HPV vaccines: Global perspectives

ABSTRACT

The discovery of HPV as the etiological factor for HPV-associated malignancies and disease has opened up several opportunities for prevention and therapy. Current commercially available HPV vaccines (Gardasil, Gardasil 9, and Cervarix) are prophylactic in nature and derived from adjuvanted L1-based virus-like particles of HPV. Globally, through several clinical trials, they were found to be very safe and efficacious. Certain limitations such as cost-effectiveness, low coverage against all HPV types and a 3-dose schedule make these vaccines difficult to use worldwide. Approaches to address these issues involve alternate expression systems using L1 or alternate antigen (L2) as well as optimizing doses and broadening protection to provide cheap and cross-protective vaccines. Additionally, promising preclinical immunogenicity results from our own studies using alternative hosts such as Pichia and an antigen delivery system-based measles vector have potential for development as next generation HPV prophylactic vaccines. Several other therapeutic approaches are also ongoing.

KEYWORDS:

• alternate expression systems and affordable
• HPV
• L1
• L2
• prophylactic vaccines
• therapeutic vaccines
• virus like particles

Introduction

The discovery of HPV as the etiological factor for HPV-associated malignancies and disease has opened up several opportunities for prevention and therapy.¹ Zur Hausen won the Nobel Prize in Physiology or Medicine in 2008 for his discovery that linked HPV with cervical cancer.

HPVs are small, 50–55 nm in diameter, non-enveloped double-stranded DNA viruses that carry out their life cycle in either mucosal or cutaneous epithelia. The virus has an icosahedral capsid composed of 72 capsomeres, surrounding a circular DNA genome of approximately 7900 bp.² Hundreds of different genotypes have been identified, and each type shares less than 90% DNA sequence homology in the region of their major capsid protein, L1.

The HPV genome is divided into an early (E) region (containing genes E1, E2, E4, E5, E6, and E7), late (L) region (containing genes L1 and L2), and an upstream regulatory region. Two major viral promoters induce transcription of polycistronic mRNAs. During the early stages of the viral life cycle, early transcripts are initiated by a promoter referred to as either p97 or p105 in the HPV-16/31 or HPV-18 subtypes, respectively. The p97 promoter is located just upstream of the E6 open reading frame. The major promoter of late genes is located further downstream and varies slightly depending on the virus subtype, but it is generally referred to as p742.³ The early genes E1–E7 play a role in regulating, promoting and supporting viral DNA transcription and replication. Therefore, they are targeted for therapeutic approaches in HPV-associated oncogenesis. The late genes, L1 and L2, are transcribed only in productively infected cells and encode the major and minor capsid proteins required for assembly of progeny virions and eventual accumulation and release into the environment. Therefore, these genes are very important targets for preventive approaches in HPV-associated infections.

Currently available HPV vaccines are preventive in nature and comprised of adjuvant HPV L1 derived virus like particles of different HPV subtypes. The 3 successful HPV vaccines commercially available to date are Gardasil, Gardasil 9, and Cervarix. All 3 vaccines prevent infections with HPV types 16 and 18, 2 high-risk HPVs that cause about 70% of cervical cancers, and an even higher percentage of some of the other HPV-associated cancers.^4,5 Gardasil also prevents infection with HPV types 6 and 11, which cause 90% of genital warts.⁶ Gardasil 9 prevents infection with the same 4 HPV types plus 5 additional high-risk HPV types i.e. 31, 33, 45, 52, and 58.⁷ All vaccines were shown to be efficacious and safe after several clinical trials globally.

In addition to providing protection against the HPV types included in these vaccines, the vaccines have been found to provide partial protection against a few additional HPV types that can cause cancer, a phenomenon called cross-protection. The vaccines do not prevent other sexually transmitted diseases, nor do they treat existing HPV infections or HPV-related diseases. Therefore they do not have therapeutic potential. Although such vaccines are not very cost effective, they do not provide protection against all types of HPV. In addition, 2–3 doses are required, which limits their worldwide application.

