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A Pan-HPV Vaccine Based on Bacteriophage PP7 VLPs Displaying Broadly Cross-Neutralizing Epitopes from the HPV Minor Capsid Protein, L2

Background Current human papillomavirus (HPV) vaccines that are based on virus-like particles (VLPs) of the major capsid protein L1 largely elicit HPV type-specific antibody responses. In contrast, immunization with the HPV minor capsid protein L2 elicits antibodies that are broadly cross-neutralizing, suggesting that a vaccine targeting L2 could provide more comprehensive protection against infection by diverse HPV types. However, L2-based immunogens typically elicit much lower neutralizing antibody titers than L1 VLPs. We previously showed that a conserved broadly neutralizing epitope near the N-terminus of L2 is highly immunogenic when displayed on the surface of VLPs derived from the bacteriophage PP7. Here, we report the development of a panel of PP7 VLP-based vaccines targeting L2 that protect mice from infection with carcinogenic and non-carcinogenic HPV types that infect the genital tract and skin. Methodology/Principal Findings L2 peptides from eight different HPV types were displayed on the surface of PP7 bacteriophage VLPs. These recombinant L2 VLPs, both individually and in combination, elicited high-titer anti-L2 IgG serum antibodies. Immunized mice were protected from high dose infection with HPV pseudovirus (PsV) encapsidating a luciferase reporter. Mice immunized with 16L2 PP7 VLPs or 18L2 PP7 VLPs were nearly completely protected from both PsV16 and PsV18 challenge. Mice immunized with the mixture of eight L2 VLPs were strongly protected from genital challenge with PsVs representing eight diverse HPV types and cutaneous challenge with HPV5 PsV. Conclusion/Significance VLP-display of a cross-neutralizing HPV L2 epitope is an effective approach for inducing high-titer protective neutralizing antibodies and is capable of offering protection from a spectrum of HPVs associated with cervical cancer as well as genital and cutaneous warts.

VLPs Displaying a Single L2 Epitope Induce Broadly Cross-Neutralizing Antibodies against Human Papillomavirus

Background Virus-like Particles (VLPs) display can be used to increase the immunogenicity of heterologous antigens. Here, we report the use of a bacteriophage MS2-based VLP display platform to develop a monovalent vaccine targeting a broadly neutralizing epitope in the minor capsid protein human papillomavirus (HPV) that provides broad protection from diverse HPV types in a mouse pseudovirus infection model. Methodology/Principal Findings Peptides spanning a previously described cross-neutralizing epitope from HPV type 16 were genetically inserted at the N-terminus of MS2 bacteriophage coat protein. Three of the four recombinant L2-coat proteins assembled into VLPs. L2-VLPs elicited high-titer anti-L2 antibodies in mice, similar to recombinant VLPs that we had previously made in which the L2 peptide was displayed on a surface-exposed loop on VLPs of a related bacteriophage, PP7. Somewhat surprisingly, L2-MS2 VLPs elicited antibodies that were much more broadly cross-reactive with L2 peptides from diverse HPV isolates than L2-PP7 VLPs. Similarly, mice immunized with L2-MS2 VLPs were protected from genital and cutaneous infection by highly diverse HPV pseudovirus types. Conclusion/Significance We show that peptides can be displayed in a highly immunogenic fashion at the N-terminus of MS2 coat protein VLPs. A VLP-based vaccine targeting HPV L2 elicits broadly cross-reactive and cross-protective antibodies to heterologous HPV types. L2-VLPs could serve as the basis of a broadly protective second generation HPV vaccine.

A Universal Virus-Like Particle-based Vaccine for Human Papillomavirus: Longevity of Protection and Role of Endogenous and Exogenous Adjuvants

Antibodies targeting epitopes within the amino terminus of the minor capsid protein L2 of human papillomavirus (HPV) are broadly neutralizing against diverse HPV isolates. We have constructed bacteriophage virus-like particle (VLP)-based vaccines that display short L2 peptides and elicit high-titer and broadly protective antibody responses. Here, we further characterize two additional features of these VLP-based vaccines; the longevity of protection and the role of endogenous and exogenous adjuvants on the magnitude and characteristics of the antibody response. We show that vaccinated mice have long-lived antibody responses against L2, persisting over 18 months after vaccination. Vaccinated mice were strongly protected against infection by diverse HPV pseudoviruses over a year after immunization. We also show that exogenous and endogenous adjuvants (LPS and encapsidated single-stranded RNA) have minor effects on antibody titers. Immunization with VLPs containing encapsidated ssRNA predominantly shifts the response to a Th1, rather than a Th2-like response. Importantly, immunization with L2-VLPs (without endogenous and exogenous adjuvants) in the presence of alum hydroxide elicited a robust antibody response.

