A Preclinical Immunogenicity Study of the Recombinant Human Papillomavirus Nine-Valent Virus-like Particle Vaccine

Abstract

Background: Cervical cancer is associated with persistent infection of high-risk human papillomaviruses (HPVs). Prophylactic HPV vaccines have been recommended and have significant efficacy in preventing cervical cancer. Multivalent HPV vaccines have a better preventative effect on HPV-related diseases. However, there is currently only one nine-valent HPV vaccine on the market: Gardasil^® 9. The development of new HPV vaccines is still urgent in order to achieve the goal of eliminating cervical cancer as proposed by the WHO.

Methods: In this study, we developed a nine-valent recombinant HPV virus-like particle (VLP) vaccine (HPV-9 vaccine) containing HPV type 6, 11, 16, 18, 31, 33, 45, 52, and 58 antigens, with an adjuvant of aluminum phosphate (AlPO₄). The type-specific L1 proteins were recombinantly expressed using Pichia pastoris, followed by self-assembly into VLPs. Immunogenicity studies of the HPV-9 vaccine were performed using rodents (mice and rats) and non-human primates (macaques) as animal models.

Results: Immunogenicity studies showed that the HPV-9 vaccine is able to elicit a robust and long-lasting neutralizing antibody response in rodents (mice and rats) and non-human primates (cynomolgus macaque) models. The HPV-9 vaccine shows immunogenicity comparable to that of Walrinvax^® and Gardasil^® 9.

Conclusions: In summary, this study provides a comprehensive investigation of the immunogenicity of the HPV-9 vaccine, including its immune persistence. These findings, derived from using models of diverse animal species, contribute valuable insights into the potential efficacy of the vaccine candidate in clinical settings.

Keywords: human papillomavirus (HPV); immunogenicity; protective efficacy; vaccines; virus-like particles (VLPs).

Figure 1

Characterization of recombinant expressed HPV L1 proteins. (A) Recombinant expressed HPV L1 proteins of each type were purified and subjected to reduced SDS-PAGE, arrowheads indicated the major band of HPV L1s. (B) Purified HPV L1 proteins were self-assembled into VLPs, and the nano-particle size was detected through using the Zetasizer Nano instrument. (C) Self-assembled VLPs of all nine types were characterized through transmission electron microscopy (TEM).

Figure 1

Characterization of recombinant expressed HPV L1 proteins. (A) Recombinant expressed HPV L1 proteins of each type were purified and subjected to reduced SDS-PAGE, arrowheads indicated the major band of HPV L1s. (B) Purified HPV L1 proteins were self-assembled into VLPs, and the nano-particle size was detected through using the Zetasizer Nano instrument. (C) Self-assembled VLPs of all nine types were characterized through transmission electron microscopy (TEM).

Figure 2

Immunogenicity study of the nine monovalent AlPO₄-adsorbed HPV VLPs. (A) Scheme of mice-based immunization routine. Briefly, female BALB/c mice were randomly divided into groups (with 10 mice/group), followed by intraperitoneal injection of 0.002 μg, 0.02 μg, or 0.2 μg monovalent AlPO₄-adsorbed VLPs twice at a 2-week interval, with injection of the adjuvant AlPO₄ alone as the placebo. Serum samples were collected for detection of binding antibody titers and neutralizing antibody titers. (B) Binding antibody titers were detected through ELISAs using HPV VLPs of each type as antigens, serum from immunized mice as the 1st antibody with series dilutions, and diluted HRP-conjugated Goat anti-Mouse (H+L) IgG as the 2nd antibody. p-value less than 0.05 was considered statistically significant (p ≤ 0.05 (*), ≤0.01 (**), ≤0.001 (***), and <0.0001 (****)). (C) Detection of neutralizing antibody titers was conducted based on the HPV pseudovirus of each type we prepared. p-value less than 0.05 was considered statistically significant (* p ≤ 0.05 (*), ≤0.01 (**), ≤0.001 (***), and <0.0001 (****)).

Figure 2

Immunogenicity study of the nine monovalent AlPO₄-adsorbed HPV VLPs. (A) Scheme of mice-based immunization routine. Briefly, female BALB/c mice were randomly divided into groups (with 10 mice/group), followed by intraperitoneal injection of 0.002 μg, 0.02 μg, or 0.2 μg monovalent AlPO₄-adsorbed VLPs twice at a 2-week interval, with injection of the adjuvant AlPO₄ alone as the placebo. Serum samples were collected for detection of binding antibody titers and neutralizing antibody titers. (B) Binding antibody titers were detected through ELISAs using HPV VLPs of each type as antigens, serum from immunized mice as the 1st antibody with series dilutions, and diluted HRP-conjugated Goat anti-Mouse (H+L) IgG as the 2nd antibody. p-value less than 0.05 was considered statistically significant (p ≤ 0.05 (*), ≤0.01 (**), ≤0.001 (***), and <0.0001 (****)). (C) Detection of neutralizing antibody titers was conducted based on the HPV pseudovirus of each type we prepared. p-value less than 0.05 was considered statistically significant (* p ≤ 0.05 (*), ≤0.01 (**), ≤0.001 (***), and <0.0001 (****)).

