On 2018 Feb 04, Sin Hang Lee commented:

The medical profession, including medical schools and hospitals, is now a part of the health care industry, and implementation of editorial policies of medical journals is commonly biased in favor of business interests. PubMed Commons has offered the only, albeit constrained, open forum to air dissenting research and opinions in science-based language. Discontinuation of PubMed Commons will silence any questioning of the industry-sponsored promotional publications indexed in PubMed.

On 2018 Jan 14, Sin Hang Lee commented:

The review by Mohsen and colleagues, titled “Major findings and recent advances in virus-like particle (VLP)-based vaccines”, correctly pointed out that bacterial or viral nucleic acids are probably the real active adjuvants through activation of various toll like receptors (TLRs) in the antigen-presenting cells (APCs) to boost innate immune responses in VLP-based vaccinations, as illustrated in Fig. 5 of the review article. However, in Section “HPV VLP-based vaccines” the authors merely mentioned the facts that Gardasil® is adjuvanted with amorphous aluminum hydroxyphosphate sulfate (AAHS) and that Cervarix® contains an aluminum hydroxide-based AS04 adjuvant which carries TLR4 agonist 3-0-descyl-4'-monophosphoryl lipid A (MPL)- a moiety of the cell wall lipopolysaccharide of Salmonella Minnesota. Since HPV vaccination is now being carried out in large scale, additional discussion on these two VLP-based vaccines is warranted.

In spite of the fact that VLPs are more immunogenic than the same epitopes displayed repetitively on a flexible polymer, both aforementioned VLP-based HPV vaccines still need special proprietary aluminum salt adjuvants to boost the immune responses of the host to reach sustained high levels of genotype-specific anti-HPV L1 antibodies for protective efficacy. Immunization with HPV L1 VLPs formulated with AAHS elicited a significantly stronger immune response with higher peak antibody titers both at four weeks post vaccination (12.7 to 41.9-fold higher) as well as in the persistent phase at week 52 (4.3 to 26.7-fold higher) than that with HPV L1 VLPs alone. [1] The AS04 formulation Cervarix® elicited an increased frequency (2.2-5.2-fold) of HPV L1 VLP specific memory B cells when compared with the aluminum salt only formulations. [2]

However, aluminum salts do not activate TLRs. They simply bind and carry the TLR agonists into the APCs. The addition of TLR agonists as adjuvant to a vaccine product is a logical choice to enhance innate immune response needed for effective vaccination. The TLR4 agonist MPL bound to aluminum hydroxide (AS04) is used in licensed vaccines against hepatitis B and human papilloma virus (Cervarix®). The effect of other TLR agonists, including TLR9 agonist CpG ODN which is known to enhance the antibody response in mice to hepatitis B antigen formulated with aluminum hydroxide, on the immune response to aluminum-adjuvanted vaccines has been investigated, but this has not yet led to licensed products. [3]

To function as an effective adjuvant in vaccination, a TLR agonist molecule either in the form of MPL or in the form of a nucleic acid must bind the cationic aluminum salt loosely or partially so that part of its linear molecule can make contact with the TLR [4]. If a bacterial or viral nucleic acid is used as the TLR9 agonist, the agonist molecule must be rendered resistant to nuclease attack, thus increasing its in vivo half-life [5].

Although both AS04 and AAHS are commonly labeled as special proprietary “vaccine adjuvants” and both prepared by partial ligand exchange of a phosphate group for a hydroxyl group on aluminum hydroxide, there is a big difference between these two “adjuvants” in that for AS04 the phosphate group is TLR4 agonist MPL while for AAHS the phosphate group is an inorganic anionic phosphate which is immunologically inert. When AAHS is used as an adjuvant in formulation of recombinant hepatitis B vaccine (Merck), some of the hydroxyl groups on the AAHS are exchanged for the phosphate groups of the phospholipid-containing hepatitis B surface antigen [6] that the phospholipid moiety of the viral surface antigen actually serves as TLR4 agonist [7]. However, for Gardasil® formulation with AAHS there is no disclosed TLR agonist.

In compliance with good manufacturing practice for vaccine production, the HPV VLPs have been rendered free of packaged DNA [8, 9]. However, Gardasil® does contain recombinant HPV L1-specific DNA fragments [10, 11], and at least some of these viral DNA fragments bound to the AAHS particles have assumed non-B topological conformations [12], thus rendered resistant to nuclease degradation after being transfected into APCs. These viral DNA fragments bound to AAHS in non-B conformations can serve as potent long-acting TLR9 agonist.