Preclinical refinements of a broadly protective VLP-based HPV vaccine targeting the minor capsid protein, L2

An ideal prophylactic human papillomavirus (HPV) vaccine would provide broadly protective and long-lasting immune responses against all high-risk HPV types, would be effective after a single dose, and would be formulated in such a manner to allow for long-term storage without the necessity for refrigeration. We have developed candidate HPV vaccines consisting of bacteriophage virus-like particles (VLPs) that display a broadly neutralizing epitope derived from the HPV16 minor capsid protein, L2. Immunization with 16L2 VLPs elicited high titer and broadly cross-reactive and cross-neutralizing antibodies against diverse HPV types. In this study we introduce two refinements for our candidate vaccines, with an eye towards enhancing efficacy and clinical applicability in the developing world. First, we assessed the role of antigen dose and boosting on immunogenicity. Mice immunized with 16L2-MS2 VLPs at doses ranging from 2 to 25 μg with or without alum were highly immunogenic at all doses; alum appeared to have an adjuvant effect at the lowest dose. Although boosting enhanced antibody titers, even a single immunization could elicit strong and long-lasting antibody responses. We also developed a method to enhance vaccine stability. Using a spray dry apparatus and a combination of sugars & an amino acid as protein stabilizers, we generated dry powder vaccine formulations of our L2 VLPs. Spray drying of our L2 VLPs did not affect the integrity or immunogenicity of VLPs upon reconstitution. Spray dried VLPs were stable at room temperature and at 37 °C for over one month and the VLPs were highly immunogenic. Taken together, these enhancements are designed to facilitate implementation of a next-generation VLP-based HPV vaccine which addresses U.S. and global disparities in vaccine affordability and access in rural/remote populations.

Gardasil-9: A global survey of projected efficacy.

Human papillomaviruses (HPVs) are the causative agents of human neoplasias such as warts and cancers. There are ∼19 HPV types associated with cancers, which has made it very challenging for first generation HPV vaccines to offer complete protection against all cancer-causing HPV types. Recently, a second generation HPV vaccine, Gardasil-9, has been approved to protect against more HPV types. Worldwide, Gardasil-9 will protect against HPV types associated with ∼90% of cervical cancer case in women and 80-95% of other HPV-associated anogenital cancers in both men and women. However, due to variation in HPV-type specific prevalence and distribution, the vaccine will offer different percentages of protection in different geographical regions; Gardasil-9 will offer protection against HPV types associated with ∼87.7% of cervical cancers in Asia, 91.7% in Africa, 92% in North America, 90.9% in Europe, 89.5% in Latin America & the Caribbean, and 86.5% in Australia. Because of this, Pap smear screening and testing for HPV types not included in Gardasil-9 will need to continue, especially in HIV/AIDS patients. In order to achieve complete protection against all HPV types that cause cervical cancer, a third-generation HPV vaccine is needed.

Second-generation prophylactic HPV vaccines: successes and challenges

The role of HPV as the causative factor in cervical cancer has led to the development of the HPV vaccines Gardasil and Cervarix. These vaccines effectively protect against two HPV types associated with 70% of cervical cancer cases. Despite this success, researchers continue to develop second-generation HPV vaccines to protect against more HPV types and allow increased uptake in developing countries. While a reformulated vaccine based on the current technology is currently in clinical trials, another strategy consists of targeting highly conserved epitopes in the minor capsid protein of HPV, L2. Vaccines targeting L2 induce broadly neutralizing antibodies, capable of blocking infection by a wide range of HPV types. Several vaccine designs have been developed to optimize the display of L2 epitopes to the immune system and to reduce the cost of manufacture and distribution. L2-based vaccines show considerable promise as a potential next-generation HPV vaccine.

