccination (1 mL) using the NZ8123-HPV16-optiE7 vaccine or a placebo at weeks 1, 2, 4, and 8.

Each dose of NZ8123-HPV16-optiE7 was administered orally, once each morning after 5 days of fasting, each treatment week. If no side effects were detected in arms given the lower dose (1 × 109 CFU/mL), the dose was escalated to 5 × 109 and 1 × 1010 CFU/mL. Both the NZ8123-HPV16-optiE7 vaccine and the placebo cohorts were administered to 26 vaccine recipients and 14 placebo recipients in each of the schedules.

Clinical safety and tolerability evaluations

Clinical safety and tolerability were assessed using the collection of adverse effects (AE) reported throughout the study. Adverse effects were monitored by the study staff in the laboratory at 1 hour and at days 2, 5, and 7 after each vaccination and by telephone for the following 7 days. Likewise, AEs that happened up to 240 days after the first programmed administration of the medication were evaluated blindly for severity and relationship to study drug. Participants were given a diary card on which they recorded systemic AEs from the day of vaccination through 7 days afterward. Toxicities were graded according to the NCI Common Terminology Criteria for Adverse Events (CTCAE) version 3.0. Blood samples were collected for clinical laboratory tests as outlined above.

Antibody assay

Blood and vaginal discharge specimens were collected from each study subject at baseline and at days 30, 60, 90, and 240. IgG- and IgA-specific HPV-16 E7-based enzyme-linked immunosorbent assays (ELISA) were tested in triplicate at dilutions of 1:50 and 1:20, respectively, in a 96-well plate format following the modification of a method described previously using goat anti-human IgG H&L (HRP) antibody (ab6858; Abcam) and goat anti-human IgA alpha chain (HRP) antibody (ab97215; Abcam). A participant who received the NZ8123-HPV16-optiE7 vaccine was deemed positive if the OD450 was greater than the mean OD450 plus three standard deviations from the mean for a panel of a participant who received a vaccine and placebo and was at least 0.100.

Detection of HPV-16 E7-specific IFNγ-secreting CD8+ CTL by IFNγ ELISPOT

Approximately 105 peripheral blood mononuclear cells (PBMC) and 105 cervical lymphocytes were isolated from patients at baseline and at days 30, 60, 90, and 240 as described previously (19). Freshly drawn peripheral blood and cervical lymphocytes were stimulated for 24 hours at 37°C with 10 μg/mL CTL epitopes bind to MHC class I including synthesized HPV‐16 E711–20 (YMLDLQPETT), E749–57 peptide (RAHYNIVTF), and E786–93 (TLGIVCPI) according to the manufacturer's protocols for the human IFN-gamma ELISPOT kit (R&D Systems, Inc.). The developed microplate was examined by calculating spots using a dissecting microscope. Quantitation of outcomes was performed by measuring the number of spot‐forming cells per number of cells added to the well. The specific HPV-16 E7-specific CD8+ CTL response was considered positive when specific T-cell frequencies were greater than 3 in 105 cervical lymphocytes and PBMCs.

Statistical analysis

The primary end points were to assess adverse side effects in order to determine the safe dosage of the vaccine, and the secondary end points were to evaluate the existence of a vaccine-specific mucosal and systemic HPV-16 E7 response in relation to the dosage. The data were analyzed in MedCalc software (version 17.6; MedCalc Software; ref. 20). Comparisons of means were done by paired samples t test for independent samples. A P value of < 0.05 was considered statistically significant, and a P value of < 0.01 was considered highly significant. In addition, risk differences and associated 95% confidence intervals (CI) were computed comparing the vaccine and placebo groups across all vaccination visits.

Study population

One hundred six subjects were screened for eligibility. Of these, 51 withdrew consent prior to randomization. Overall, 55 (51.88%) of the 106 study participants received all scheduled doses of the study medication, and ultimately, 40 subjects completed the vaccination program (Fig. 1). Patients who were included in the study had a mean age of 34.5250 years (range, 30.6607–38.3893 years; P < 0.0001). The placebo cohorts, n = 14, mean age = 36.3571 years (range, 29.3344–43.3799 years with 95% CI), were compared with the vaccine cohorts, n = 26, mean age = 33.5385 years (range, 28.6257–38.4512 years with 95% CI). Comparison of the two groups indicated that the placebo cohorts were somewhat older than the vaccine cohorts, but the value was not statistically significant (P = 0.959). The mean body mass index (BMI) of participants was 26.8775 (range, 24.8017–28.9533, P < 0.0001) at the start of the study. Two subjects had no sex partner, and 34 participants had one sex partner during the year before enrollment; 3 volunteers had 2 partners in the past 12 months, and 1 had 6 partners during that period. The demographic characteristics of the study population are shown in Table 1.

Figure 1.

Flowchart of participants enrolled in this study and reasons for exclusion.

Figure 1.

Flowchart of participants enrolled in this study and reasons for exclusion.

Close modal
Table 1.

Baseline characteristics of healthy women subjects by vaccination dose group at enrollment visit (per-protocol population).

