THERAPEUTIC vaccines for HPV (human papillomavirus)

HPV, short for "human papillomavirus," is responsible for several cancer types. Examples include cervical cancer, as well as anal cancers and head and neck cancers. In previous articles, we discussed what HPV is, how it spreads and the mechanisms it uses to hide from our immune system. In this article, we will discuss therapeutic (treatment) vaccines used to treat HPV.

Note: In general, there are 2 types of vaccine: Preventive and therapeutic vaccines. When vaccines are mentioned, we all think of preventive vaccines (childhood vaccines, coronavirus vaccines and HPV vaccines such as Gardasil). Therapeutic vaccines, however, are a new concept: examples include Lmlygic for melanoma and Provenge for prostate cancer.

What is a therapeutic HPV vaccine? 

Therapeutic HPV vaccines aim to develop cell-mediated immunity against HPV-infected cells to eliminate precursor lesions and malignant (cancerous) tumors. They therefore target HPV antigens. Vaccine approaches vary:

1. Live-vector-based vaccines,
2. Peptide-protein-based vaccines,
3. Nucleic-acid-based vaccines, and
4. Cell-based vaccines.

What is an antigen?

An antigen is a protein substance that, on entering the body or undergoing a change within it (for example, in cancer cells), is recognized as foreign by the immune system and leads to antibody formation. Antigens are mostly distinctive components of microorganisms such as viruses, bacteria and parasites.

HPV biology: understanding how vaccines work

• HPV is a nonenveloped virus with a double-stranded, circular DNA molecule.
• A member of the Papillomaviridae family, HPV encodes 6 early proteins (E1, E2, E4, E5, E6, E7) and 2 late proteins (L1, L2).
• Early proteins facilitate viral DNA replication and produce cellular proteins, while late proteins form the viral capsid structure that encloses new viruses.
• E6 and E7, among the genes encoding these proteins, are more highly expressed in virus-infected cells. As a result, E6 and E7 proteins increase.
• E6 suppresses the p53 protein; E7 suppresses the RB gene, disrupting the normal cell cycle and enabling infected cells to become cancerous.
• The E2 gene also suppresses E6 and E7. In infected cells, E2 decreases, increasing E6 and E7 protein synthesis.
• Vaccines seek to treat HPV by acting on these genes in various ways.

Types of therapeutic HPV vaccine

1. Live-vector-based vaccines

These vaccines use viruses or bacteria as carriers. Vector-based vaccines rely on antigen presentation to dendritic cells. Many live-vector vaccines are highly immunogenic because they replicate rapidly within host cells, allowing antigens to spread between cells. However, factors such as potential toxicity can limit their use. Live-vector vaccines can be considered in 2 classes:

1. Bacterial vectors
2. Viral vectors

Bacterial vectors

Attenuated bacteria can be used to present antigens to dendritic cells. L.monocytogenes, Salmonella, Lactococcus lactis are examples of bacteria used. Bacterial-vector vaccines have been shown to produce a strong E7-specific T-cell mediated immune response.

Viral vectors

Viral vectors are also highly immunogenic. Various types, such as vaccinia virus, adenovirus and adeno-associated virus, may be used in vaccines (the Oxford-AstraZeneca vaccine developed against coronavirus also uses adenovirus as a viral vector).

Among these viruses, vaccinia is thought to hold promise for antigen-specific immunotherapy and to be more effective against infections. A recombinant vaccinia virus called MVA-E2 can synthesize the E2 protein. A vaccine using this vector has been tested in patients with CIN (cervical intraepithelial neoplasia) and condyloma lesions. Studies showed that patients developed antibodies to the MVA-E2 vaccine and that a specific T-lymphocyte response occurred in cancerous cells.