Current status and future scope of new HPV vaccines

Based on the current challenges faced by the available HPV vaccines, there is a continuous need for their improvement, as well as the introduction of the next generation HPV vaccines. There are several ongoing studies in early preclinical and various stages of clinical trials aimed at producing new HPV vaccines with value additions (fewer doses, affordable, therapeutic, safer and providing broader protection).⁸ Few of the important approaches in practice and for future applications in HPV vaccines are described as follows.

Making affordable, safe, and effective HPV vaccines by reducing the cost of production in alternative high-yielding host systems for producing HPV L1 virus-like particles

The most prominent factor affecting current HPV vaccines for public health worldwide is their high cost. Gardasil and Cervarix vaccination costs can vary by country. In developed countries, Gardasil costs approximately $120 per dose, and Cervarix, $100 per dose.⁹ The vaccines offered in developing countries are still expensive to afford even at half price. Furthermore, HPV vaccines are not currently offered by the government in the National Immunization Program.

Therefore, multiple approaches are being tested currently using alternative expression systems to clone and express L1-based virus like particles (VLPs). The advantages with such newer HPV vaccines compared with Gardasil and Cervarix based on equivalent pseudovirus neutralizing antibody levels¹⁰ are always the lower cost of production and higher chance of success in clinical trials if comparable HPV immune responses are observed in preclinical animal studies. Alternative expression hosts such as E. coli,¹¹ Pichia,^12,13 Hansenula,¹⁴ and plants¹⁵ are being used to express VLPs in higher concentration, and are still under preclinical or clinical evaluation. Live viral and bacterial vectors such as Measles,¹⁶ adeno-associated viruses,¹⁷ and Salmonella Typhi¹⁸ have shown promising results in preclinical studies for immunogenicity. They have shown higher expression and relatively cheaper cost of production than current hosts such as the baculovirus expression system in Cervarix and Saccharomyces cerevisiae in Gardasil. Using alternative hosts also overcome patent infringement with the current vaccines and allow for more variety of new HPV vaccines.

We used Pichia pastoris as an expression host, and have shown very high expression levels for HPV16L1 and HPV18L1 VLPs of 1.4–1.8 g/L and 1.1–1.4 g/L, respectively. These values were higher than earlier reported yields.¹² Our methods could quickly be altered for high scale production. Therefore, it can be applied directly for low cost HPV vaccine production in the future, following clinical trials.

We have also compared another novel bivalent Measles vectored HPV (16 L1 and 18 L1) vaccine candidate and found a strong immune response against both Measles and HPV as measured by ELISA and neutralizing antibody response. Both vaccines have shown comparable immune responses in primates until day 586, as a standalone vaccine or as a prime boost combination. We also observed that anti-Measles antibodies do not interfere with the anti-HPV immune response in Measles vectored HPV vaccines.¹⁹ These successful preclinical results need to be confirmed in future clinical trials.

Alternative novel peptides or proteins based subunit vaccines for simpler process design, higher recovery, and easy scale up in high volumes for mass vaccination

Using subunits of VLP proteins or antigenic peptides is also one way to avoid infringement by current patents of commercial vaccines. However, it will also be very challenging to explore and prove immune mechanisms for efficient delivery of such new HPV antigens. The major advantage here is fewer production process losses and a reduced number of steps that HPV L1 VLPs require on the other side. VLPs are produced in very complex multistep processes that lead to tremendous in process yield losses, which is one reason for their high cost. Proteins are much simpler to purify with fewer losses. Furthermore, peptides are easy to synthesize at mass production scale. Using peptides or proteins as target antigens for HPV vaccine is a novel approach that has much broader future scope to ease the methods of production, cost, and scalability for mass production. Many subunit vaccines are used worldwide for influenza, Haemophilus influenzae Type B, pneumococcal, meningococcal, tetanus, typhoid, and acellular pertussis, and these vaccines are very successful with regards to safety and efficacy.

The HPV subunits used, whether L1 based epitopes, proteins or its capsomeres,^20,21 must be carefully selected to elicit a strong immune response of similar or higher magnitude to the VLPs as well as for the same or longer duration. This can be achieved by preparing formulations using different adjuvants or fusion proteins with each other, HSPs^22,23,24 or alternative delivery systems as well as optimizing their dose to generate strong immune responses.

