Minor capsid protein L2 polytope induces broad protection against oncogenic and mucosal human papillomaviruses

The amino terminus of the human papillomavirus minor capsid protein L2 contains a major cross-neutralization epitope which provides the basis for the development of a broadly protecting HPV vaccine. Wide range of protection against different HPV types would eliminate one of the major drawbacks of the commercial, L1 based prophylactic vaccines. Previously, we have reported that insertion of the L2 epitope into a scaffold composed of bacterial thioredoxin protein generates a potent antigen inducing comprehensive protection against different animal and human papillomaviruses. We also reported, however, that although protection is broad, some oncogenic HPV types escape the neutralizing antibody response, if L2 epitopes from single HPV types are used as immunogen. We were able to compensate for this by applying a mix of thioredoxin proteins carrying L2 epitopes from HPV types 16, 31, and 51. As the development of a cost-efficient HPV prophylactic vaccines is one of our objectives, this approach is not feasible as it requires the development of multiple good manufacturing production processes in combination with a complex vaccine formulation. Here we report the development of a thermostable thioredoxin based single peptide vaccine carrying an L2 polytope of up to 11 different HPV types. The L2 polytope antigens have excellent abilities in respect to broadness of protection and robustness of induced immune responses. To further increase immunogenicity, we fused the thioredoxin L2 polytope antigen with a heptamerization domain. In the final vaccine design, we achieve protective responses against all 14 oncogenic HPV types we have analyzed plus the low risk HPV types 6 and 11 and a number of cutaneous HPVs.ImportanceInfections by a large number of human papillomaviruses lead to malignant and non-malignant disease. Current commercial vaccines based on virus-like particles effectively protect against some HPV types but fail to do so for most others. Further, only about a third of all countries have access to the VLP vaccines. The minor capsid protein L2 has been shown to contain so called neutralization epitopes within its N-terminus. We designed polytopes comprising the L2 epitope amino acids 20-38 of up to 11 different mucosal HPV types and inserted them into the scaffold of thioredoxin derived from a thermophile achaebacterium. The antigen induced neutralizing antibody responses in mice and guinea pigs against 26 mucosal and cutaneous HPV types. Further, addition of a heptamerization domain significantly increased the immunogenicity. The final vaccine design comprising an heptamerized L2 8mer thioredoxin single peptide antigen with excellent thermal stability might overcome some of the limitations of the current VLP vaccines.

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Broad Neutralization Responses Against Oncogenic Human Papillomaviruses Induced by a Minor Capsid L2 Polytope Genetically Incorporated Into Bacterial Ferritin Nanoparticles

Frontiers in Immunology ◽

10.3389/fimmu.2020.606569 ◽

2020 ◽

Vol 11 ◽

Author(s):

Fan Yang ◽

Filipe C. Mariz ◽

Xueer Zhao ◽

Gloria Spagnoli ◽

Simone Ottonello ◽

...

Keyword(s):

Cervical Cancer ◽

Capsid Protein ◽

Pyrococcus Furiosus ◽

Neutralizing Antibody ◽

Hpv Infection ◽

Cross Reactivity ◽

Human Papillomaviruses ◽

Promising Alternative ◽

Peptide Epitope ◽

Hpv Types

Cervical cancer remains a global health burden despite the introduction of highly effective vaccines for the prophylaxis of causative human papillomavirus infection (HPV). Current efforts to eradicate cervical cancer focus on the development of broadly protective, cost-effective approaches. HPV minor capsid protein L2 is being recognized as a promising alternative to the major capsid protein L1 because of its ability to induce responses against a wider range of different HPV types. However, a major limitation of L2 as a source of cross-neutralizing epitopes is its lower immunogenicity compared to L1 when assembled into VLPs. Various approaches have been proposed to overcome this limitation, we developed and tested ferritin-based bio-nanoparticles displaying tandemly repeated L2 epitopes from eight different HPV types grafted onto the surface of Pyrococcus furiosus thioredoxin (Pf Trx). Genetic fusion of the Pf Trx-L2(8x) module to P. furiosus ferritin (Pf Fe) did not interfere with ferritin self-assembly into an octahedral structure composed by 24 protomers. In guinea pigs and mice, the ferritin super-scaffolded, L2 antigen induced a broadly neutralizing antibody response covering 14 oncogenic and two non-oncogenic HPV types. Immune-responsiveness lasted for at least one year and the resulting antibodies also conferred protection in a cervico-vaginal mouse model of HPV infection. Given the broad organism distribution of thioredoxin and ferritin, we also verified the lack of cross-reactivity of the antibodies elicited against the scaffolds with human thioredoxin or ferritin. Altogether, the results of this study point to P. furiosus ferritin nanoparticles as a robust platform for the construction of peptide-epitope-based HPV vaccines.