Study arm
Vaccine groups (CFU/mL dose)Placebo groups (CFU/mL dose)
1 × 1095 × 1091 × 10101 × 1095 × 1091 × 1010
(n = 8)(n = 9)(n = 9)(n = 5)(n = 4)(n = 5)
DemographicStatusNumber (percentage)
Age 18–25 years 2 (25%) 3 (33.33%) 3 (33.33%) 3 (60%) 1 (25%) 0 (0%) 
 26–35 years 2 (25%) 2 (22.22%) 3 (33.33%) 0 (0%) 1 (25%) 3 (60%) 
 36–46 years 3 (37.50%) 1 (11.11%) 1 (11.11%) 1 (20%) 1 (25%) 0 (0%) 
 47–59 years 1 (11.50%) 3 (33.33%) 2 (22.22%) 1 (20%) 1 (25%) 2 (40%) 
Marital status Married 6 (75%) 6 (66.66%) 4 (44.44%) 3 (60%) 1 (25%) 3 (60%) 
 Divorce—widow 2 (25%) 1 (11.11%) 1 (11.11%) 1 (20%) 2 (40%) 2 (40%) 
 Single 0 (0%) 2 (22.22%) 4 (44.44%) 1 (20%) 1 (25%) 0 (0%) 
BMI Underweight = <18.5 1 (11.50%) 1 (11.11%) 3 (33.33%) 1 (20%) 1 (25%) 0 (0%) 
 Normal weight = 18.5–24.9 1 (11.50%) 4 (44.44%) 0 (0%) 2 (40%) 0 (0%) 0 (0%) 
 Overweight = 25–29.9 3 (37.50%) 1 (11.11%) 2 (22.22%) 0 (0%) 2 (50%) 3 (60%) 
 Obesity = BMI of 30 or greater 3 (37.50%) 3 (33.33%) 4 (44.44%) 2 (40%) 1 (25%) 2 (40%) 
Age at first sexual intercourse ≤16 0 (0%) 0 (0%) 1 (11.11%) 0 (0%) 1 (25%) 2 (40%) 
 17 4 (50%) 5 (55.55%) 6 (66.66%) 2 (40%) 0 (0%) 0 (0%) 
 18 1 (12.50%) 4 (44.44%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 
 19 3 (37.50%) 0 (0%) 2 (22.22%) 2 (40%) 2 (40%) 3 (60%) 
 20≤ 0 (0%) 0 (0%) 0 (0%) 1 (20%) 1 (25%) 0 (0%) 
Smoking status Never smoked 7 (87.50%) 6 (66.66%) 7 (77.77%) 5 (100%) 3 (75%) 3 (60%) 
 Ex-smoker 0 (0%) 1 (11.11%) 0 (0%) 0 (0%) 0 (0%) 1 (20%) 
 Current smoker 1 (12.50%) 2 (22.22%) 2 (22.22%) 0 (0%) 1 (25%) 1 (20%) 
Study arm
Vaccine groups (CFU/mL dose)Placebo groups (CFU/mL dose)
1 × 1095 × 1091 × 10101 × 1095 × 1091 × 1010
(n = 8)(n = 9)(n = 9)(n = 5)(n = 4)(n = 5)
DemographicStatusNumber (percentage)
Age 18–25 years 2 (25%) 3 (33.33%) 3 (33.33%) 3 (60%) 1 (25%) 0 (0%) 
 26–35 years 2 (25%) 2 (22.22%) 3 (33.33%) 0 (0%) 1 (25%) 3 (60%) 
 36–46 years 3 (37.50%) 1 (11.11%) 1 (11.11%) 1 (20%) 1 (25%) 0 (0%) 
 47–59 years 1 (11.50%) 3 (33.33%) 2 (22.22%) 1 (20%) 1 (25%) 2 (40%) 
Marital status Married 6 (75%) 6 (66.66%) 4 (44.44%) 3 (60%) 1 (25%) 3 (60%) 
 Divorce—widow 2 (25%) 1 (11.11%) 1 (11.11%) 1 (20%) 2 (40%) 2 (40%) 
 Single 0 (0%) 2 (22.22%) 4 (44.44%) 1 (20%) 1 (25%) 0 (0%) 
BMI Underweight = <18.5 1 (11.50%) 1 (11.11%) 3 (33.33%) 1 (20%) 1 (25%) 0 (0%) 
 Normal weight = 18.5–24.9 1 (11.50%) 4 (44.44%) 0 (0%) 2 (40%) 0 (0%) 0 (0%) 
 Overweight = 25–29.9 3 (37.50%) 1 (11.11%) 2 (22.22%) 0 (0%) 2 (50%) 3 (60%) 
 Obesity = BMI of 30 or greater 3 (37.50%) 3 (33.33%) 4 (44.44%) 2 (40%) 1 (25%) 2 (40%) 
Age at first sexual intercourse ≤16 0 (0%) 0 (0%) 1 (11.11%) 0 (0%) 1 (25%) 2 (40%) 
 17 4 (50%) 5 (55.55%) 6 (66.66%) 2 (40%) 0 (0%) 0 (0%) 
 18 1 (12.50%) 4 (44.44%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 
 19 3 (37.50%) 0 (0%) 2 (22.22%) 2 (40%) 2 (40%) 3 (60%) 
 20≤ 0 (0%) 0 (0%) 0 (0%) 1 (20%) 1 (25%) 0 (0%) 
Smoking status Never smoked 7 (87.50%) 6 (66.66%) 7 (77.77%) 5 (100%) 3 (75%) 3 (60%) 
 Ex-smoker 0 (0%) 1 (11.11%) 0 (0%) 0 (0%) 0 (0%) 1 (20%) 
 Current smoker 1 (12.50%) 2 (22.22%) 2 (22.22%) 0 (0%) 1 (25%) 1 (20%) 

Primary endpoints: safety and tolerability

The vaccine was well tolerated at all dosage levels, and no serious vaccine-related AEs occurred according to CTCAE (version 3.0). Hence, a wide range of AEs, including colitis, constipation, diarrhea, distension/bloating (abdominal), esophagitis, gastritis (including bile reflux gastritis), heartburn/dyspepsia, hemorrhoids, nausea, salivary gland changes/saliva, taste alteration (dysgeusia), and vomiting were graded from 1 to 5 with unique clinical descriptions of severity for each adverse event (AE) based on this general guideline: grade 1, mild AE; grade 2, moderate AE; grade 3, severe AE; grade 4, life-threatening or disabling AE; grade 5, death related to AE. The most common systemically clinical AE was nausea and vomiting with mild to moderate intensity. A few of the volunteers at both doses experienced adverse side effects; one subject had nausea (20%, P = 0.3739) in cohort 1, and one subject had vomiting (12.5%, P = 0.3506) in cohort 4, expressed during history taking or after the physical examination conducted before and after each successive vaccination. Four subjects from cohort 6 did experience nausea (3 subjects, 33.33%, P = 0.0805) and vomiting (1 subject, 11.11%, P = 0.3466) after vaccination with the dose of 1 × 1010 CFU/mL, but this was not significantly different (P = 0.6213) from cohort 3 in which 2 subjects experienced nausea (40%, P = 0.1778). Correspondingly, no adverse side effects were recorded in any group (vaccine or placebo) 6 months after the last vaccination.

Secondary endpoints: HPV-16 E7-specific IgG antibody

The volunteers were evaluated for serum IgG responses to the vaccine using an indirect ELISA. Overall, the administration of each of the all doses of the NZ8123-HPV16-optiE7 vaccine produced dramatic increases in serum antibody. There was no statistically significant difference in the baseline antibody amounts between the vaccine and placebo groups (P = 0.7990, P = 1.0000, and P = 1.0000 for the 1 × 109, 5 × 109, and 1 × 1010 CFU/mL cohorts, respectively). In comparison with the placebo cohorts, all active study vaccines except cohort 1 × 109 CFU/mL (P = 0.0059) experienced the induction of a high level of HPV-16 E7-specific IgG antibody response 30 days after administration of the NZ8123-HPV16-optiE7 vaccine (P = 0.0137 and P = 0.0018 for 5 × 109 and 1 × 1010 CFU/mL cohorts, respectively). An additional increase was noted 8 weeks after the initial vaccination, and the levels of IgG antibody against HPV-16 E7 peaked at day 60 in each group receiving the NZ8123-HPV16-optiE7 vaccine in comparison with the placebo cohorts, ranging from 0.2370 to 0.6310 in the 1 × 109 CFU/mL dose group (mean difference: 0.4340, P = 0.0036 with 95% CI), 0.2536 to 1.5614 in the 5 × 109 CFU/mL dose group (mean difference: 0.9075, P = 0.0215 with 95% CI), and 0.6383 to 1.3177 in the 1 × 1010 CFU/mL dose group (mean difference: 0.9780, P = 0.0013 with 95% CI). Compared with month 2, IgG antibody levels were markedly higher in the 5 × 109 CFU/mL (ranging from 0.1031 to 0.7294, mean difference: 0.4163, P = 0.0163 with 95% CI) and 1 × 1010 CFU/mL (ranging from 0.2291 to 0.7159, mean difference: 0.4725, P = 0.0025 with 95% CI) vaccine groups than in recipients of the 1 × 109 CFU/mL dose. As was noted for antibodies measured by ELISA, IgG antibody levels decreased one (day 90) and 6 (day 240) month after the last vaccination (Fig. 2, top).

Figure 2.

HPV-16 E7-specific serum IgG antibody (1, top) and E7-specific vaginal IgA antibody (2, bottom) levels by placebo groups [cohorts 1 (A, left); 1 × 109 CFU/mL dose, cohorts 2 (B, center); 5 × 109 CFU/mL dose, and cohorts 3 (C, right); 1 × 1010 CFU/mL dose] and vaccine group [cohorts 4 (A, left); 1 × 109 CFU/mL dose, cohorts 5 (B, center); 5 × 109 CFU/mL dose, and cohorts 6 (C, right); 1 × 1010 CFU/mL dose] measured by ELISA at a serum dilution of 1:100 using goat anti-human IgG H&L (HRP) antibody and at a vaginal fluid dilution of 1:10 using goat anti-human IgA alpha chain (HRP) antibody, respectively. Results are expressed with 95% CIs for the following time points: prevaccination (day 0), days 30, 60, 90, and 240. The absorbance of each well was measured at 450 nm. Bars represent the mean ± standard error value of each group. Statistically significant differences are denoted by an asterisk between T60 and T30, T90, and T240 of vaccine groups (*, P ≤ 0.0001; **, P ≤ 0.001; ***, P ≤ 0.05; ****, P ≤ 0.1).