The progression from HPV infection to cancer is illustrated below:

2. Peptide / protein-based vaccines

Peptide-based vaccines

Peptides derived from HPV antigens can also be used in vaccination against HPV. The mechanism is as follows: when antigenic HPV proteins enter the body, dendritic cells take them up and present them through MHC-1- or MHC-2-associated pathways on the HLA molecule (human leukocyte antigen), producing an immune response. These vaccines are generally safe and easy to manufacture, but their use remains limited. They are MHC-specific, and the existence of many HLA types makes developing a vaccine effective in every patient difficult. They also provide low levels of immunity. Many studies therefore focus on supportive molecules such as chemokines and cytokines to increase their potential.

For example, in a Phase 1/2 human study testing a lipidated HPV-E7 peptide-based vaccine, 3 of the 18 vaccinated patients had dysplasia disappear, while lesions regressed markedly in 6 patients.

Protein-based vaccines

Like peptide-based vaccines, protein-based vaccines are safe and easy to produce. Unlike peptide vaccines, they are not MHC-specific. Protein antigens can be processed and presented on dendritic-cell surfaces and contain every possible HLA epitope. Nevertheless, they have low immunogenic activity, so supportive molecules are used to enhance their potential. Another limitation is that, after vaccination, antigen-presenting cells (APCs) sometimes encounter and engulf the injected proteins for MHC 1 presentation, inactivating them. Examples of protein-based vaccine studies include:

• The TA-CIN vaccine, consisting of a fusion of HPV-16 L2, E6 and E7, induced antibody production in all women who received it and was shown to be safe.
• Another study investigated a vaccine combining HPV-16 E7 with the M.bovis hsp16 protein. The vaccine was shown to be well tolerated in all patients with high-grade anal intraepithelial neoplasia (AIN).

3. Nucleic-acid-based vaccines

There are 2 types of nucleic-acid-based vaccine:

1. Vaccines using DNA molecules and
2. RNA replicon vaccines.

We will discuss these vaccine types in detail.

DNA-based vaccines

DNA vaccines have potential efficacy in antigen-specific immunotherapy strategies. Naked DNA, not accompanied by cells or proteins, is safe, stable and easy to produce.

Compared with other vaccines, DNA vaccines:

• Provide longer-lasting antigen expression than RNA or protein vaccines.
• Do not induce neutralizing antibody production, unlike live-vector vaccines, allowing repeated vaccination of the same patient.

Although DNA vaccines are safer than other vaccines, some concerns remain. For example, DNA could integrate into the host genome and disrupt it, although no data support this. Another concern is weak immunogenic activity, because the vaccine DNA cannot spread and replicate from HPV-infected cells into surrounding cells. Strategies have therefore been developed to increase DNA vaccine efficacy.

The first strategy is to increase the number of antigen-expressing dendritic cells. One method used to achieve this is the “gene gun” technique, which is highly potent. A “gene gun” fires DNA-coated gold particles, transfecting dendritic cells that migrate to lymphoid organs to mature into T cells (transfection means introducing a piece of DNA into a cell from outside). Naked DNA thus enters the dendritic cells. Another way to increase dendritic cells is to “promote the spread of encoded antigen between dendritic cells.” This requires linking the antigen to a protein capable of transport between cells.

Another strategy is to increase antigen expression, processing and presentation in dendritic cells. This can be achieved through a technique called "codon optimization," which replaces rarely used codons with common ones, changing the antigen gene sequence and increasing translation of the DNA vaccine in dendritic cells. (Translation is polypeptide synthesis under mRNA control.) Linking the antigen to a protein targeting proteasomal degradation or the endoplasmic reticulum also increases cytotoxic T-lymphocyte (CTL) responses through increased MHC-1 antigen presentation. For example, a vaccine containing E7 linked to calreticulin (CRT), a protein that enhances MHC-1 presentation, was shown to increase E-7-specific CD8 T-cell responses and be effective against E7-expressing tumors.

Another technique is the "MHC-1 single-chain trimer (SCT) technique." It bypasses antigen processing and provides MHC-1 presentation of the peptide encoded by the DNA vaccine.