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Protective Immunity to Rabbit Oral and Cutaneous Papillomaviruses by Immunization with Short Peptides of L2, the Minor Capsid Protein

Journal of Virology ◽

10.1128/jvi.76.19.9798-9805.2002 ◽

2002 ◽

Vol 76 (19) ◽

pp. 9798-9805 ◽

Cited By ~ 73

Author(s):

Monica E. Embers ◽

Lynn R. Budgeon ◽

Martin Pickel ◽

Neil D. Christensen

Keyword(s):

Capsid Protein ◽

Protective Immunity ◽

Neutralizing Antibody ◽

Distinct Region ◽

Minor Capsid Protein ◽

Cottontail Rabbit ◽

Infected Cells ◽

Hpv Types

ABSTRACT The papillomavirus minor capsid protein, L2, has been shown to exhibit immunogenicity, whereby a variety of B-cell epitopes, predominantly in the amino terminus of L2, have been deduced. However, immunity to L2 in vivo has not been examined extensively. Notably, a common neutralization epitope for human papillomavirus (HPV) types 6 and 16 was mapped to amino acids (aa) 108 to 120. The objectives of this study were to derive antisera from rabbits using the corresponding sequences from rabbit viruses and to assess the ability of these peptides to protect against infection. Synthetic peptides consisting of two overlapping sequences each in the region of aa 94 to 122 of the rabbit oral (ROPV) and cottontail rabbit (CRPV) papillomaviruses were used to immunize rabbits. Rabbits were then infected with both ROPV and CRPV and monitored for the development of oral and cutaneous papillomas, respectively. Serum derived from rabbits immunized with either of the two peptides was shown to (i) react to purified L2 from the cognate virus, (ii) specifically recognize L2 within virus-infected cells, and (iii) neutralize virus in vitro. Following viral challenge, cutaneous papilloma growth was completely absent in rabbits immunized with either CRPV peptide. Likewise, ROPV peptide-immunized rabbits were protected from oral papillomatosis. Challenge of CRPV peptide-immune rabbits with the viral genome resulted in efficient papilloma growth, suggesting a neutralizing antibody-mediated mechanism of protection. These results afford in vivo evidence for the immunogenicity provided by a distinct region of L2 and further support previous evidence for the ability of this region to elicit antiviral immunity.

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Papillomavirus Prophylactic Vaccines: Established Successes, New Approaches

Journal of Virology ◽

10.1128/jvi.01927-09 ◽

2009 ◽

Vol 84 (3) ◽

pp. 1214-1220 ◽

Cited By ~ 38

Author(s):

M. Saveria Campo ◽

Richard B. S. Roden

Keyword(s):

Animal Models ◽

Capsid Protein ◽

Human Papillomaviruses ◽

Cold Chain ◽

Minor Capsid Protein ◽

Virus Like Particles ◽

Good Level ◽

New Approaches ◽

Prophylactic Vaccines ◽

Cancer Of The Cervix

ABSTRACT Vaccines against the human papillomaviruses (HPVs) most frequently associated with cancer of the cervix are now available. These prophylactic vaccines, based on virus-like particles (VLPs), are extremely effective, providing protection from infection in almost 100% of cases. However, the vaccines present some limitations: they are effective primarily against the HPV type present in the vaccine, are expensive to produce, and need a cold chain. Vaccines based on the minor capsid protein L2 have been very successful in animal models and have been shown to provide a good level of protection against different papillomavirus types. The potential of L2-based vaccines to protect against many types of HPVs is discussed.