In theory, the major difference between Cervarix® and Gardasil® in the mechanism of stimulating immune response seems to be that Cervarix® depends on using an MPL bound to cationic aluminum to activate the TLR4 located on the surface of innate immune cells [13] while Gardasil® uses HPV L1 gene DNA fragments also bound to cationic aluminum to activate the TLR9 located in the endosome of the immune cells [13]. In general, TLR4 activation by MPL (AS04) induces cytokine cascades of both Th1 and Th2 type responses with a probably preferential bias toward induction of a Th2 phenotype while TLR9 activation favors induction of pro-inflammatory and Th1 cytokines (for example, IL-6, IL-1, TNFα, IFNγ and IL-12). [14] However, Cervarix® may contain HPV L1 DNA fragments as well, and both the DNA and MPL molecules may attach side-by-side to the same cationic aluminum; the juxtaposition of these two linear molecules may change the shape of the DNA and interfere with conventional PCR amplification for DNA detection. In addition, TLR9 activation may be effected by more than one category of agonists. Therefore, additional research on TLR activation is needed for further improvement of safety and efficacy in HPV vaccination.

References

[1] Ruiz W, et al. Kinetics and isotype profile of antibody responses in rhesus macaques induced following vaccination with HPV 6, 11, 16 and 18 L1-virus-like particles formulated with or without Merck aluminum adjuvant. J Immune Based Ther Vaccines. 2005;3:2.

[2] Giannini SL, et al. Enhanced humoral and memory B cellular immunity using HPV16/18 L1 VLP vaccine formulated with the MPL/aluminium salt combination (AS04) compared to aluminium salt only. Vaccine. 2006;24:5937-49.

[3] Hogenesch H. Mechanism of immunopotentiation and safety of aluminum adjuvants. Front Immunol. 2013;3:406.

[4] Tagliabue A, Rappuoli R. Vaccine adjuvants: the dream becomes real. Hum Vaccin. 2008;4:347-9.

[5] Aebig JA, et al. Formulation of vaccines containing CpG oligonucleotides and alum. J Immunol Methods. 2007;323:139-46.

[6] Egan PM, et al. Relationship between tightness of binding and immunogenicity in an aluminum-containing adjuvant-adsorbed hepatitis B vaccine. Vaccine. 2009;27:3175-80.

[7] Wong-Baeza C, et al. Nonbilayer Phospholipid Arrangements Are Toll-Like Receptor-2/6 and TLR-4 Agonists and Trigger Inflammation in a Mouse Model Resembling Human Lupus. J Immunol Res. 2015;2015:369462.

[8] Frazer IH. Eradicating HPV-Associated Cancer Through Immunization: A Glass Half Full…. Viral Immunol. 2018 Jan 3. doi: 10.1089/vim.2017.0119.

[9] Mach H, et al. Disassembly and reassembly of yeast-derived recombinant human papillomavirus virus-like particles (HPV VLPs). J Pharm Sci. 2006 Oct;95(10):2195-206.

[10] Lee SH. Detection of human papillomavirus (HPV) L1 gene DNA possibly bound to particulate aluminum adjuvant in the HPV vaccine Gardasil. J Inorg Biochem. 2012;117:85-92.

[11] FDA Information on Gardasil – Presence of DNA Fragments Expected, No Safety Risk. http://www.fda.gov/BiologicsBloodVaccines/Vaccines/ApprovedProducts/ucm276859.htm

[12] Lee SH. Melting profiles may affect detection of residual HPV L1 gene DNA fragments in Gardasil®. Curr Med Chem. 2014;21:932-40.

[13] O'Neill LA, et al. The history of Toll-like receptors - redefining innate immunity. Nat Rev Immunol. 2013;13:453-60.

[14] Dowling JK, Mansell A. Toll-like receptors: the swiss army knife of immunity and vaccine development. Clin Transl Immunology. 2016;5(5):e85.

Conflicts of Interest: Sin Hang Lee, MD is the director of Milford Molecular Diagnostics Laboratory specialized in developing DNA sequencing-based diagnostic tests.