Figure 2.

HPV-16 E7-specific serum IgG antibody (1, top) and E7-specific vaginal IgA antibody (2, bottom) levels by placebo groups [cohorts 1 (A, left); 1 × 109 CFU/mL dose, cohorts 2 (B, center); 5 × 109 CFU/mL dose, and cohorts 3 (C, right); 1 × 1010 CFU/mL dose] and vaccine group [cohorts 4 (A, left); 1 × 109 CFU/mL dose, cohorts 5 (B, center); 5 × 109 CFU/mL dose, and cohorts 6 (C, right); 1 × 1010 CFU/mL dose] measured by ELISA at a serum dilution of 1:100 using goat anti-human IgG H&L (HRP) antibody and at a vaginal fluid dilution of 1:10 using goat anti-human IgA alpha chain (HRP) antibody, respectively. Results are expressed with 95% CIs for the following time points: prevaccination (day 0), days 30, 60, 90, and 240. The absorbance of each well was measured at 450 nm. Bars represent the mean ± standard error value of each group. Statistically significant differences are denoted by an asterisk between T60 and T30, T90, and T240 of vaccine groups (*, P ≤ 0.0001; **, P ≤ 0.001; ***, P ≤ 0.05; ****, P ≤ 0.1).

Close modal

Secondary endpoints: HPV-16 E7-specific IgA antibody

Samples of the vaginal discharge of subjects who completed the vaccination program were also analyzed for IgA-specific antibodies. As expected, most vaccine recipients also became seropositive for vaginal IgA; however, relative to IgG responses, the responses varied considerably and were generally weaker. The paired sample t test analysis showed that IgA antibodies peaked at day 60 in the vaccine groups and ranged from 0.07343 to 0.1946 in the 1 × 109 CFU/mL dose (mean difference: 0.1340, P = 0.0036 with 95% CI), 0.1767 to 0.6833 in the 5 × 109 CFU/mL dose (mean difference: 0.4300, P = 0.0124 with 95% CI), and 0.3532 to 0.5628 in the 1 × 1010 CFU/mL dose (mean difference: 0.4580, P = 0.0003 with 95% CI) compared with the placebo groups. In comparison with the 1 × 109 CFU/mL cohort, statistical analysis indicated that IgA antibody level after 60 days of 5 × 109 CFU/mL (ranging from 0.1371 to 0.3654, mean difference: 0.2513, P = 0.0012 with 95% CI) and 1 × 1010 CFU/mL (ranging from 0.2360 to 0.3665, mean difference: 0.3013, P < 0.0001 with 95% CI) in the vaccination groups had statistically significant fold increases, whereas IgA antibody levels for the 5 × 109 CFU/mL and 1 × 1010 CFU/mL groups were similar (P = 0.2345). The IgA ELISA levels decreased 1 (day 90) and 6 (day 240) months after the last vaccination (Fig. 2. bottom).

Secondary endpoints: cell-mediated immune responses

E7-specific IFNγ-secreting CD8+ CTL responses to the NZ8123-HPV16-optiE7 vaccine were measured in cervical lymphocytes and PBMCs after in vitro stimulation. The mean cytokine levels for each group are presented in Figs. 3, 4, and 5. The results indicated that E711-20‐, E749-57-, and E786-93-specific IFNγ-secreting CD8+ CTL responses were higher in all the active vaccination groups than they were in the placebo group at 30 days after vaccination (P = 0.0093, P = 0.0098, and P = 0.0093 in the 1 × 109 CFU/mL dose group; P = 0.0067, P = 0.0279, and P = 0.0056 in the 5 × 109 CFU/mL dose group; and P = 0.0056, P = 0.0024, and P = 0.0249 in the 1 × 1010 CFU/mL dose group, respectively) in cervical lymphocytes and (P = 0.0516, P = 0.0240, and P = 1.0000 in the 1 × 109 CFU/mL dose group; P = 0.1612, P = 0.0632, and P = 0.6042 in the 5 × 109 CFU/mL dose group; and P = 0.0349, P = 0.0004, and P = 0.8149 in the 1 × 1010 CFU/mL dose group, respectively) in PBMCs, but this was not statistically significant in all cohorts, especially in the PBMC.

Figure 3.

E711–20-specific IFNγ-secreting CD8+ CTL responses in cervical lymphocytes (1, top) and PBMCs (2, bottom) for samples from the same donor; placebo groups [cohorts 1 (A, left); 1 × 109 CFU/mL dose, cohorts 2 (B, center); 5 × 109 CFU/mL dose, and cohorts 3 (C, right); 1 × 1010 CFU/mL dose] and vaccine group [cohorts 4 (A, left); 1 × 109 CFU/mL dose, cohorts 5 (B, center); 5 × 109 CFU/mL dose, and cohorts 6 (C, right); 1 × 1010 CFU/mL dose] at different time points; before (day 0) and after immunizations (days 30, 60, 90, and 240). Each sign represents one woman. Data are reported as mean ± SD. Results are expressed as spot‐forming cells per number of cells with 95% CIs. Statistically significant differences are denoted by an asterisk between T90 and T30, T60, and T240 of vaccine groups (*, P ≤ 0.0001; **, P ≤ 0.001; ***, P ≤ 0.05; ****, P ≤ 0.1; *****, P ≤ 1).

Figure 3.

E711–20-specific IFNγ-secreting CD8+ CTL responses in cervical lymphocytes (1, top) and PBMCs (2, bottom) for samples from the same donor; placebo groups [cohorts 1 (A, left); 1 × 109 CFU/mL dose, cohorts 2 (B, center); 5 × 109 CFU/mL dose, and cohorts 3 (C, right); 1 × 1010 CFU/mL dose] and vaccine group [cohorts 4 (A, left); 1 × 109 CFU/mL dose, cohorts 5 (B, center); 5 × 109 CFU/mL dose, and cohorts 6 (C, right); 1 × 1010 CFU/mL dose] at different time points; before (day 0) and after immunizations (days 30, 60, 90, and 240). Each sign represents one woman. Data are reported as mean ± SD. Results are expressed as spot‐forming cells per number of cells with 95% CIs. Statistically significant differences are denoted by an asterisk between T90 and T30, T60, and T240 of vaccine groups (*, P ≤ 0.0001; **, P ≤ 0.001; ***, P ≤ 0.05; ****, P ≤ 0.1; *****, P ≤ 1).

Close modal
Figure 4.

E749–57-specific IFNγ-secreting CD8+ CTL responses in cervical lymphocytes (1, top) and PBMCs (2, bottom) for samples from the same donor; placebo groups [cohorts 1 (A, left); 1 × 109 CFU/mL dose, cohorts 2 (B, center); 5 × 109 CFU/mL dose, and cohorts 3 (C, right); 1 × 1010 CFU/mL dose] and vaccine group [cohorts 4 (A, left); 1 × 109 CFU/mL dose, cohorts 5 (B, center); 5 × 109 CFU/mL dose, and cohorts 6 (C, right); 1 × 1010 CFU/mL dose] at different time points; before (day 0) and after immunizations (days 30, 60, 90, and 240). Each sign represents one woman. Data are reported as mean ± SD. Results are expressed as spot‐forming cells per number of cells with 95% CIs. Statistically significant differences are denoted by an asterisk between T90 and T30, T60, and T240 of vaccine groups (*, P ≤ 0.0001; **, P ≤ 0.001; ***, P ≤ 0.05; ****, P ≤ 0.1; *****, P ≤ 1).