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Virus-like Particle-Based L2 Vaccines against HPVs: Where Are We Today?

Viruses ◽

10.3390/v12010018 ◽

2019 ◽

Vol 12 (1) ◽

pp. 18 ◽

Cited By ~ 5

Author(s):

Rashi Yadav ◽

Lukai Zhai ◽

Ebenezer Tumban

Keyword(s):

Amino Acids ◽

Capsid Protein ◽

Hpv Vaccine ◽

Genital Warts ◽

Human Papillomaviruses ◽

Target Antigen ◽

Sexually Transmitted ◽

Virus Like Particle ◽

Protective Antibodies ◽

Hpv Types

Human papillomaviruses (HPVs) are the most common sexually transmitted infections worldwide. Ninety percent of infected individuals clear the infection within two years; however, in the remaining 10% of infected individuals, the infection(s) persists and ultimately leads to cancers (anogenital cancers and head and neck cancers) and genital warts. Fortunately, three prophylactic vaccines have been approved to protect against HPV infections. The most recent HPV vaccine, Gardasil-9 (a nonavalent vaccine), protects against seven HPV types associated with ~90% of cervical cancer and against two HPV types associated with ~90% genital warts with little cross-protection against non-vaccine HPV types. The current vaccines are based on virus-like particles (VLPs) derived from the major capsid protein, L1. The L1 protein is not conserved among HPV types. The minor capsid protein, L2, on the other hand, is highly conserved among HPV types and has been an alternative target antigen, for over two decades, to develop a broadly protective HPV vaccine. The L2 protein, unlike the L1, cannot form VLPs and as such, it is less immunogenic. This review summarizes current studies aimed at developing HPV L2 vaccines by multivalently displaying L2 peptides on VLPs derived from bacteriophages and eukaryotic viruses. Recent data show that a monovalent HPV L1 VLP as well as bivalent MS2 VLPs displaying HPV L2 peptides (representing amino acids 17–36 and/or consensus amino acids 69–86) elicit robust broadly protective antibodies against diverse HPV types (6/11/16/18/26/31/33/34/35/39/43/44/45/51/52/53/56/58/59/66/68/73) associated with cancers and genital warts. Thus, VLP-based L2 vaccines look promising and may be favorable, in the near future, over current L1-based HPV vaccines and should be explored further.

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Seroprevalences of Herpes Simplex Virus Type 2, Five Oncogenic Human Papillomaviruses, and Chlamydia trachomatis in Katowice, Poland

Clinical and Vaccine Immunology ◽

10.1128/cvi.00260-07 ◽

2008 ◽

Vol 15 (4) ◽

pp. 675-680 ◽

Cited By ~ 5

Author(s):

Staffan Görander ◽

Teresa Lagergård ◽

Malgorzata Romanik ◽

Raphael P. Viscidi ◽

Gayane Martirosian ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Chlamydia Trachomatis ◽

Blood Donors ◽

Human Papillomaviruses ◽

Igg Antibodies ◽

Hpv Types ◽

Simplex Virus ◽

Oncogenic Hpv

ABSTRACT Herpes simplex virus type 2 (HSV-2), human papillomaviruses (HPVs), and Chlamydia trachomatis are the most common pathogens causing sexually transmitted infections (STIs). There is limited information about the prevalences of these STIs in Poland. Here, we estimated the occurrence of immunoglobulin G (IgG) antibodies against HSV-2, HPV, and C. trachomatis in 199 blood donors and 110 patients of both genders attending an STI clinic in Katowice in southern Poland. The seroprevalences of HSV-2 were 5% for blood donors and 14% in the STI cohort. The seroprevalences of the five potentially oncogenic HPV types 16, 18, 31, 35, and 51 were 15%, 7%, 5%, 5%, and 17%, respectively, in blood donors and 37%, 8%, 12%, 5%, and 21%, respectively, in the STI cohort. The majority of HPV-infected individuals showed antibodies against more than one type, i.e., had been infected with multiple HPV types. Anti-C. trachomatis IgG antibodies were detected in 6% of blood donors and 13% of individuals attending the STI clinic. The relatively high prevalence of HPV-51 may have implications for future vaccine programs, as the newly introduced HPV vaccines are based on the potentially oncogenic HPV types 16 and 18.

