Safety and immunogenicity of an Escherichia coli-produced 9-valent human papillomavirus L1 virus-like particle vaccine (types 6/11/16/18/31/33/45/52/58) in healthy adults: an open-label, dose-escalation phase 1 clinical trial

Summary

Background

A safe and highly efficacious Escherichia coli (E. coli)-produced HPV 16/18 bivalent vaccine has been prequalified by the World Health Organization. Here, we conducted a single-center, open-label, dose-escalation phase 1 clinical trial to evaluate the safety and immunogenicity of the second-generation nonavalent HPV 6/11/16/18/31/33/45/52/58 vaccine.

Method

Twenty-four eligible volunteers aged 18–45 years were enrolled in January 2019 in Dongtai, China and received 0.5 mL (135 μg) or 1.0 mL (270 μg) of the candidate vaccine with a 0/1/6-month dose-escalation schedule. Local and systemic adverse events (AEs) occurring within 30 days after each vaccination and serious adverse events (SAEs) occurring within 7 months were recorded. Blood samples from each participant were collected before and 2 days after the first and third vaccinations to determine changes in laboratory parameters. Serum IgG and neutralizing antibody (nAb) levels against each HPV type at month 7 were analyzed (ClinicalTrials.gov: NCT03813940).

Findings

The incidences of total AEs in the 135 μg and 270 μg groups were 66.7% and 83.3%, respectively. All AEs were mild or moderate, and no SAEs were reported. No clinically significant changes were found in paired blood indices before or after any of the vaccinations. All the participants in the per-protocol set except for two who failed to seroconvert for HPV 11 or 58 in the 135 μg group seroconverted at month 7 for both IgG and nAbs.

Interpretation

The candidate E. coli-produced 9vHPV vaccine has been preliminarily proven to be well tolerated and immunogenic, which encourages further studies in large cohorts with a wider age range.

Funding

This study was supported by the National Natural Science Foundation of China, Fujian Provincial Natural Science Foundation, Fujian Province Health and Education Joint Research Program, Xiamen Science and Technology Plan Project, Fundamental Research Funds for the Central Universities, CAMS Innovation Fund for Medical Sciences of China, and Xiamen Innovax Biotechnology Co., Ltd.

Research in context.

Evidence before this study

We have systematically followed the scientific literature and public accessible reports from PubMed and regulatory agencies (US Food and Drug Administration, European Medicines Agency, National Medical Products Administration, and WHO) related to studies of human papillomavirus (HPV) prophylactic vaccines published between January 1, 2000 and February 6, 2023, with the terms (“Human Papillomavirus” OR “HPV”) AND “vaccine”; (“Human Papillomavirus” OR “HPV”) AND “vaccine” AND “bivalent”; (“Human Papillomavirus” OR “HPV”) AND “vaccine” AND “quadrivalent”; (“Human Papillomavirus” OR “HPV”) AND “vaccine” AND “nonavalent” without any language restrictions. We focused on RCTs for the safety, immunogenicity, and efficacy of the licensed and novel candidate HPV vaccines, and the manufacturing process of HPV virus-like particles (VLPs) vaccines. Six prophylactic HPV vaccines are currently available and marketed worldwide, including the newly marketed HPV bivalent vaccine, Walrinvax, manufactured by Yuxi Zerun (Yuxi, China), and HPV quadrivalent vaccine, Cervavax, manufactured by Serum Institute of India. Substantial evidence regarding the safety and long-term efficacy/effectiveness profile of the licensed HPV vaccines had been accumulated, except for the two new vaccines based on yeast expression systems which had no detailed clinical trial data disclosed yet. To our knowledge, no data from human clinical trials of novel HPV vaccine candidates had been reported at the time of the search. As the only HPV vaccine produced by prokaryotic expression system, Cecolin (Xiamen Innovax, China) has been widely recognized for its good safety and efficacy, thus, the safety and immunogenicity of the second-generation vaccine developed based on the same production system have received wide attention.

Added value of this study

To our knowledge, this is the first study to report clinical data for a novel prophylactic nine-valent HPV vaccine. We evaluated the safety and immunogenicity of the second-generation Escherichia coli-produced 9vHPV (types 6/11/16/18/31/33/45/52/58) vaccine candidate in a single-center, open-label, dose-escalation phase 1 clinical trial. The candidate 9vHPV vaccine has been preliminarily proven to be well tolerated and immunogenic in this study, which encourages further efficacy studies in large cohorts.