Optimized Formulation of a Thermostable Spray-Dried Virus-Like Particle Vaccine against Human Papillomavirus

Existing vaccines against human papillomavirus (HPV) require continuous cold-chain storage. Previously, we developed a bacteriophage virus-like particle (VLP)-based vaccine for HPV infection, which elicits broadly neutralizing antibodies against diverse HPV types. Here, we formulated these VLPs into a thermostable dry powder using a multicomponent excipient system and by optimizing the spray-drying parameters using a half-factorial design approach. Dry-powder VLPs were stable after spray drying and after long-term storage at elevated temperatures. Immunization of mice with a single dose of reconstituted dry-powder VLPs that were stored at 37 °C for more than a year elicited high anti-L2 IgG antibody titers. Spray-dried thermostable, broadly protective L2 bacteriophage VLPs vaccine could be accessible to remote regions of the world (where ∼84% of cervical cancer patients reside) by eliminating the cold-chain requirement during transportation and storage.

Immunization with a consensus epitope from Human Papillomavirus L2 induces antibodies that are broadly neutralizing

Vaccines targeting conserved epitopes in the HPV minor capsid protein, L2, can elicit antibodies that can protect against a broad spectrum of HPV types that are associated with cervical cancer and other HPV malignancies. Thus, L2 vaccines have been explored as alternatives to the current HPV vaccines, which are largely type-specific. In this study we assessed the immunogenicity of peptides spanning the N-terminal domain of L2 linked to the surface of a highly immunogenic bacteriophage virus-like particle (VLP) platform. Although all of the HPV16 L2 peptide-displaying VLPs elicited high-titer anti-peptide antibody responses, only a subset of the immunogens elicited antibody responses that were strongly protective from HPV16 pseudovirus (PsV) infection in a mouse genital challenge model. One of these peptides, mapping to HPV16 L2 amino acids 65-85, strongly neutralized HPV16 PsV but showed little ability to cross-neutralize other high-risk HPV types. In an attempt to broaden the protection generated through vaccination with this peptide, we immunized mice with VLPs displaying a peptide that represented a consensus sequence from high-risk and other HPV types. Vaccinated mice produced antibodies with broad, high-titer neutralizing activity against all of the HPV types that we tested. Therefore, immunization with virus-like particles displaying a consensus HPV sequence is an effective method to broaden neutralizing antibody responses against a type-specific epitope.

The use of hybrid virus-like particles to enhance the immunogenicity of a broadly protective HPV vaccine

Virus‐like particles (VLPs) can serve as a highly immunogenic vaccine platform for the multivalent display of epitopes from pathogens. We have used bacteriophage VLPs to develop vaccines that target a highly conserved epitope from the human papillomavirus (HPV) minor capsid protein, L2.VLPs displaying an L2‐peptide from HPV16 elicit antibodies that broadly neutralize infection by HPV types associated with the development of cervical cancer. To broaden the cross‐neutralization further, we have developed a strategy to display two different peptides on a single, hybrid VLP in a multivalent, highly immunogenic fashion. In general, hybrid VLPs elicited high‐titer antibody responses against both targets, although in one case we observed an immunodominant response against only one of the displayed epitopes. Immunization with hybrid particles elicited antibodies that were able to neutralize heterologous HPV types at higher titers than those elicited by particles displaying one epitope alone, indicating that the hybrid VLP approach may be an effective technique to target epitopes that undergo antigenic variation. Biotechnol. Bioeng. 2014;111: 2398–2406.

Aerosol delivery of virus-like particles to the genital tract induces local and systemic antibody responses

The induction of mucosal immune responses in the genital tract may be important for increasing the effectiveness of vaccines for sexually transmitted infections (STIs). In this study, we asked whether direct immunization of the mouse genital tract with a non-replicating virus-like particle (VLP)-based vaccine could induce local mucosal as well as systemic antibody responses. Using VLPs derived from two bacteriophages, Qβ and PP7, and from a mammalian virus that normally infects the genital tract, human papillomavirus (HPV), we show that intravaginal aerosol administration of VLPs can induce high titer IgG and IgA antibodies in the female genital tract as well as IgG in the sera. Using a mouse model for HPV infection, we show that intravaginal immunization with either HPV type 16 VLPs or with PP7 bacteriophage VLPs displaying a peptide derived from the HPV minor capsid protein L2 could protect mice from genital infection with an HPV16 pseudovirus. These results provide a general method for inducing genital mucosal and systemic antibody responses using VLP-based immunogens.