Figure 4.

E749–57-specific IFNγ-secreting CD8+ CTL responses in cervical lymphocytes (1, top) and PBMCs (2, bottom) for samples from the same donor; placebo groups [cohorts 1 (A, left); 1 × 109 CFU/mL dose, cohorts 2 (B, center); 5 × 109 CFU/mL dose, and cohorts 3 (C, right); 1 × 1010 CFU/mL dose] and vaccine group [cohorts 4 (A, left); 1 × 109 CFU/mL dose, cohorts 5 (B, center); 5 × 109 CFU/mL dose, and cohorts 6 (C, right); 1 × 1010 CFU/mL dose] at different time points; before (day 0) and after immunizations (days 30, 60, 90, and 240). Each sign represents one woman. Data are reported as mean ± SD. Results are expressed as spot‐forming cells per number of cells with 95% CIs. Statistically significant differences are denoted by an asterisk between T90 and T30, T60, and T240 of vaccine groups (*, P ≤ 0.0001; **, P ≤ 0.001; ***, P ≤ 0.05; ****, P ≤ 0.1; *****, P ≤ 1).

Close modal
Figure 5.

E786–93-specific IFNγ-secreting CD8+ CTL responses in cervical lymphocytes (1, top) and PBMCs (2, bottom) for samples from the same donor; placebo groups [cohorts 1 (A, left); 1 × 109 CFU/mL dose, cohorts 2 (B, center); 5 × 109 CFU/mL dose, and cohorts 3 (C, right); 1 × 1010 CFU/mL dose] and vaccine group [cohorts 4 (A, left); 1 × 109 CFU/mL dose, cohorts 5 (B, center); 5 × 109 CFU/mL dose, and cohorts 6 (C, right); 1 × 1010 CFU/mL dose] at different time points; before (day 0) and after immunizations (days 30, 60, 90, and 240). Each sign represents one woman. Data are reported as mean ± SD. Results are expressed as spot‐forming cells per number of cells with 95% CIs. Statistically significant differences are denoted by an asterisk between T90 and T30, T60, and T240 of vaccine groups (**, P ≤ 0.001; ***, P ≤ 0.05; ****, P ≤ 0.1; *****, P ≤ 1).

Figure 5.

E786–93-specific IFNγ-secreting CD8+ CTL responses in cervical lymphocytes (1, top) and PBMCs (2, bottom) for samples from the same donor; placebo groups [cohorts 1 (A, left); 1 × 109 CFU/mL dose, cohorts 2 (B, center); 5 × 109 CFU/mL dose, and cohorts 3 (C, right); 1 × 1010 CFU/mL dose] and vaccine group [cohorts 4 (A, left); 1 × 109 CFU/mL dose, cohorts 5 (B, center); 5 × 109 CFU/mL dose, and cohorts 6 (C, right); 1 × 1010 CFU/mL dose] at different time points; before (day 0) and after immunizations (days 30, 60, 90, and 240). Each sign represents one woman. Data are reported as mean ± SD. Results are expressed as spot‐forming cells per number of cells with 95% CIs. Statistically significant differences are denoted by an asterisk between T90 and T30, T60, and T240 of vaccine groups (**, P ≤ 0.001; ***, P ≤ 0.05; ****, P ≤ 0.1; *****, P ≤ 1).

Close modal

Time-to-peak response in E711-20‐, E749-57-, and E786-93-specific IFNγ-secreting CD8+ CTL responses was seen and statistically significant in cervical lymphocytes 90 days after vaccination for all active vaccine groups compared with the placebo groups, ranging from 14.0581 to 37.9419, 16.4845 to 46.3155, and 8.1081 to 13.4919 in the 1 × 109 CFU/mL dose group (mean difference: 26.0000, P = 0.0038; mean difference: 31.4000, P = 0.0043; and mean difference: 10.8000, P = 0.0004 with 95% CI, respectively); 23.5699 to 71.9301, 25.3944 to 45.6056, and 4.7455 to 34.7545 in the 5 × 1010 CFU/mL dose group (mean difference: 47.7500, P = 0.0081; mean difference: 35.5000, P = 0.0015; and mean difference: 19.7500, P = 0.0248 with 95% CI, respectively); and 28.9365 to 73.8635, 37.0894 to 61.7106, and 8.5332 to 27.0668 in the 1 × 1010 CFU/mL dose group (mean difference: 51.4000, P = 0.0031; mean difference: 49.4000, P = 0.0004; and mean difference: 17.8000, P = 0.0060 with 95% CI, respectively).

Again, no significant increase was seen in any active vaccine group (ranging from 1.5811 to 4.8189, mean difference: 3.2000, P = 0.0054 with 95% CI; ranging from 2.5169 to 6.2831, mean difference: 4.4000, P = 0.0029 with 95% CI; and ranging from −1.4831 to 2.2831, mean difference: 0.4000, P = 0.5870 with 95% CI for the 1 × 109 CFU/mL dose group; from 0.3575 to 15.1425, mean difference: 7.7500, P = 0.0445 with 95% CI; ranging from 2.7217 to 9.7783, mean difference: 6.2500, P = 0.0110 with 95% CI; ranging from 0.2478 to 4.2522, mean difference: 2.2500, P = 0.0374 with 95% CI for the 5 × 109 CFU/mL dose group; and from 2.7299 to 15.2701, mean difference: 9.0000, P = 0.0163 with 95% CI; ranging from 1.4151 to 9.3849, mean difference: 5.4000, P = 0.0197 with 95% CI; and ranging from 0.7611 to 2.8389, mean difference: 1.8000, P = 0.0086 with 95% CI for the 1 × 1010 CFU/mL dose group) compared with the placebo control recipients in the PBMCs of the same volunteers 90 days after vaccination.

The results demonstrated that oral administration of NZ8123-HPV16-optiE7 elicited a dose-dependent increase in E7-specific IFNγ-secreting T-cell responses in cervical lymphocytes and PBMC. The day-90 HPV-16 E711-20‐, E749-57-, and E786-93-specific IFNγ-secreting CD8+ CTL responses for the vaccine groups that received 5 × 109 and 1 × 1010 CFU/mL of the NZ8123-HPV-16-optiE7 vaccine were somewhat similar in cervical lymphocytes (range, −7.1801 to 12.0690, mean difference: 2.4444, P = 0.5742 with 95% CI, range −10.0095 to 11.5650, mean difference: 0.7778, P = 0.8721 with 95% CI, and range −4.5885 to 5.2552, mean difference: 0.3333, P = 0.8798 with 95% CI, respectively) and PBMCs (range, −4.1394 to 4.1394, mean difference: 0.0000, P = 1.0000 with 95% CI, range, −0.8882 to 1.9993, mean difference: 0.5556, P = 0.4008 with 95% CI, and range −0.6022 to 0.8244, mean difference: 0.1111, P = 0.7287 with 95% CI, respectively). In contrast, these responses were statistically significantly higher than the day-90 IFN‐γ secreting T-cell responses of the cervical lymphocytes (range, 10.7696 to 38.7304, mean difference: 24.7500, P = 0.0041 with 95% CI, range, 3.0583 to 30.4417, mean difference: 16.7500, P = 0.0232 with 95% CI, and range 0.8215 to 16.4285, mean difference: 8.6250, P = 0.0347 with 95% CI for 5 × 109; range, 9.4761 to 40.2739, mean difference: 24.8750, P = 0.0065 with 95% CI, range, 8.1933 to 25.5567, mean difference: 16.8750, P = 0.0025 with 95% CI, and range 1.0673 to 15.1827, mean difference: 8.1250, P = 0.0297 with 95% CI for 1 × 1010 CFU/mL cohorts, respectively) and the PBMCs (range, 2.4357 to 8.8143, mean difference: 5.6250, P = 0.0042 with 95% CI; range, 1.4303 to 5.3197, mean difference: 3.3750, P = 0.0046 with 95% CI, and range, 0.5022 to 3.2478, mean difference: 1.8750, P = 0.0145 with 95% CI for 5 × 109; and range, 3.4682 to 9.0318, mean difference: 6.2500, P = 0.0011 with 95% CI, range, 0.4522 to 4.5478, mean difference: 2.5000, P = 0.0234 with 95% CI, and range, 0.8330 to 2.9170, mean difference: 1.8750, P = 0.0038 with 95% CI for 1 × 1010 CFU/mL cohorts, respectively) in the groups that received 1 × 109 CFU/mL of vaccine.