Implications of all the available evidence

In 2018, the WHO Director-General called for actions to eliminate cervical cancer. One key strategy is the implementation of massively scaled up vaccination worldwide. The scaling up of a broad-spectrum vaccine covering 9 HPV types will achieve the greatest impact on the elimination of cervical cancer. However, there is only one commercial 9vHPV vaccine available with limited supplies. Based on the same production system of Cecolin, which has been proven to be safe and highly efficacious, the second generation of a 9vHPV (types 6/11/16/18/31/33/45/52/58) vaccine candidate has been preliminarily proven to be safe and immunogenic. Considering the characteristics of inexpensive culturing, short turnaround time, and easy to mass production of the E. coli production system, it holds the promise of contributing to eliminating global cervical cancers.

Introduction

Human papillomavirus (HPV) causes almost all cervical cancers and a substantial fraction of vulva, vagina, penis, anus, and oropharynx cancers, as well as genital warts.^1,2 It is estimated that approximately 4.5% of new cancer cases are associated with high-risk HPV infection worldwide, the majority of which occur in less developed regions. Among the above HPV infection-related cancers, HPV 16 and 18 are the most predominant types, accounting for approximately 70% of cervical cancers, while HPV 31/33/45/52/58 account for approximately another 20%.^1,3 In addition, more than 90% of genital warts are associated with HPV 6 and 11 infections.⁴

Prophylactic HPV vaccination can substantially reduce the disease burden of cervical cancer and other HPV-associated diseases and is recommended as a primary preventive intervention by the World Health Organization (WHO).⁵ HPV vaccine type-related persistent infection, anogenital warts, high-grade cervical abnormalities and cervical cancer decreased considerably after the introduction of HPV vaccination in real-world settings.^6,7 Currently, six commercial prophylactic HPV vaccines are available, including three bivalent vaccines (HPV 16 and 18), two quadrivalent (HPV 6, 11, 16, and 18) vaccine (qHPV) and a nine-valent (HPV 6, 11, 16, 18, 31, 33, 45, 52, and 58) vaccine (9vHPV).^5,8,9

Five of the commercial HPV vaccines are composed of L1 virus-like particles (VLPs) produced in eukaryotic cells. The remaining HPV vaccine is an Escherichia coli (E. coli)-produced L1 VLP-based bivalent HPV 16/18 vaccine (Cecolin, Xiamen Innovax, Xiamen), which has been proven to be safe and highly efficacious against HPV 16/18-associated high-grade genital lesions and persistent infection in women and was licensed in China and received WHO prequalification in 2019 and 2021, respectively,9, 10, 11 and was thereafter licensed in Morocco, Nepal, Thailand, and Congo (DRC) in 2022. The E. coli production system is easy to scale up and cost-effective, which has great potential to alleviate supply constraints and affordability of the HPV vaccine. Consequently, based on the same production system, the second generation of HPV vaccine with an additional 7 HPV types, a 9vHPV (types 6/11/16/18/31/33/45/52/58) vaccine candidate, has been developed and demonstrated to be safe and immunogenic in preclinical studies.¹² This first-in-human phase 1 clinical trial aimed to preliminarily determine the safety and immunogenicity of the candidate E. coli-produced L1 VLP-based 9vHPV vaccine in healthy adults.

Methods

Study design and participants

This study was a single-center, open-label, dose-escalation phase 1 trial (ClinicalTrials.gov: NCT 03813940) to evaluate the safety and immunogenicity of the E. coli-produced 9vHPV vaccine in adults aged 18–45 years from January 2019 to August 2019 in Dongtai, Jiangsu, China. This study was approved by the Ethics Committee of the Jiangsu Provincial Centre for Disease Control and Prevention (JSJK2018-A023). As this is an open-label study, an independent safety monitoring committee was not set. All subjects signed written informed consent forms before enrollment. According to guidelines from the National Medical Products Administration (NMPA) of China, new vaccine candidate should be firstly tested in adults and then go down to adolescents and children. Therefore, we restricted the age of the participants to be over 18 years old in this study. Healthy adults were enrolled if they met the following inclusion criteria: aged 18–45 years; axillary temperature lower than 37.0 °C; negative urine pregnancy test (only for women); and willingness to comply with the procedure of the study. Participants were excluded if they had any preexisting severe, acute or chronic disease, history of severe anaphylaxis with vaccines or history of any HPV vaccination.