Other than L1, immunization with proteins derived from the minor capsid protein L2 elicits broadly cross-neutralizing antibody responses.^25-30 However, L2 is poorly immunogenic since conserved regions eliciting cross-protective responses are made of linear peptides and, unlike L1, it cannot self-assemble into VLPs. Various strategies have been adapted to increase the immunogenicity of L2 neutralization epitopes, most notably amino acids (aa) 17–36.³¹ Most of these rely on presenting neutralization epitopes as repetitive, high molecular weight entities. For example, cross-protective responses have been elicited by virus-like display of L2 epitopes on bacteriophage PP7 and MS2 VLPs,^32,33 or on HPV16 L1 VLPs.^34,35 Another potent, non VLP-display approach is based on bacterially-produced, multi-type L2 fusion proteins.^36,37 Furthermore, the common L2 epitope selected from highly divergent HPV31 and HPV51 types in addition to HPV16 was fused to bacterial thioredoxin to improve cross protection. These generated similar anti-HPV16 neutralization titers and cross-reactive responses against HPV31 and HPV51 of higher magnitude and were more robust than their previous L2 fusion vaccine candidates.³⁸ This provides strong evidence for L2 as a potent antigen for cross protection against different HPV types.

Reducing or increasing the dose of vaccines

The other aspect of HPV vaccination with current HPV vaccines is reducing the number of doses from 3 to 2 or 1. The Centers for Disease Control (CDC) recommended in 2016 2 doses of HPV vaccines at least 6 months apart to 11- to 12-year-olds. The recommendation for teens and young adults ages 15–26 y was 3 doses to protect against cancer-causing HPV infections.³⁹ To reduce the number of vaccine doses, the antigenic dose in higher concentrations could be tested. Furthermore, it has been demonstrated that the expression system can have an influence on immunogenicity.^12,19 This could also be another way to reduce the number of doses.

Broadening protection by adding new HPV types

After covering major types of HPV causing cancers using Gardasil 9, still more types remain to be included in vaccine composition for full coverage against all high risk HPV infections. Gardasil was first launched covering 4 HPV types, and was followed by Gardasil 9. The question arises as to “what is next, and who can afford such vaccines.” Adding more HPV types to vaccines could solve the issue but would increase the cost of production. Researchers have determined that using alternate antigen (L2) based vaccines induces broader protection.^27-38

Therapeutic HPV vaccines

In many HPV-associated lesions that lead to cancers, the HPV viral DNA genome has been found to be integrated into the host's genome. This process often leads to the deletion of many early (E1, E2, E4, and E5) and late (L1 and L2) genes. The deletion of L1 and L2 during the integration process is what renders prophylactic vaccines useless against HPV-associated cancers. E2 is a negative regulator of the HPV oncogenes E6 and E7. The deletion of E2 during integration leads to elevated expression of E6 and E7 and is thought to contribute to the carcinogenesis of HPV-associated lesions.^1,40 Oncoproteins E6 and E7 are required for the initiation and upkeep of HPV-associated malignancies and are expressed in transformed cells.⁴¹ Furthermore, therapeutic HPV vaccines targeting E6 and E7 can circumvent the problem of immune tolerance against self-antigens because these virus encoded oncogenic proteins are foreign proteins for humans. Therefore, HPV oncoproteins E6 and E7 serve as ideal targets for therapeutic HPV vaccines.⁴² The multiple approaches used for therapeutic vaccines were recently reviewed by Yang et al.⁴³

Conclusions

Future HPV vaccines need to provide cross protection, include better production systems to reduce cost of production, therapeutic potential, early age protection, life-long protection, protection of both sexes, and the possibility of combination with other vaccines. Researchers have established new antigens and alternate delivery systems such as viral vectors, which have shown significant promise in preclinical and clinical studies. Therefore, the next generation HPV vaccines could be developed in a much shorter time than classical HPV vaccines, possibly in the near future.

Abbreviations

AA	=	Amino acids
CDC	=	Center for Disease Control
HPV	=	Human Papilloma virus
HSP	=	Heat Shock proteins
VLPs	=	Virus like particles

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.