Based on the analysis results, statistically significant increases in E711-20‐, E749-57-, and E786-93-specific IFNγ-secreting CD8+ CTL responses were seen in cervical lymphocytes compared with PBMCs 90 days after vaccination (ranging from 16.2826 to 32.4674, mean difference: 24.3750, P = 0.0002 with 95% CI, ranging from 15.5507 to 33.1993, mean difference: 24.3750, P = 0.0003 with 95% CI, and ranging from 7.4040 to 11.5960, mean difference: 9.5000, P < 0.0001 with 95% CI for the 1 × 109 CFU/mL dose; from 32.5169 to 53.0387, mean difference: 42.7778, P < 0.0001 with 95% CI, ranging from 30.1318 to 45.4238, mean difference: 37.7778, P < 0.0001 with 95% CI, and ranging from 9.7945 to 21.9833, mean difference: 15.8889, P = 0.0003 with 95% CI for the 5 × 109 CFU/mL dose; and ranging from 24.3985 to 56.2681, mean difference: 40.3333, P = 0.0004 with 95% CI, ranging from 28.7652 to 46.3459, mean difference: 37.5556, P < 0.0001 with 95% CI, and ranging from 10.2860 to 21.0473, mean difference: 15.6667, P = 0.0002 with 95% CI for the 1 × 1010 CFU/mL dose, respectively).

Surprisingly, statistically significant long-term E7-specific IFNγ-secreting CD8+ CTL responses to the NZ8123-HPV16-optiE7 vaccine were observed at 6 months after the last vaccination (day 240) in the cervical lymphocytes (P = 0.0056, P = 0.0026, and P < 0.0001 for 1 × 109 CFU/mL dose; P = 0.0118, P = 0.0001, and P = 0.0249 for the 5 × 109 CFU/mL dose; and P = 0.0038, P = 0.0005, and P = 0.0118 for the 1 × 1010 CFU/mL dose, respectively). Similarly, statistically significant long-term E7-specific IFN‐γ-secreting CD8+ CTL responses were recorded in the PBMCs except in the 1 × 109 CFU/mL dose group (P = 0.1596, P = 0.1599, and P = 0.7489 for the 1 × 109 CFU/mL dose; P = 0.0005, P = 0.0012, and P = 0.0663 for the 5 × 109 CFU/mL dose; and P = 0.0023, P = 0.0111, and P = 0.0111 for the 1 × 1010 CFU/mL dose, respectively) in comparison with the placebo groups.

Current therapeutic strategies remain expensive due to systems of manufacture, and the cold chain is necessary to preserve the potency of substances while they are in transit. Therefore, these limitations contribute to the inaccessibility of vaccine products in developing countries. To overcome these limitations, probiotic-based oral vaccines are recommended, because with them, there is no need for injection or the cold chain of vaccine transport. By storing the vaccine antigens in cytoplasm and cell walls, these microorganisms could effectively deliver antigens to the gut without degradation; therefore, they are an encouraging technology to agreement cost-effective products. Although no lactococcal-based therapeutic vaccine for HPV-16–related disease has reached clinical trial, few strategies have proven the potential of L. lactis and other strains of lactic acid bacteria as vaccine candidates, as they have been shown to provoke HPV-specific CTL responses and result in tumor regression in mice models. Bermudez-Humaran and colleagues used the nasal or oral administration to mice of L. lactis and Lactobacillus plantarum producing HPV E7 and IL12 and confirmed the stimulation of CTL activity and the prevention of tumor formation (21, 22). Poo and colleagues showed a reduction in tumor formation following oral immunization of C57BL/6 mice with Lactobacillus casei–expressing HPV-16 E7 (23). In our previous animal study, the oral administration of L. lactis harboring pNZ8123‐rE7 elicited the highest levels of HPV-16 E7‐specific antibody and specific IL2 and IFNγ in antigen-stimulated splenocytes and intestinal mucosal lymphocytes. Our preclinical data also confirmed that vaccinated C57BL/6 mice were able to generate potent protective and strong therapeutic antitumor effects against challenged murine models (12). Our preclinical data supported the results obtained in the current clinical trials phase I. Thus, the therapeutic activity of the NZ8123-HPV16-optiE7 vaccine is consistent with our findings in animal models, in which the vaccination of sexually active females with NZ8123-HPV16-optiE7 vaccine-induced strong virus-specific T-cell immunity.

To date, no therapeutic vaccine has been approved by the FDA for commercial use in the treatment of HPV-related disease. Nevertheless, previous clinical trials in humans have suggested the evolution of vaccine strategies in this regard, including the use of DNA vaccines (24), nucleic acid vaccines (25), subunit vaccines (26), minimal epitope peptides vaccines (27, 28), protein-based vaccines (29), polynucleotide-encoding papillomavirus proteins (30), dendritic cells (31), virus proteins (32), and RNA replicon–based DNA vaccines (33), which have all been shown to induce an HPV-specific immune response and the development of a cytotoxic T-cell response. Some clinical advantage was detected in the mentioned studies against HPV-16 oncoproteins E6 and E7, but a long-lasting complete response has been reported only occasionally. Based on these observances, the next generations of HPV vaccines, such as live vector vaccines (viral and bacterial vaccines), were developed.

Toso and colleagues conducted a phase I study of the intravenous administration of attenuated Salmonella typhimurium. They reported that the maximum-tolerated dose was 3 × 108 cfu/m², which was accompanied by increased serum levels of IL6, IL1, and TNF. Dose-limiting toxicity was observed in their study in patients receiving 1 × 109 cfu/m², which included a range of adverse effects such as thrombocytopenia, anemia, persistent bacteremia, hyperbilirubinemia, diarrhea, vomiting, nausea, elevated alkaline phosphatase, and hypophosphatemia (34).

In the phase I trial reported by Maciag and colleagues, the safety of Listeria monocytogenes harboring HPV-16 E7 antigen was assessed in 15 patients by intravenous infusion of vaccine (1 × 109 CFU, 3.3 × 109 CFU, or 1 × 1010 CFU). The results showed an increase in E7-specific IFNγ+ T cells in 3 patients and a reduction in tumor size in 4 patients along with flu-like symptoms as a side effect (35).

In contrast with the adverse effects recorded in the current study (only nausea and vomiting with mild to moderate intensity), a comprehensive range of side effects was reported after vaccination with Salmonella typhimurium and Listeria monocytogenes. The outcomes indicated that administration of all doses of NZ8123-HPV16-optiE7 vaccine was generally safe, well tolerated, and highly immunogenic in healthy seronegative volunteers. A smaller proportion of subjects who received the NZ8123-HPV16-optiE7 vaccine and placebo cohorts experienced adverse nausea and vomiting experiences. Of the adverse reactions reported, only nausea and vomiting were significantly more common in study participants receiving the 1 × 1010 CFU/mL vaccine dose than in participants receiving the 1 × 109 CFU/mL and 5 × 109 CFU/mL vaccine doses. Also, we found that no study participant withdrew from either study due to an adverse experience.