Procedures

Two groups of participants were sequentially enrolled with a sex ratio of 1:1 in each group. Participants in the 135 μg dose group (Group 1) and 270 μg dose group (Group 2) received 0.5 mL or 1.0 mL of the candidate 9vHPV vaccine intramuscularly in the deltoid muscle in the upper arm at months 0, 1 and 6, respectively. Enrollment of the Group 2 could not start unless no vaccine-related serious adverse events (SAEs) occurred within 7 days after administering the first dose of the participants in Group 1.

The test 9vHPV vaccine (Cecolin9, Xiamen Innovax, Xiamen) is the second-generation HPV vaccine of the licensed E. coli-produced HPV 16/18 bivalent vaccine. One dose of 9vHPV vaccine contained a total of 270 μg of HPV L1 VLPs, including 30 μg of HPV 6, 40 μg of HPV 11, 60 μg of HPV 16, 40 μg of HPV 18, 20 μg of HPV 31, 20 μg of HPV 33, 20 μg of HPV 45, 20 μg of HPV 52, and 20 μg of HPV 58 L1 VLPs, absorbed into 0.42 mg of aluminum hydroxide adjuvant suspended in 1.0 mL of phosphate buffered saline. All vaccines for the clinical trial were manufactured by Xiamen Innovax, Xiamen, China, under good manufacturing practices according to the requirements of NMPA.

Participants were observed for 30 min after each vaccination and were trained to record solicited local and systemic adverse events (AEs) for 7 days and unsolicited adverse events for 30 days after each vaccination in a vaccination diary. SAEs and pregnancy events were recorded throughout the study. Paired blood samples from each participant were collected for routine blood tests and biochemical tests before and 2 days after the first and third vaccinations. Laboratory indices included four types of routine blood tests: white blood cell count (WBC), red blood cells (RBCs), platelets (PLTs), and hemoglobin (HGB) and seven types of biochemical tests: alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), direct bilirubin (DBIL), indirect bilirubin (IBIL), urea nitrogen (UREA) and creatinine (CREA). AEs and abnormal laboratory indices were graded by the investigators according to the Guidelines for Adverse Event Classification Standards for Clinical Trials of Preventive Vaccines (2019) issued by the NMPA.

Serum samples were collected from all participants before the first vaccination and 1 month after the third vaccination (month 7) to evaluate HPV 6/11/16/18/31/33/45/52/58 type-specific IgG and neutralizing antibody (nAb), which were both performed at the China National Institute for Food and Drug Control (NIFDC).

Neutralizing antibodies were measured by a modified triple-color pseudovirion-based neutralization assay (PBNA), which was reported in the previous study.¹³ Samples of serially diluted serum duplicates and negative controls were added in parallel to three 384-well plates (labeled as Plates A, B and C), and then the corresponding pseudovirions were added and mixed by centrifugation. Subsequently, the 293FT cell suspension was added and cocultured for 60–96 h (37 °C). The 384-well flat-bottom plates were scanned by a high-throughput cell imaging microplate detection system. For each of the three 384-well plates, three types of HPV pseudovirions harboring different fluorescence protein-expressing genes were added (Plate A: HPV types 6, 33 and 45; Plate B: HPV types 11, 31 and 58; Plate C: HPV types 16, 18 and 52); thus, a total of 9 types of HPV-specific nAbs were measured. The neutralization titers of positive samples were calculated as the highest serum dilution with a percent infection inhibition higher than 50%. The cutoff titers of nAbs for HPV 6/11/16/18/31/33/45/52/58 were 177, 97, 85, 100, 77, 112, 148, 84 and 125, respectively.