Figure 3

The immunogenicity and immune persistence of the HPV-9 vaccine in BALB/c mice. (A) As the immunization routine scheme shows, female BALB/c mice were randomly divided into groups, with 10 mice per group. The mice were intraperitoneally injected with a 1/100 clinical dose of Gardasil^® 9 and HPV-9 vaccine or Walrinvax^® or the adjuvant as the placebo at weeks 0, 2, and 6; neutralizing assays were conducted at weeks 2, 4, 8, 12, 20, 24 and 28. (B) Two weeks after the 3rd vaccination, the neutralizing antibody titers tested through neutralizing experiments demonstrated that the HPV-9 vaccine’s effectiveness is comparable to that of Gardasil^® 9 and Walrinvax^® (p ≤ 0.01 (**), ≤0.001 (***), and <0.0001 (****)). (C) To evaluate the long-term protection induced by the HPV-9 vaccine in comparison with Gardasil^® 9 or Walrinvax^®, neutralizing assays were conducted to test the antibody-specific neutralizing antibodies in serum from vaccinated mice. The arrowheads represent the immunization timepoints.

Figure 4

The immunogenicity and immune persistence of the HPV-9 vaccine in SD rats. (A) As the immunization routine scheme shows, female SD rats were randomly divided into groups (5 rats per group) and then intramuscularly injected with vaccines, namely the HPV-9 vaccine, Walrinvax^®, and Gardasil^® 9, at 1/10, 1/1000, or 1/10,000 of the clinical dose, respectively, at weeks 0, 2, and 6. Rats injected with the AlPO₄ adjuvant alone were used as the placebo control. Neutralizing assays of serum samples were conducted at weeks 2, 4, 8, 12, 20, 24, and 28. (B) Two weeks after the 3rd vaccination, the neutralizing antibody levels tested through neutralizing experiments showed that all vaccines induced dose-dependent immunogenicity, which was comparable among the vaccines (p ≤ 0.05 (*), ≤0.01 (**), and ≤0.001 (***)). (C) To evaluate the long-term protective effect induced by the HPV-9 vaccine compared to Gardasil^® 9 or Walrinvax^®, neutralizing assays were conducted to test the antibody-specific neutralizing antibodies in serum from vaccinated rats. Durable immune protection was induced with 1/10 of the clinical dose. The arrowheads represent the vaccination timepoints.

Figure 4

The immunogenicity and immune persistence of the HPV-9 vaccine in SD rats. (A) As the immunization routine scheme shows, female SD rats were randomly divided into groups (5 rats per group) and then intramuscularly injected with vaccines, namely the HPV-9 vaccine, Walrinvax^®, and Gardasil^® 9, at 1/10, 1/1000, or 1/10,000 of the clinical dose, respectively, at weeks 0, 2, and 6. Rats injected with the AlPO₄ adjuvant alone were used as the placebo control. Neutralizing assays of serum samples were conducted at weeks 2, 4, 8, 12, 20, 24, and 28. (B) Two weeks after the 3rd vaccination, the neutralizing antibody levels tested through neutralizing experiments showed that all vaccines induced dose-dependent immunogenicity, which was comparable among the vaccines (p ≤ 0.05 (*), ≤0.01 (**), and ≤0.001 (***)). (C) To evaluate the long-term protective effect induced by the HPV-9 vaccine compared to Gardasil^® 9 or Walrinvax^®, neutralizing assays were conducted to test the antibody-specific neutralizing antibodies in serum from vaccinated rats. Durable immune protection was induced with 1/10 of the clinical dose. The arrowheads represent the vaccination timepoints.

Figure 5

Comparing the immunogenicity of the HPV-9 vaccine with that of Gardasil^® 9 in macaques. (A) Macaques were randomly divided into groups (3 females and 3 males in each group), as the immunization routine scheme shows. Serum samples were collected as the control, followed by intramuscular injection of 1 clinical dose of the HPV-9 vaccine or Gardasil at weeks 0, 8, and 24 and the collection of blood at weeks 4, 12, and 26. (B) After the 2nd or 3rd vaccination, neutralizing antibody titers were detected at weeks 12 and 26, which indicated that the immunogenicity of the HPV-9 vaccine is comparative to that of Gardasil^® 9 (p > 0.05 (ns), and ≤0.001 (***)). (C) Trend regarding neutralizing antibodies at each timepoint compared with Gardasil^® 9. The arrowheads represent the vaccination timepoints.

Figure 5

Comparing the immunogenicity of the HPV-9 vaccine with that of Gardasil^® 9 in macaques. (A) Macaques were randomly divided into groups (3 females and 3 males in each group), as the immunization routine scheme shows. Serum samples were collected as the control, followed by intramuscular injection of 1 clinical dose of the HPV-9 vaccine or Gardasil at weeks 0, 8, and 24 and the collection of blood at weeks 4, 12, and 26. (B) After the 2nd or 3rd vaccination, neutralizing antibody titers were detected at weeks 12 and 26, which indicated that the immunogenicity of the HPV-9 vaccine is comparative to that of Gardasil^® 9 (p > 0.05 (ns), and ≤0.001 (***)). (C) Trend regarding neutralizing antibodies at each timepoint compared with Gardasil^® 9. The arrowheads represent the vaccination timepoints.