On 2018 Jan 13, Sin Hang Lee commented:

None

On 2018 Jan 13, Sin Hang Lee commented:

None

On 2018 Jan 14, Sin Hang Lee commented:

The review by Mohsen and colleagues, titled “Major findings and recent advances in virus-like particle (VLP)-based vaccines”, correctly pointed out that bacterial or viral nucleic acids are probably the real active adjuvants through activation of various toll like receptors (TLRs) in the antigen-presenting cells (APCs) to boost innate immune responses in VLP-based vaccinations, as illustrated in Fig. 5 of the review article. However, in Section “HPV VLP-based vaccines” the authors merely mentioned the facts that Gardasil® is adjuvanted with amorphous aluminum hydroxyphosphate sulfate (AAHS) and that Cervarix® contains an aluminum hydroxide-based AS04 adjuvant which carries TLR4 agonist 3-0-descyl-4'-monophosphoryl lipid A (MPL)- a moiety of the cell wall lipopolysaccharide of Salmonella Minnesota. Since HPV vaccination is now being carried out in large scale, additional discussion on these two VLP-based vaccines is warranted.

In spite of the fact that VLPs are more immunogenic than the same epitopes displayed repetitively on a flexible polymer, both aforementioned VLP-based HPV vaccines still need special proprietary aluminum salt adjuvants to boost the immune responses of the host to reach sustained high levels of genotype-specific anti-HPV L1 antibodies for protective efficacy. Immunization with HPV L1 VLPs formulated with AAHS elicited a significantly stronger immune response with higher peak antibody titers both at four weeks post vaccination (12.7 to 41.9-fold higher) as well as in the persistent phase at week 52 (4.3 to 26.7-fold higher) than that with HPV L1 VLPs alone. [1] The AS04 formulation Cervarix® elicited an increased frequency (2.2-5.2-fold) of HPV L1 VLP specific memory B cells when compared with the aluminum salt only formulations. [2]

However, aluminum salts do not activate TLRs. They simply bind and carry the TLR agonists into the APCs. The addition of TLR agonists as adjuvant to a vaccine product is a logical choice to enhance innate immune response needed for effective vaccination. The TLR4 agonist MPL bound to aluminum hydroxide (AS04) is used in licensed vaccines against hepatitis B and human papilloma virus (Cervarix®). The effect of other TLR agonists, including TLR9 agonist CpG ODN which is known to enhance the antibody response in mice to hepatitis B antigen formulated with aluminum hydroxide, on the immune response to aluminum-adjuvanted vaccines has been investigated, but this has not yet led to licensed products. [3]

To function as an effective adjuvant in vaccination, a TLR agonist molecule either in the form of MPL or in the form of a nucleic acid must bind the cationic aluminum salt loosely or partially so that part of its linear molecule can make contact with the TLR [4]. If a bacterial or viral nucleic acid is used as the TLR9 agonist, the agonist molecule must be rendered resistant to nuclease attack, thus increasing its in vivo half-life [5].

Although both AS04 and AAHS are commonly labeled as special proprietary “vaccine adjuvants” and both prepared by partial ligand exchange of a phosphate group for a hydroxyl group on aluminum hydroxide, there is a big difference between these two “adjuvants” in that for AS04 the phosphate group is TLR4 agonist MPL while for AAHS the phosphate group is an inorganic anionic phosphate which is immunologically inert. When AAHS is used as an adjuvant in formulation of recombinant hepatitis B vaccine (Merck), some of the hydroxyl groups on the AAHS are exchanged for the phosphate groups of the phospholipid-containing hepatitis B surface antigen [6] that the phospholipid moiety of the viral surface antigen actually serves as TLR4 agonist [7]. However, for Gardasil® formulation with AAHS there is no disclosed TLR agonist.

In compliance with good manufacturing practice for vaccine production, the HPV VLPs have been rendered free of packaged DNA [8, 9]. However, Gardasil® does contain recombinant HPV L1-specific DNA fragments [10, 11], and at least some of these viral DNA fragments bound to the AAHS particles have assumed non-B topological conformations [12], thus rendered resistant to nuclease degradation after being transfected into APCs. These viral DNA fragments bound to AAHS in non-B conformations can serve as potent long-acting TLR9 agonist.