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Common Neutralization Epitope in Minor Capsid Protein L2 of Human Papillomavirus Types 16 and 6

Journal of Virology ◽

10.1128/jvi.73.7.6188-6190.1999 ◽

1999 ◽

Vol 73 (7) ◽

pp. 6188-6190 ◽

Cited By ~ 95

Author(s):

Kei Kawana ◽

Hiroyuki Yoshikawa ◽

Yuji Taketani ◽

Kunito Yoshiike ◽

Tadahito Kanda

Keyword(s):

Capsid Protein ◽

Synthetic Peptide ◽

Human Papillomaviruses ◽

Prophylactic Vaccine ◽

Vaccine Strategy ◽

Neutralization Epitope ◽

Minor Capsid Protein ◽

Neutralizing Activity ◽

Infected Monkey ◽

Cross Neutralization

ABSTRACT Studies of virus neutralization by antibody are a prerequisite for development of a prophylactic vaccine strategy against human papillomaviruses (HPVs). Using HPV16 and -6 pseudovirions capable of inducing β-galactosidase in infected monkey COS-1 cells, we examined the neutralizing activity of mouse monoclonal antibodies (MAbs) that recognize surface epitopes in HPV16 minor capsid protein L2. Two MAbs binding to a synthetic peptide with the HPV16 L2 sequence of amino acids (aa) 108 to 120 were found to inhibit pseudoinfections with HPV16 as well as HPV6. Antisera raised by immunizing BALB/c mice with the synthetic peptide had a cross-neutralizing activity similar to that of the MAb. The data indicate that HPV16 and -6 have a common cross-neutralization epitope (located within aa 108 to 120 of L2 in HPV16), suggesting that this epitope may be shared by other genital HPVs.

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Progress in L2-Based Prophylactic Vaccine Development for Protection against Diverse Human Papillomavirus Genotypes and Associated Diseases

Vaccines ◽

10.3390/vaccines8040568 ◽

2020 ◽

Vol 8 (4) ◽

pp. 568

Author(s):

Pola Olczak ◽

Richard B.S. Roden

Keyword(s):

Vaccine Development ◽

Humoral Response ◽

Neutralizing Antibody ◽

Human Papillomaviruses ◽

Benign Diseases ◽

Viral Capsids ◽

Skin Cancers ◽

Tumor Viruses ◽

Hpv Types ◽

Early Phase Clinical Trials

The human papillomaviruses (HPVs) are a family of small DNA tumor viruses including over 200 genotypes classified by phylogeny into several genera. Different genera of HPVs cause ano-genital and oropharyngeal cancers, skin cancers, as well as benign diseases including skin and genital warts. Licensed vaccines composed of L1 virus-like particles (VLPs) confer protection generally restricted to the ≤9 HPV types targeted. Here, we examine approaches aimed at broadening the protection against diverse HPV types by targeting conserved epitopes of the minor capsid protein, L2. Compared to L1 VLP, L2 is less immunogenic. However, with appropriate presentation to the immune system, L2 can elicit durable, broadly cross-neutralizing antibody responses and protection against skin and genital challenge with diverse HPV types. Such approaches to enhance the strength and breadth of the humoral response include the display of L2 peptides on VLPs or viral capsids, bacteria, thioredoxin and other platforms for multimerization. Neither L2 nor L1 vaccinations elicit a therapeutic response. However, fusion of L2 with early viral antigens has the potential to elicit both prophylactic and therapeutic immunity. This review of cross-protective HPV vaccines based on L2 is timely as several candidates have recently entered early-phase clinical trials.

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Mutation of the N-Terminal Region of Chikungunya Virus Capsid Protein: Implications for Vaccine Design

mBio ◽

10.1128/mbio.01970-16 ◽

2017 ◽

Vol 8 (1) ◽

Cited By ~ 15

Author(s):

Adam Taylor ◽

Xiang Liu ◽

Ali Zaid ◽

Lucas Y. H. Goh ◽

Jody Hobson-Peters ◽

...