Moreover, in the studies of Maciag and colleagues and Toso and colleagues, specific systemic cellular immune responses were assessed, and their findings provided no insight into mucosal cellular immune responses after vaccination (34, 35).

Oral presentation of antigens by L. lactis to gut mucosa offers certain advantages over other routes such as intravenous administration (intrinsic adjuvant properties, cost effectiveness, and reduction in hypersensitivity; refs. 36, 37). Furthermore, mucosal vaccination via the oral route is an effective method for the stimulation of mucosal immunity. It has been previously reported that the administration of antigens through probiotic bacteria such as L. lactis to gut mucosa through the oral route is the greatest significant noninvasive alternative to systemic vaccination that provokes the host mucosal as well as humoral and cellular immune systems (22).

Kawana and colleagues evaluated the oral administration of a L. casei vaccine expressing a modified HPV-16 E7 oncoprotein in a phase I/IIa clinical trial in 17 patients with HPV-16–positive CIN3. They reported a significant increase in E7 cell–mediated immunity and no adverse effects experienced by their patients. They further reported that patients using 4 capsules/day showed increased E7-specific cell-mediated immune responses in cervical lymphocytes at week 9, whereas no patient demonstrated an increase in E7 cell–mediated immunity in their PBMCs (19).

Until now, we are not aware of any publication regarding the stimulation of immune responses by recombinant L. lactis to HPV-16 in humans.

To the best of our knowledge, this article is the first to report data on the effect of recombinant L. lactis expressing HPV-16 E7 oncoprotein on the immune response in 40 healthy women.

It was observed that oral administration of NZ8123-HPV16-optiE7 vaccines induced robust humoral and mucosal cell-mediated immune responses against the transforming protein E7 in the serum and cervical secretions of healthy women compared with those who received the placebo.

Data from the current study indicate that the vaccine formula containing L. lactis expressing HPV-16 E7 significantly increased serum IgG, vaginal IgA, and E7-specific IFNγ-secreting T-cell responses in cervical lymphocytes from baseline in healthy women after 4 weeks of vaccination and had a very strong trend toward increasing serum IgG and vaginal IgA after 60 days of oral immunization, and E7-specific IFNγ-secreting T-cell responses in cervical lymphocytes after 90 days of oral immunization from baseline in participants receiving the 5 × 109 CFU/mL and 1 × 1010 CFU/mL vaccine doses. Nonetheless, in similar data reported by Kawanaa and colleagues, a negligible amount of E7-specific IFNγ was produced in PBMCs after oral immunization.

HLA class I–restricted HPV-16 E7 peptides most likely to produce a cytotoxic T-cell response have been defined elsewhere (38). Bauer and colleagues and Chu and colleagues concluded that, respectively, the HPV16 E786–93 and E749–57 peptides are able to induce CTL responses, and the induced IFNγ is closely associated with the effects of Th1/CTLs (39, 40). Riemer and colleagues revealed that epitope E711–19 is highly conserved among HPV-16 strains and serves to direct cytolysis by T-cell lines (41).

In our patients, there was stimulation of CTL response to the E711–20, E749–57, and E786–93, proving that the use of well-defined minimal CTL peptide epitopes results in strong responses of CD8+ T cells.

We observed that the introduction of L. lactis expressing HPV-16 E7 oncoprotein to the gut can prime the immune system and develop potent antibody and cytokine responses. On the basis of this fact, our data displayed that NZ8123-HPV16-optiE7 vaccine had poor ability to stimulate systemic cell-mediated immune responses. This result may be due to dilution and low concentration of lymphocytes in the circulation. Although oral vaccination with the NZ8123-HPV16-optiE7 vaccine could provoke strong mucosal cell-mediated immune responses in the cervix at intestinal mucosal inductive sites, e.g., Peyer's patches, suggesting that induced mucosal effector T cells in the gut enter the peripheral circulation and finally migrate and settle in the cervical mucosa.

Consistent with a previous dose-escalation study, a dose-dependent response in the groups receiving vaccines was observed in the current study. Specifically, as determined by ELISA and ELISPOT results, no difference was seen between the recipients of the 5 × 109 CFU/mL and the 1 × 1010 CFU/mL doses, but a significantly lower response was found in recipients of the 1 × 109 CFU/mL dose. This is in accordance with our finding that, because the consumption of the 1 × 1010 CFU/mL dose was associated with increased nausea and vomiting in recipients of the NZ8123-HPV16-optiE7 vaccine, the 5 × 109 CFU/mL dose could be considered as a candidate for further study.

Regardless of the dose present in the vaccine, the results showed that the NZ8123-HPV16-optiE7 vaccine was highly immunogenic, and persistent immune responses were observed in the volunteers through 1 and 6 months after the last immunization.

The results of the current study suggest that advantage can be taken of the better immunogenicity afforded young women compared with older women by receiving the 3-dose schedule. Accordingly, vaccine-induced responses were substantially higher in 18- to 25-year-olds than those observed in 26- to 59-year-old women. Cost-effective implementation of current vaccine studies implies that immunization develops less cost-savings with the growth age of subjects up to 25 years, as is recommended by other public health organizations (refs. 42, 43; Fig. 6). Nevertheless, sexually active women above the age of 25 years (as documented in the current trial) correspondingly have the potential to benefit from vaccination and should be permitted the chance to select to be vaccinated on this schedule.

Figure 6.

Assessment of logarithm 10 E711–20‐ (1-A), E749–57- (1-B), and E786–93- (1-C) specific IFNγ-secreting CD8+ CTL in cervical lymphocytes, and serum IgG (2-A), and vaginal IgA (2-B) responding to NZ8123-HPV16-optiE7 vaccine between participants of two age arms (A: 20–25 years and B: 26–59 years). Statistically significant differences are denoted by an asterisk between subjects ages 20 to 25 years and subjects ages 26 to 59 years (**, P ≤ 0.001; ***, P ≤ 0.05; ****, P ≤ 0.1; *****, P ≤ 1).

Figure 6.

Assessment of logarithm 10 E711–20‐ (1-A), E749–57- (1-B), and E786–93- (1-C) specific IFNγ-secreting CD8+ CTL in cervical lymphocytes, and serum IgG (2-A), and vaginal IgA (2-B) responding to NZ8123-HPV16-optiE7 vaccine between participants of two age arms (A: 20–25 years and B: 26–59 years). Statistically significant differences are denoted by an asterisk between subjects ages 20 to 25 years and subjects ages 26 to 59 years (**, P ≤ 0.001; ***, P ≤ 0.05; ****, P ≤ 0.1; *****, P ≤ 1).

Close modal

The data generated in this study will be critically important in powering future studies investigating the use of NZ8123-HPV16-optiE7 as an immune agent for mucosal vaccines after solving the limitations of the current study. The pending results from follow-up at months 12, 18, 24, and 48 will also be important.

Furthermore, the first limitation of the current study was the low number of study participants, which may have resulted in insufficient statistical power to detect differences between the vaccine and placebo groups for some of the outcome variables, as the main focus of the study was the safety and immunogenicity of the HPV immunotherapeutics. The second limitation was the potential differences in the sensitivity of serological assays that test for HPV antibodies and cytokines. The key question of whether mucosal administration with the NZ8123-HPV16-optiE7 vaccine can confer protection and therapy under natural conditions must await the outcome of controlled efficacy trials. Taken together, these findings suggest that the clinical efficacy of the NZ8123-HPV16-optiE7 oral vaccine for the treatment of HPV-16–associated cervical cancer must be determined by an appropriately conducted field phase II trial.

The data presented herein strengthen the results of the previous study and reaffirm the previous conclusions on the immunogenicity, safety, and high efficacy of the HPV-16 vaccine in sexually active women.