IgG antibodies were detected by E. coli-produced HPV VLP-based enzyme-linked immunosorbent assay (ELISA). For the VLP-based ELISA, samples of serially diluted serum, serially diluted reference serum and negative controls were added to 96-well microtiter plates coated with HPV types 6, 11, 16, 18, 31, 33, 45, 52 or 58 VLPs and then incubated (45 min, 37 °C), and the plates were washed before adding horseradish peroxidase-conjugated goat anti-human IgG. After another round of incubation (45 min, 37 °C) and washing, tetramethylbenzidine solution was added and further incubated for 15 min at 37 °C. Finally, the reactions were stopped with the addition of H₂SO₄ solution, and the optical density (OD) was read at 450/620 nm. The reference standard curve was generated from the serially diluted reference serum pool from HPV vaccine recipients. The positive samples for HPV 16 or 18 were further quantified using references traceable to the WHO international standards for antibodies against HPV 16 (NIBSC code 05/134) or HPV 18 (NIBSC code 10/140), expressed in international units (IUs). Because the international reference standard for HPV 6/11/31/33/45/52/58 antibodies is not yet available, the quantification of HPV 6/33/45/52/58 IgG antibodies used the NIFDC national standards for antibodies against HPV 6 (NIFDC code 220027), HPV 33 (code 220023), HPV 45 (code 220024), HPV 52 (code 220025) and HPV 58 (code 220026), expressed in U/mL, while the reference standard for HPV 11 and 31 were from Xiamen Innovax, expressed in YU/mL. The cutoffs for HPV 6/11/16/18/31/33/45/52/58 IgG antibody were 8.1 U/mL, 6.4 YU/mL, 3.0 IU/mL, 2.1 IU/mL, 281 YU/mL, 6.6 U/mL, 171 U/mL, 94 U/mL, and 78.5 U/mL, respectively. The antibody concentration of seronegative samples was artificially set as half of the cutoff value.

Outcomes

The primary outcomes included solicited local or general symptoms within seven days after each vaccination; unsolicited adverse events within 30 days after each vaccination; and, SAEs throughout the entire study period.

The secondary outcome included the changes in routine blood tests and biochemical tests before and two days after vaccination. Exploratory outcome pre-specified in the protocol included seroconversion rate and level of HPV 6, 11, 16, 18, 31, 33, 45, 52, and 58 type-specific neutralizing antibodies (nAbs) and IgG antibodies at month 7.

Statistical analysis

The primary objective of this study was to evaluate the safety and tolerability of the candidate 9vHPV vaccine in adults aged 18–45 years. All participants who received at least one vaccination were included in the safety analyses. All AEs were summarized as frequencies and percentages by dose groups. McNemar's test was used to compare the changes in laboratory indices before and 2 days after vaccination.

Immunogenicity analyses were mainly based on the per-protocol set (PPS), which included participants who received three doses of vaccine, had no major deviation of the protocol, were seronegative for corresponding IgG antibody or nAb against the relevant types of HPV at Day 0, and had antibody results at month 7. The Intention-to-treat (ITT) set included participants who received at least one vaccination, had antibody results at Day 0 and month 7, was also analyzed for immunogenicity. Seroconversion was defined as at least a four fold increase in antibody titers or concentrations over baseline, and the geometric mean of fold increase of the antibody titers/concentrations was defined as the geometric mean increase (GMI). The geometric mean concentration (GMC) for IgG and geometric mean titer (GMT) for nAb, as well as GMI for IgG and nAb, with 95% confidential intervals (CIs) were calculated based on Student's t distribution of the log-transformed values. All statistical analyses were performed by SAS 9.4.

Role of the funding source

The study sponsor (Xiamen Innovax) had no role in the data analysis, data interpretation, or writing of the report. The corresponding authors and the core writing team had full access to all data, as well as the final responsibility to submit for publication.

Results

Characteristics of study participants

A total of 27 adults aged 18–45 years old were sequentially recruited, among which one refused to participate and two were excluded due to abnormal platelet levels. The first participant in Group 2 was enrolled in the absence of vaccine-related SAEs occurred within 7 days after administering the first dose of the participants in Group 1. Finally, 24 healthy volunteers were enrolled in this study, with the first 12 participants in the low-dose group (135 μg, 0.5 mL) and the second 12 participants in the high-dose group (270 μg, 1.0 mL) (Fig. 1). The ratio of males to females was 1:1, and the number of participants in the 18–25, 26–35 and 36–45 age groups was also the same in each group. The mean age (SD) of the two groups was similar at 30.4 (6.9) years and 30.8 (6.5) years, respectively. The majority of participants were seronegative for neutralizing and IgG antibodies at baseline (Table 1).

Fig. 1.

Trial profile. This dose-escalation phase 1 study was carried out in two stages. Enrollment of the Group 2 could not start unless no vaccine-related serious adverse events (SAEs) occurred within 7 days after administering the first dose of the participants in Group 1. All the participants received three doses of the corresponding vaccine according to the protocol.

Table 1.

Baseline demographic characteristics of the participants.