The final strategy used to enhance DNA vaccine efficacy is to increase interaction between dendritic cells and T cells. After an antigen is processed and presented, interactions between dendritic cells and T cells are crucial for T-cell activation. Following priming, dendritic cells become targets for activated effector T cells. To prevent this T-cell-mediated apoptosis in dendritic cells, DNA can encode antiapoptotic proteins that prolong dendritic-cell survival. A mouse study showed that a vaccine combining E7-encoding DNA with DNA encoding antiapoptotic proteins such as Bcl-2 increased E7-specific T-cell responses. DNA vaccines also enhance antigen presentation by prolonging dendritic-cell lifespan.

RNA replicon vaccines

The genomic unit in which replication takes place is called a replicon. RNA replicons are naked RNA molecules capable of replication in transfected cells. They can be derived from alphaviruses or Semliki forest viruses. RNA-replicon-based vectors have several advantages for cancer vaccine development. They can replicate in many cell types and provide continuous antigen presentation, making them more immunogenic than conventional DNA vaccines. Many replicons contain no viral genes, so no infectious particles are produced and immune responses to the vector are limited. RNA replicon vaccines can therefore be reused in the same individuals. They also reduce the chromosomal-integration risk associated with DNA vaccines, but are less stable. An alternative combining the advantages of RNA replicon molecules and DNA vaccines is the DNA-launched RNA replicon, called “suisidal DNA.” Suicidal DNA has been used in preclinical HPV vaccine development. In transfected cells, suicidal DNA turned into RNA and enabled effective tumor-antigen expression.

Another replicon strategy is the “Kunjin (KUN)” method, using the flavivirus Kunjin to distribute replicons. A study of mice vaccinated with KUN replicons expressing HPV-16 E7 showed stimulation of specific T-cell responses and protection from tumor transformation. Although RNA replicon molecules have many advantages, clinical studies and available data on RNA-replicon-based vaccines remain limited, so more studies need examination.

4. Whole-cell-based vaccines

Dendritic-cell-based vaccines

Using dendritic cells in HPV vaccines is another reasonable strategy. These cells must be loaded with viral antigen to make such a vaccine. Dendritic cells can be prepared ex vivo (outside the living body) by physically transferring MHC-1 and 2 molecules. Antigen loading can use peptide or protein antigens or transfer of HPV-antigen-encoding DNA / RNA. Although effective vaccines have been developed using autologous dendritic cells, producing individualized vaccines is difficult. The culture technique used to prepare dendritic cells also affects vaccine quality.

Several methods have been proposed to improve dendritic-cell-based vaccine efficacy, including prolonging dendritic-cell survival as discussed above. For example, one study tested E7-loaded dendritic cells infected with small interfering RNA (siRNA) targeting proapoptotic proteins such as Bak and Bax. The study showed suppression of Bak and Bax in dendritic cells, increasing resistance to T-cell-mediated apoptosis.

Clinical trials of dendritic-cell-based vaccines are ongoing. In one trial, a patient with metastatic cervical cancer received subcutaneous injections of dendritic cells loaded with HPV-18 E7. Tumor progression was subsequently shown to be suppressed. Although complete remission (regression) was not achieved, the patient’s health improved and no side effects were reported.

Tumor-cell-based vaccines

The 2nd approach to whole-cell-based vaccine development uses tumor cells. Their immunogenic effects can be enhanced by stimulating them ex vivo (outside the living body) to produce immunomodulatory proteins such as cytokines. Cytokine genes such as IL-2, IL-12 and GM-CSF are used in these vaccines. One advantage is that tumor antigens cannot be clearly identified and they are easy to produce.

Tumor-cell-based vaccines are being tested in preclinical models. For example, vaccination using GM-CSF-producing E7-positive tumor cells in mice was shown to induce E7-specific T-cell responses and enhance antitumor activity.

However, these vaccines also carry a risk of causing new cancers, which limits their development.