Keyword(s):

Host Cell ◽

Capsid Protein ◽

Chikungunya Virus ◽

Fluorescent Protein ◽

Nuclear Translocation ◽

Vaccine Design ◽

Terminal Region ◽

Site Directed Mutagenesis ◽

Nucleolar Localization ◽

Localization Sequence

ABSTRACTMosquito-transmitted chikungunya virus (CHIKV) is an arthritogenic alphavirus of theTogaviridaefamily responsible for frequent outbreaks of arthritic disease in humans. Capsid protein, a structural protein encoded by the CHIKV RNA genome, is able to translocate to the host cell nucleolus. In encephalitic alphaviruses, nuclear translocation induces host cell transcriptional shutoff; however, the role of capsid protein nucleolar localization in arthritogenic alphaviruses remains unclear. Using recombinant enhanced green fluorescent protein (EGFP)-tagged expression constructs and CHIKV infectious clones, we describe a nucleolar localization sequence (NoLS) in the N-terminal region of capsid protein, previously uncharacterized in CHIKV. Mutation of the NoLS by site-directed mutagenesis reduced efficiency of nuclear import of CHIKV capsid protein. In the virus, mutation of the capsid protein NoLS (CHIKV-NoLS) attenuated replication in mammalian and mosquito cells, producing a small-plaque phenotype. Attenuation of CHIKV-NoLS is likely due to disruption of the viral replication cycle downstream of viral RNA synthesis. In mice, CHIKV-NoLS infection caused no disease signs compared to wild-type CHIKV (CHIKV-WT)-infected mice; lack of disease signs correlated with significantly reduced viremia and decreased expression of proinflammatory factors. Mice immunized with CHIKV-NoLS, challenged with CHIKV-WT at 30 days postimmunization, develop no disease signs and no detectable viremia. Serum from CHIKV-NoLS-immunized mice is able to efficiently neutralize CHIKV infectionin vitro. Additionally, CHIKV-NoLS-immunized mice challenged with the related alphavirus Ross River virus showed reduced early and peak viremia postchallenge, indicating a cross-protective effect. The high degree of CHIKV-NoLS attenuation may improve CHIKV antiviral and rational vaccine design.IMPORTANCECHIKV is a mosquito-borne pathogen capable of causing explosive epidemics of incapacitating joint pain affecting millions of people. After a series of major outbreaks over the last 10 years, CHIKV and its mosquito vectors have been able to expand their range extensively, now making CHIKV a human pathogen of global importance. With no licensed vaccine or antiviral therapy for the treatment of CHIKV disease, there is a growing need to understand the molecular determinants of viral pathogenesis. These studies identify a previously uncharacterized nucleolar localization sequence (NoLS) in CHIKV capsid protein, begin a functional analysis of site-directed mutants of the capsid protein NoLS, and examine the effect of the NoLS mutation on CHIKV pathogenesisin vivoand its potential to influence CHIKV vaccine design. A better understanding of the pathobiology of CHIKV disease will aid the development of effective therapeutic strategies.

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Genetic diversity of cutaneous human papillomaviruses

Journal of General Virology ◽

10.1099/vir.0.82911-0 ◽

2007 ◽

Vol 88 (10) ◽

pp. 2662-2669 ◽

Cited By ~ 50

Author(s):

Ola Forslund

Keyword(s):

Genetic Diversity ◽

Phylogenetic Analysis ◽

Human Skin ◽

Human Papillomaviruses ◽

Sequence Identity ◽

Hpv Types

Human papillomaviruses (HPVs) of the genera Betapapillomavirus and Gammapapillomavirus are common on human skin. Sequencing of subgenomic amplicons of cutaneous HPVs has revealed a large number of novel putative HPV types within these genera. Phylogenetic analysis based on these amplicons revealed 133 putative HPV types with <90 % sequence identity to any known HPV type or to each other. As there are already 34 characterized HPV types described within the genera Betapapillomavirus and Gammapapillomavirus, they appear to be the most genetically diverse of the HPVs, apparently comprising at least 167 different HPV types.

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