Characteristics	Group 1 (135 μg)	Group 2 (270 μg)	Total
	n (%)	n (%)	n (%)
N	12	12	24
Sex
Male	6 (50.0)	6 (50.0)	12 (50.0)
Female	6 (50.0)	6 (50.0)	12 (50.0)
Age group
18-25	4 (33.3)	4 (33.3)	4 (33.3)
26-35	4 (33.3)	4 (33.3)	4 (33.3)
36-45	4 (33.3)	4 (33.3)	4 (33.3)
Mean ± SD	30.4 ± 6.9	30.8 ± 6.5	30.6 ± 6.6
Neutralizing antibody seropositive
HPV 6	0 (0.0)	0 (0.0)	0 (0.0)
HPV 11	0 (0.0)	0 (0.0)	0 (0.0)
HPV 16	1 (8.3)	0 (0.0)	1 (4.2)
HPV 18	1 (8.3)	0 (0.0)	1 (4.2)
HPV 31	0 (0.0)	0 (0.0)	0 (0.0)
HPV 33	0 (0.0)	1 (8.3)	1 (4.2)
HPV 45	0 (0.0)	0 (0.0)	0 (0.0)
HPV 52	0 (0.0)	0 (0.0)	0 (0.0)
HPV 58	1 (8.3)	2 (16.7)	3 (12.5)
IgG antibody seropositive
HPV 6	0 (0.0)	0 (0.0)	0 (0.0)
HPV 11	0 (0.0)	0 (0.0)	0 (0.0)
HPV 16	2 (16.7)	3 (25.0)	5 (20.8)
HPV 18	4 (33.3)	3 (25.0)	7 (29.2)
HPV 31	0 (0.0)	0 (0.0)	0 (0.0)
HPV 33	1 (8.3)	0 (0.0)	1 (4.2)
HPV 45	0 (0.0)	0 (0.0)	0 (0.0)
HPV 52	1 (8.3)	1 (8.3)	2 (8.3)
HPV 58	1 (8.3)	1 (8.3)	2 (8.3)

Safety and tolerability

All the subjects received 3 doses of the vaccine in the prespecified time window and completed the final follow-up visit at month 7. Although with no significant difference (P = 0.64), slightly higher incidence of AEs was observed in the high-dose group, with eight (66.7%, 23 events) and ten participants (83.3%, 31 events) reporting at least one AE in the 135 μg group and 270 μg group, respectively. All the reported AEs were mild or moderate, and no serious adverse events were reported during the 7-month follow-up (Table 2).

Table 2.

Adverse events after vaccination.

	Group 1 (135 μg)		Group 2 (270 μg)
	Number of participants (%)	Number of events	Number of participants (%)	Number of events
Total adverse events	8 (66.7)	23	10 (83.3)	31
Adverse reactionsa	7 (58.3)	17	9 (75.0)	24
Local adverse reactions	6 (50.0)	8	7 (58.3)	16
Systemic adverse reactions	4 (33.3)	9	4 (33.3)	8
Vaccine-unrelated eventsb	4 (33.3)	6	6 (50.0)	7
Adverse events ≥ grade 3b	0 (0.0)	0	0 (0.0)	0
Serious adverse eventsc	0 (0.0)	0	0 (0.0)	0
Discontinuation due to AE	0 (0.0)	0	0 (0.0)	0

^aAny vaccine-related adverse events.

^bOccurred during days 1–30 following any vaccination.

^cOccurred thoughout the study (months 0–7). AE: adverse event; SAE: serious adverse event.

The incidences of injection-site AEs did not differ significantly between the two HPV vaccine dose groups (P = 1.00). The most common (incidence>10%) local reaction was injection site pain in the 135 μg group (5/12, 41.7%) and 270 μg group (7/12, 58.3%). The most common systemic reactions were fatigue (2/12, 16.7%) and cough (2/12, 16.7%) among subjects in the 135 μg group and fever (3/12, 25.0%) and fatigue (2/12, 16.7%) in the 270 μg group (Fig. 2). All vaccine-unrelated events that occurred within 30 days following each vaccination are listed in Supplementary Table S1.

Fig. 2.

The incidences of adverse reactions.