In theory, the major difference between Cervarix® and Gardasil® in the mechanism of stimulating immune response seems to be that Cervarix® depends on using an MPL bound to cationic aluminum to activate the TLR4 located on the surface of innate immune cells [13] while Gardasil® uses HPV L1 gene DNA fragments also bound to cationic aluminum to activate the TLR9 located in the endosome of the immune cells [13]. In general, TLR4 activation by MPL (AS04) induces cytokine cascades of both Th1 and Th2 type responses with a probably preferential bias toward induction of a Th2 phenotype while TLR9 activation favors induction of pro-inflammatory and Th1 cytokines (for example, IL-6, IL-1, TNFα, IFNγ and IL-12). [14] However, Cervarix® may contain HPV L1 DNA fragments as well, and both the DNA and MPL molecules may attach side-by-side to the same cationic aluminum; the juxtaposition of these two linear molecules may change the shape of the DNA and interfere with conventional PCR amplification for DNA detection. In addition, TLR9 activation may be effected by more than one category of agonists. Therefore, additional research on TLR activation is needed for further improvement of safety and efficacy in HPV vaccination.

References

[1] Ruiz W, et al. Kinetics and isotype profile of antibody responses in rhesus macaques induced following vaccination with HPV 6, 11, 16 and 18 L1-virus-like particles formulated with or without Merck aluminum adjuvant. J Immune Based Ther Vaccines. 2005;3:2.

[2] Giannini SL, et al. Enhanced humoral and memory B cellular immunity using HPV16/18 L1 VLP vaccine formulated with the MPL/aluminium salt combination (AS04) compared to aluminium salt only. Vaccine. 2006;24:5937-49.

[3] Hogenesch H. Mechanism of immunopotentiation and safety of aluminum adjuvants. Front Immunol. 2013;3:406.

[4] Tagliabue A, Rappuoli R. Vaccine adjuvants: the dream becomes real. Hum Vaccin. 2008;4:347-9.

[5] Aebig JA, et al. Formulation of vaccines containing CpG oligonucleotides and alum. J Immunol Methods. 2007;323:139-46.

[6] Egan PM, et al. Relationship between tightness of binding and immunogenicity in an aluminum-containing adjuvant-adsorbed hepatitis B vaccine. Vaccine. 2009;27:3175-80.

[7] Wong-Baeza C, et al. Nonbilayer Phospholipid Arrangements Are Toll-Like Receptor-2/6 and TLR-4 Agonists and Trigger Inflammation in a Mouse Model Resembling Human Lupus. J Immunol Res. 2015;2015:369462.

[8] Frazer IH. Eradicating HPV-Associated Cancer Through Immunization: A Glass Half Full…. Viral Immunol. 2018 Jan 3. doi: 10.1089/vim.2017.0119.

[9] Mach H, et al. Disassembly and reassembly of yeast-derived recombinant human papillomavirus virus-like particles (HPV VLPs). J Pharm Sci. 2006 Oct;95(10):2195-206.

[10] Lee SH. Detection of human papillomavirus (HPV) L1 gene DNA possibly bound to particulate aluminum adjuvant in the HPV vaccine Gardasil. J Inorg Biochem. 2012;117:85-92.

[11] FDA Information on Gardasil – Presence of DNA Fragments Expected, No Safety Risk. http://www.fda.gov/BiologicsBloodVaccines/Vaccines/ApprovedProducts/ucm276859.htm

[12] Lee SH. Melting profiles may affect detection of residual HPV L1 gene DNA fragments in Gardasil®. Curr Med Chem. 2014;21:932-40.

[13] O'Neill LA, et al. The history of Toll-like receptors - redefining innate immunity. Nat Rev Immunol. 2013;13:453-60.

[14] Dowling JK, Mansell A. Toll-like receptors: the swiss army knife of immunity and vaccine development. Clin Transl Immunology. 2016;5(5):e85.

Conflicts of Interest: Sin Hang Lee, MD is the director of Milford Molecular Diagnostics Laboratory specialized in developing DNA sequencing-based diagnostic tests.

On 2018 Feb 04, Sin Hang Lee commented:

The medical profession, including medical schools and hospitals, is now a part of the health care industry, and implementation of editorial policies of medical journals is commonly biased in favor of business interests. PubMed Commons has offered the only, albeit constrained, open forum to air dissenting research and opinions in science-based language. Discontinuation of PubMed Commons will silence any questioning of the industry-sponsored promotional publications indexed in PubMed.