This phase I safety study shows that the candidate HPV-16 vaccines based on L. lactis–produced E7 oncoproteins were safe, reasonably tolerated, can modulate immune response to the oral vaccination, and can induce a faster increase in serum IgG, vaginal IgA, and vaginal IFNγ concentrations in healthy volunteer women. On the basis of the safety and immunogenicity profile obtained in this study, further randomized trial phase II controlled studies including a larger number of patients with HPV-16 cervical carcinoma could be performed with the 5 × 109 CFU/mL dose of NZ8123-HPV16-optiE7 vaccines to understand the immunomodulatory effect.

No potential conflicts of interest were disclosed.

This randomized, double-blind, placebo-controlled clinical trial was registered in the Iranian website as https://www.irct.ir/trial/39227: IRCT (20190504043464N1). It was carried out in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the IUMS (Tehran, Iran).

Conception and design: S. Taghinezhad-S, H. Keyvani

Development of methodology: A.H. Mohseni, H. Keyvani

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): A.H. Mohseni

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S. Taghinezhad-S

Writing, review, and/or revision of the manuscript: S. Taghinezhad-S

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): A.H. Mohseni

Study supervision: H. Keyvani

The authors would like to thank all the volunteers and their families for their tireless cooperation in this study and the support provided by the KVSL staff involved in collecting and analyzing the blood and vaginal samples for assistance with subject monitoring. In addition, we are grateful to Narges Ghobadi for her input, her essential work in the overseeing of this clinical trial, and for making this study possible.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Gellin
B
,
Modlin
JF
,
Tamms
G
,
Barr
E
. 
Quadrivalent human papillomavirus vaccine
.
Clin Infect Dis
2007
;
45
:
609
17
.
2.
Schiffman
M
,
Doorbar
J
,
Wentzensen
N
,
De Sanjosé
S
,
Fakhry
C
,
Monk
BJ
, et al
Carcinogenic human papillomavirus infection
.
Nat Rev Dis Primers
2016
;
2
:
16086
.
3.
Taghinezhad-S
S
,
Razavilar
V
,
Keyvani
H
,
Razavi
MR
,
Nejadsattari
T
. 
Extracellular overproduction of recombinant Iranian HPV-16 E6 oncoprotein in Lactococcus lactis using the NICE system
.
Future Virol
2018
;
13
:
697
710
.
4.
Mohseni
AH
,
Taghinezhad-S
S
,
Keyvani
H
,
Razavilar
V
. 
Extracellular overproduction of E7 oncoprotein of Iranian human papillomavirus type 16 by genetically engineered Lactococcus lactis
.
BMC Biotech
2019
;
19
:
8
.
5.
Tulay
P
,
Serakinci
N
. 
The role of human papillomaviruses in cancer progression
.
J Cancer Metastasis Treat
2016
;
2
:
202
.
6.
Devaraj
K
,
Gillison
ML
,
Wu
TC
. 
Development of HPV vaccines for HPV-associated head and neck squamous cell carcinoma
.
Crit Rev Oral Biol Med
2003
;
14
:
345
62
.
7.
Stanley
M
. 
Prophylactic HPV vaccines
.
J Clin Pathol
2007
;
60
:
961
5
.
8.
Tsang
KY
,
Fantini
M
,
Fernando
RI
,
Palena
C
,
David
JM
,
Hodge
JW
, et al
Identification and characterization of enhancer agonist human cytotoxic T-cell epitopes of the human papillomavirus type 16 (HPV16) E6/E7
.
Vaccine
2017
;
35
:
2605
11
.
9.
van der Sluis
TC
,
van der Burg
SH
,
Arens
R
,
Melief
CJ
. 
New approaches in vaccine-based immunotherapy for human papillomavirus-induced cancer
.
Curr Opin Immunol
2015
;
35
:
9
14
.
10.
Taghinezhad-S
S
,
Mohseni
AH
,
Keyvani
H
,
Razavilar
V
. 
Protection against human papillomavirus type 16-induced tumors in C57BL/6 mice by mucosal vaccination with Lactococcus lactis NZ9000 expressing E6 oncoprotein
.
Microb Pathog
2019
;
126
:
149
56
.
11.
Kechagia
M
,
Basoulis
D
,
Konstantopoulou
S
,
Dimitriadi
D
,
Gyftopoulou
K
,
Skarmoutsou
N
, et al
Health benefits of probiotics: a review
.
ISRN Nutr
2013
;
2013
:
481651
.
12.
Mohseni
AH
,
Razavilar
V
,
Keyvani
H
,
Razavi
MR
,
Khavari-Nejad
RA
. 
Oral immunization with recombinant Lactococcus lactis NZ9000 expressing human papillomavirus type 16 E7 antigen and evaluation of its immune effects in female C57BL/6 mice
.
J Med Virol
2019
;
91
:
296
307
.
13.
Bermúdez-Humarán
LG
,
Kharrat
P
,
Chatel
J-M
,
Langella
P
. 
Lactococci and lactobacilli as mucosal delivery vectors for therapeutic proteins and DNA vaccines
.
Microb Cell Fact
2011
;
10
Suppl 1
:
S4
.
14.
Mohseni
AH
,
Razavilar
V
,
Keyvani
H
,
Razavi
MR
,
Khavari-Nejad
RA
. 
Efficient production and optimization of E7 oncoprotein from Iranian human papillomavirus type 16 in Lactococcus lactis using nisin-controlled gene expression (NICE) system
.
Microb Pathog
2017
;
110
:
554
60
.
15.
Taghinezhad-S
S
,
Razavilar
V
,
Keyvani
H
,
Razavi
MR
,
Nejadsattari
T
. 
Codon optimization of Iranian human papillomavirus Type 16 E6 oncogene for Lactococcus lactis subsp. cremoris MG1363
.
Future Virol
2017
;
12
:
499
511
.
16.
Mohseni
AH
,
Razavilar
V
,
Keyvani
H
,
Razavi
MR
,
Khavari Nejad
RA
. 
Codon usage optimization and construction of plasmid encoding Iranian human papillomavirus type 16 E7 oncogene for Lactococcus lactis Subsp. Cremoris MG1363
.
Asian Pac J Cancer Prev
2017
;
18
:
783
.
17.
Keyvani
H
,
Saroukalaei
ST
,
Mohseni
AH
. 
Assessment of the human cytomegalovirus UL97 gene for identification of resistance to ganciclovir in Iranian immunosuppressed patients
.
Jundishapur J Microbiol
2016
;
9
:
e31733
.
18.
Monographs
NPG
.
United States Pharmacopeia and National Formulary USP 41–NF 36
.
Rockville, MD
:
The United States Pharmacopeia Convention, Inc
.; 
2018
.
19.
Kawana
K
,
Adachi
K
,
Kojima
S
,
Taguchi
A
,
Tomio
K
,
Yamashita
A
, et al
Oral vaccination against HPV E7 for treatment of cervical intraepithelial neoplasia grade 3 (CIN3) elicits E7-specific mucosal immunity in the cervix of CIN3 patients
.
Vaccine
2014
;
32
:
6233
9
.
20.
Mohseni
AH
,
Taghinezhad-S
S
,
Keyvani
H
,
Ghobadi
N
. 
Comparison of acyclovir and multistrain Lactobacillus brevis in women with recurrent genital herpes infections: a double-blind, randomized, controlled study