Laboratory parameters

A total of 11 hematological parameters were analyzed for changes before and 2 days after the first and third vaccinations, resulting in a total of 264 pairs of results in each group. Three pairs (1.1%) and five pairs of indices shifted from normal to abnormal ranges after vaccination in the 135 μg and 270 μg groups, respectively (Table 3). There was also 1 pair of indices that shifted from abnormal to normal after vaccination in both groups. All the abnormal indices post-vaccination were mild or moderate without clinical significance as determined by the clinician (Supplementary Table S2).

Table 3.

Laboratory parameters evaluated pre- and post-vaccination.

Post-vaccination	Group 1 (135 μg)			Group 2 (270 μg)
	Pre-vaccination		P value by McNemar Test	Pre-vaccination		P value by McNemar Test
	Normal	Abnormal		Normal	Abnormal
Any indicesa
Normal	258	1	0.63	256	1	0.13
Abnormal	3	2		6	1
Blood routine indices
Normal	94	0	NA	92	1	1.0
Abnormal	2	0		2	1
Serum biochemical indices
Normal	164	1	1.0	164	0	NA
Abnormal	1	2		4	0

^aLaboratory indices included four types of routine blood tests: white blood cell count (WBC), red blood cells (RBC), platelets (PLT), hemoglobin (HGB), and seven types of biochemical tests: alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), direct bilirubin (DBIL), indirect bilirubin (IBIL), urea nitrogen (UREA) and creatinine (CREA).

Immunogenicity

The seroconversion rates and the GMTs of nAbs and GMCs of IgG for HPV types 6/11/16/18/31/33/45/52/58 at one month after the whole vaccination course (month 7) in the PPS cohorts are shown in Supplementary Table S3. In the 270 μg group, all participants in the PPS achieved seroconversion of both nAbs and IgG antibodies to all nine vaccine HPV types. In the 135 μg group, one participant in the PPS cohort failed to seroconvert for nAbs of HPV 58 (seroconversion rate 90.9%) and two failed to seroconvert for nAbs and IgG of HPV 11 (seroconversion rates were 83.8% for both nAbs and IgG). In addition, the GMT of nAbs for HPV 6 was significantly lower in the 135 μg group than in the 270 μg group (P = 0.0315), and the GMC of IgG for HPV 58 was significantly lower in the 135 μg group than in the 270 μg group (P = 0.0473). The distributions of nAbs and IgG are plotted in Fig. 3A and B. The GMIs of the nAbs and IgG of the participants who were seropositive at baseline as well as the ITT cohorts were shown in Supplementary Tables S4 and S5.

Fig. 3.

Levels of neutralizing and IgG antibodies in the per-protocol set (PPS). A. Neutralizing antibodies. B. IgG antibodies. The dotted lines indicate the cutoff values for relevant HPV types. nAb: neutralizing antibody. ID50: half maximal inhibitory concentration. The PPS included participants who received three doses of vaccine, had no major deviation of the protocol, and were seronegative for corresponding IgG antibody or nAb against the relevant types of HPV at Day 0.

Discussion

This first-in-human, open-label, dose-escalation phase 1 clinical trial preliminarily indicated that the candidate E. coli-produced 9vHPV vaccine is well tolerated and immunogenic in healthy males and females aged 18–45 years old. All the reported AEs were mild or moderate, and no SAE occurred during the trial. The incidences of adverse reactions between the two dose groups were similar, whereas the immune responses were higher in the 270 μg group, suggesting that the 270 μg dose may be a better choice for further immunogenicity and efficacy studies of this vaccine.

Although the incidence of AEs was slightly higher in the 270 μg dose group, all AEs were mild or moderate, and no serious adverse events were reported in either group. The incidence of AEs was 83.3% in the 270 μg dose group, which was similar to that of the commercial 9vHPV vaccine (93.9%).¹⁴ The most common adverse reactions were injection site pain, fever and fatigue in the 270 μg dose group. Due to the increased antigen content of the 9vHPV vaccine, more attention was focused on the injection site symptoms. However, no participants reported injection site swelling or erythema in either dose group. The laboratory results also proved the safety of the candidate vaccine. All the changes in paired blood indices were mild and without clinical significance.

Robust immunogenicity was observed after three vaccination doses. All participants in the 270 μg dose group seroconverted and developed high levels of nAbs and IgG antibodies to all vaccine types. In the 135 μg group, a small number of participants failed to seroconvert for nAbs or IgG against HPV 11 and HPV 58, and the levels of nAbs for HPV 6 and IgG antibody for HPV 58 were lower than in the 270 μg group. These data indicate that with similarly good safety, 270 μg might be a better choice for further evaluation. On the other hand, due to the different tests for antibodies, the antibody levels in this study cannot be directly compared to those induced by the commercial 9vHPV vaccine, which warrants further head-to-head studies to directly compare the immunogenicity of the two 9vHPV vaccines.

E. coli has been shown to be an efficient and versatile tool for producing recombinant proteins. Except for Cecolin, another E. coli-produced recombinant vaccine, the hepatitis E vaccine (trade name Hecolin, Xiamen Innovax, Xiamen), which has also been proven to have good safety and extremely high efficacy for at least 4.5 years, was licensed in 2011 in China and 2020 in Pakistan.^15,16 The efficacy of Cecolin against high-grade genital lesions and persistent infection associated with HPV 16 and 18 was 100.0% (95% CI: 67.2–100.0) and 97.3% (95% CI: 89.9–99.7), respectively.¹⁰ Containing the same HPV type 16 and 18 L1 VLP antigens with increased doses (20 μg more for both HPV types) as Cecolin, the candidate HPV 9-valent vaccine was supplemented with 7 additional types of E. coli-produced HPV L1 VLPs and has the potential to prevent more than 90% of high-grade cervical lesions. A double-blind, randomized and Cecolin-controlled phase 3 study of Cecolin9 of 270 μg dose is currently ongoing, in which non-inferiority of anti-HPV types 16 and 18 antibodies induced by Cecolin9 compared with Cecolin has been set as one of the primary endpoints.

In 2018, the WHO Director-General called for all countries to take action to eliminate cervical cancer. One key strategy is the implementation of massively scaled up vaccination worldwide.¹⁷ The scaling up of a broad-spectrum 9vHPV vaccine will achieve the greatest impact on the elimination of cervical cancer.¹⁸ However, there is only one commercial 9vHPV vaccine available on the market, and the supplies are currently very limited. The success of this candidate E. coli-produced 9vHPV vaccine will effectively alleviate the current 9vHPV vaccine supply shortage.

Our study has some limitations. One limitation is the small sample size. However, this sample size was in compliance with Chinese regulatory requirements for phase 1 clinical trials. Safety and immunogenicity will be further evaluated in larger cohorts. The second limitation is that due to the lack of an international standard for quantifying antibodies against HPV 6/11/31/33/45/52/58, the measured antibody levels of these types could not directly compare with those of antibodies induced by other similar products. Another head-to-head immunogenicity non-inferiority study directly compared with Gardasil 9 is currently ongoing. In addition, it's regretful that the immunogenicity of 2-dose schedule was not parallelly explored in this study. WHO has recently updated its recommendation to 2-dose schedule in adults,⁵ although most countries such as China and the United States have not officially approved the schedule with reduced doses in the adult women yet, 2-dose schedule in adults deserves to be studied in future to confer more evidence for policy making.

In conclusion, the new candidate E. coli-produced 9vHPV vaccine has been preliminarily proven to be well tolerated and immunogenic in a phase 1 clinical study, which encourages further safety, immunogenicity and efficacy studies of this vaccine in large cohorts with a wider age range.

Contributors

J Z, T W, S-J H, H-R P, Y-M H, F-C Z, and N-S X contributed to the study design. K C, C-L Y, H-M J, X Z, D-L L, H-X P, Z-F B, Y-F L, Q-F Z, and G S contributed to data collection, or interpretation; W-J H, L Z, Q C, F-Z Z contributed to antibody detection. Z-F B, Y H, Y-Y S, S-J H, T W, and J Z were the core team for data analysis and manuscript preparation. H-R P, Y-M H, F-C Z, S-J H, T W, J Z and N-S X were responsible for supervision of the study and decision to submit for publication. All authors critically reviewed the manuscript and approved the final version.

Data sharing statement

We will share individual participant data that underlie the results reported in this article beginning from 6 months post the major findings from the final analysis of the study were published, ending 2 years later. Proposals should be directed to wuting@xmu.edu.cn. To gain access, data requestors will need to sign a data access agreement.

Declaration of interests

F-Z Z (Feng-Zhu Zheng), and Q-FZ (Qiu-Fen Zhang) report being either current employees of Xiamen Innovax; G S (Guang-Sun) and H-R P (Hui-Rong Pan) report being current employees of and have stock options in Xiamen Innovax. No other potential conflicts of interest relevant to this article were reported.