.
Probiotics Antimicrob Proteins
2018
;
10
:
740
7
.
21.
Bermudez-Humaran
L
,
Langella
P
,
Cortes-Perez
N
,
Gruss
A
,
Alcocer-Gonzales
J
,
Tamez-Guerra
R
, et al
Intranasal immunization in mice with recombinant lactococci expressing the interleukin-12 and the HPV-16 E7 antigen
.
Lait
2004
;
84
:
191
206
.
22.
Bermúdez-Humarán
LG
,
Cortes-Perez
NG
,
Lefèvre
F
,
Guimarães
V
,
Rabot
S
,
Alcocer-Gonzalez
JM
, et al
A novel mucosal vaccine based on live Lactococci expressing E7 antigen and IL-12 induces systemic and mucosal immune responses and protects mice against human papillomavirus type 16-induced tumors
.
J Immunol
2005
;
175
:
7297
302
.
23.
Poo
H
,
Pyo
HM
,
Lee
TY
,
Yoon
SW
,
Lee
JS
,
Kim
CJ
, et al
Oral administration of human papillomavirus type 16 E7 displayed on Lactobacillus casei induces E7‐specific antitumor effects in C57/BL6 mice
.
Int J Cancer
2006
;
119
:
1702
9
.
24.
Klencke
B
,
Matijevic
M
,
Urban
RG
,
Lathey
JL
,
Hedley
ML
,
Berry
M
, et al
Encapsulated plasmid DNA treatment for human papillomavirus 16-associated anal dysplasia: a phase I study of ZYC101
.
Clin Cancer Res
2002
;
8
:
1028
37
.
25.
Vici
P
,
Pizzuti
L
,
Mariani
L
,
Zampa
G
,
Santini
D
,
Di Lauro
L
, et al
Targeting immune response with therapeutic vaccines in premalignant lesions and cervical cancer: hope or reality from clinical studies
.
Expert Rev Vaccines
2016
;
15
:
1327
36
.
26.
Chabeda
A
,
Yanez
RJ
,
Lamprecht
R
,
Meyers
AE
,
Rybicki
EP
,
Hitzeroth
II
. 
Therapeutic vaccines for high-risk HPV-associated diseases
.
Papillomavirus Res
2018
;
5
:
46
58
.
27.
van der Burg
SH
,
Ressing
ME
,
Kwappenberg
KM
,
de Jong
A
,
Straathof
K
,
de Jong
J
, et al
Natural T‐helper immunity against human papillomavirus type 16 (hpv16) e7–derived peptide epitopes in patients with hpv16‐positive cervical lesions: identification of 3 human leukocyte antigen class ii–restricted epitopes
.
Int J Cancer
2001
;
91
:
612
8
.
28.
Muderspach
L
,
Wilczynski
S
,
Roman
L
,
Bade
L
,
Felix
J
,
Small
L
, et al
A phase I trial of a human papillomavirus (HPV) peptide vaccine for women with high-grade cervical and vulvar intraepithelial neoplasia who are HPV 16 positive
.
Clin Cancer Res
2000
;
6
:
3406
16
.
29.
Goldstone
S
,
Thompson
J
,
Neefe
J
. 
Clinical and pathological response in a phase II trial (SGN-00101-9902) of HspE7 in high grade anal dysplasia. In: Proceedings of the 18th International Papillomavirus Conference
; 
2000
.
Jul 20–28; Barcelona, Spain. Geneva, Switzerland: International Papillomavirus Society; 2000
.
30.
Sheets
EE
,
Urban
RG
,
Crum
CP
,
Hedley
ML
,
Politch
JA
,
Gold
MA
, et al
Immunotherapy of human cervical high-grade cervical intraepithelial neoplasia with microparticle-delivered human papillomavirus 16 E7 plasmid DNA
.
Am J Obstet Gynecol
2003
;
188
:
916
26
.
31.
Santin
AD
,
Hermonat
PL
,
Ravaggi
A
,
Chiriva-Internati
M
,
Zhan
D
,
Pecorelli
S
, et al
Induction of human papillomavirus-specific CD4+ and CD8+ lymphocytes by E7-pulsed autologous dendritic cells in patients with human papillomavirus type 16-and 18-positive cervical cancer
.
J Virol
1999
;
73
:
5402
10
.
32.
Kaufmann
AM
,
Stern
PL
,
Rankin
EM
,
Sommer
H
,
Nuessler
V
,
Schneider
A
, et al
Safety and immunogenicity of TA-HPV, a recombinant vaccinia virus expressing modified human papillomavirus (HPV)-16 and HPV-18 E6 and E7 genes, in women with progressive cervical cancer
.
Clin Cancer Res
2002
;
8
:
3676
85
.
33.
Lundstrom
K
. 
Replicon RNA viral vectors as vaccines
.
Vaccines
2016
;
4
:
39
.
34.
Toso
JF
,
Gill
VJ
,
Hwu
P
,
Marincola
FM
,
Restifo
NP
,
Schwartzentruber
DJ
, et al
Phase I study of the intravenous administration of attenuated Salmonella typhimurium to patients with metastatic melanoma
.
J Clin Oncol
2002
;
20
:
142
.
35.
Maciag
PC
,
Radulovic
S
,
Rothman
J
. 
The first clinical use of a live-attenuated Listeria monocytogenes vaccine: a phase I safety study of Lm-LLO-E7 in patients with advanced carcinoma of the cervix
.
Vaccine
2009
;
27
:
3975
83
.
36.
Lei
H
,
Sheng
Z
,
Ding
Q
,
Chen
J
,
Wei
X
,
Lam
DM-K
, et al
Evaluation of oral immunization with recombinant avian influenza virus HA1 displayed on the Lactococcus lactis surface and combined with the mucosal adjuvant cholera toxin subunit B
.
Clin Vaccine Immunol
2011
;
18
:
1046
51
.
37.
Chamcha
V
,
Jones
A
,
Quigley
BR
,
Scott
JR
,
Amara
RR
. 
Oral immunization with a recombinant Lactococcus lactis-expressing HIV-1 antigen on group a Streptococcus pilus induces strong mucosal immunity in the gut
.
J Immunol
2015
;
195
:
5025
34
.
38.
Ressing
ME
,
Sette
A
,
Brandt
R
,
Ruppert
J
,
Wentworth
PA
,
Hartman
M
, et al
Human CTL epitopes encoded by human papillomavirus type 16 E6 and E7 identified through in vivo and in vitro immunogenicity studies of HLA-A* 0201-binding peptides
.
J Immunol
1995
;
154
:
5934
43
.
39.
Bauer
M
,
Wagner
H
,
Lipford
GB
. 
HPV type 16 protein E7 HLA-A2 binding peptides are immunogenic but not processed and presented
.
Immunol Lett
2000
;
71
:
55
9
.
40.
Chu
X
,
Li
Y
,
Long
Q
,
Xia
Y
,
Yao
Y
,
Sun
W
, et al
Chimeric HBcAg virus-like particles presenting a HPV 16 E7 epitope significantly suppressed tumor progression through preventive or therapeutic immunization in a TC-1-grafted mouse model
.
Int J Nanomed
2016
;
11
:
2417
.
41.
Riemer
AB
,
Keskin
DB
,
Zhang
G
,
Handley
M
,
Anderson
KS
,
Brusic
V
, et al
A conserved E7-derived cytotoxic T lymphocyte epitope expressed on human papillomavirus 16-transformed HLA-A2+ epithelial cancers
.
J Biol Chem
2010
;
285
:
29608
22
.
42.
Bonanni
P
,
Bechini
A
,
Donato
R
,
Capei
R
,
Sacco
C
,
Levi
M
, et al
Human papilloma virus vaccination: impact and recommendations across the world
.
Ther Adv Vaccines
2015
;
3
:
3
12
.
43.
Liu
YJ
,
Zhang
Q
,
Hu
SY
,
Zhao
FH
. 
Effect of vaccination age on cost-effectiveness of human papillomavirus vaccination against cervical cancer in China
.
BMC Cancer
2016
;
16
:
164
.