Human Papillomavirus Pseudovirions Research Articles

Expression of human papillomavirus genotype 52 pseudovirus in HEK-293T cells

Human papillomavirus (HPV) is the predominant cause of cervical cancer globally. HPV genotype 52 is categorized as a high-risk type and holds the highest prevalence in Indonesia (23.2 %). The HPV pseudovirus can be used to test the immunogenicity of HPV vaccine candidates and evaluate the neutralization efficiency of the antibodies against the virus. The aim of this study was to express the HPV52 L1 and L2 capsid protein into pseudovirion containing the GFP reporter plasmid (pfwB) in HEK-293T cells. HPV52 L1 and L2 coding plasmid (pVITRO52) along with pfwB were amplified in E. coli. The plasmids were extracted and co-transfected using polyethyleneimine (PEI)-based transfection method into the HEK-293T cells. The expression of HPV52 pseudoviruses were confirmed by fluorescence microscopy and western blot method. Co-transfection of HPV52 L1 and L2 coding plasmid (pVITRO52) along with pfWB into the HEK-293T cells was successfully carried out. The co–transfected HEK-293T cells showed fluorescence. The western blot assay using HPV52 L1 protein primary antibody showed a band around ∼55 kDa. In the future, the results of this study will be used to evaluate the immunogenicity and neutralization assay of HPV vaccine candidates.

Alum and a TLR7 agonist combined with built-in TLR4 and 5 agonists synergistically enhance immune responses against HPV RG1 epitope

To relieve the limitations of the human papillomavirus (HPV) vaccines based on L1 capsid protein, vaccine formulations based on RG1 epitope of HPV L2 using various built-in adjuvants are under study. Herein, we describe design and construction of a rejoined peptide (RP) harboring HPV16 RG1 epitope fused to TLR4/5 agonists and a tetanus toxoid epitope, which were linked by the (GGGS)3 linker in tandem. In silico analyses indicated the proper physicochemical, immunogenic and safety profile of the RP. Docking analyses on predicted 3D model suggested the effective interaction of TLR4/5 agonists within RP with their corresponding TLRs. Expressing the 1206 bp RP-coding DNA in E. coli produced a 46 kDa protein, and immunization of mice by natively-purified RP in different adjuvant formulations indicated the crucial role of the built-in adjuvants for induction of anti-RG1 responses that could be further enhanced by combination of TLR7 agonist/alum adjuvants. While the TLR4/5 agonists contributed in the elicitation of the Th2-polarized immune responses, combination with TLR7 agonist changed the polarization to the balanced Th1/Th2 immune responses. Indeed, RP + TLR7 agonist/alum adjuvants induced the strongest immune responses that could efficiently neutralize the HPV pseudoviruses, and thus might be a promising formulation for an inexpensive and cross-reactive HPV vaccine.

Cross-neutralizing protection of vaginal and oral mucosa from HPV challenge by vaccination in a mouse model

The species and tissue specificities of HPV (human papillomavirus) for human infection and disease complicates the process of prophylactic vaccine development in animal models. HPV pseudoviruses (PsV) that carry only a reporter plasmid have been utilized in vivo to demonstrate cell internalization in mouse mucosal epithelium. The current study sought to expand the application of this HPV PsV challenge model with both oral and vaginal inoculation and to demonstrate its utility for testing vaccine-mediated dual-site immune protection against several HPV PsV types. We observed that passive transfer of sera from mice vaccinated with the novel experimental HPV prophylactic vaccine RG1-VLPs (virus-like particles) conferred HPV16-neutralizing as well as cross-neutralizing Abs against HPV39 in naïve recipient mice. Moreover, active vaccination with RG1-VLPs also conferred protection to challenge with either HPV16 or HPV39 PsVs at both vaginal and oral sites of mucosal inoculation. These data support the use of the HPV PsV challenge model as suitable for testing against diverse HPV types at two sites of challenge (vaginal vault and oral cavity) associated with the origin of the most common HPV-associated cancers, cervical cancer and oropharyngeal cancer.

Optimal size of DNA encapsidated by plant produced human papillomavirus pseudovirions

Human papillomaviruses (HPVs) are known to be the cause of anogenital and oropharyngeal cancers as well as genital and common warts. HPV pseudovirions (PsVs) are synthetic viral particles that are made up of the L1 major and L2 minor HPV capsid proteins and up to 8 Kb of encapsidated pseudogenome dsDNA. HPV PsVs are used to test novel neutralising antibodies elicited by vaccines, for studying the virus life cycle, and potentially for the delivery of therapeutic DNA vaccines. HPV PsVs are typically produced in mammalian cells, however, it has recently been shown that Papillomavirus PsVs can be produced in plants, a potentially safer, cheaper and more easily scalable means of production. We analysed the encapsidation frequencies of pseudogenomes expressing EGFP, ranging in size from 4.8 Kb to 7.8 Kb, by plant-made HPV-35 L1/L2 particles. The smaller pseudogenomes were found to be packaged more efficiently into PsVs as higher concentrations of encapsidated DNA and higher levels of EGFP expression were obtained with the 4.8 Kb pseudogenome, compared to the larger 5.8–7.8 Kb pseudogenomes. Thus, smaller pseudogenomes, of 4.8 Kb, should be used for efficient plant production of HPV-35 PsVs.

The Activity of Chelidonium majus L. Latex and Its Components on HPV Reveal Insights into the Antiviral Molecular Mechanism.

Yellow-orange latex of Chelidonium majus L. has been used in folk medicine as a therapeutic agent against warts and other visible symptoms of human papillomavirus (HPV) infections for centuries. The observed antiviral and antitumor properties of C. majus latex are often attributed to alkaloids contained therein, but recent studies indicate that latex proteins may also play an important role in its pharmacological activities. Therefore, the aim of the study was to investigate the effect of the crude C. majus latex and its protein and alkaloid-rich fractions on different stages of the HPV replication cycle. The results showed that the latex components, such as alkaloids and proteins, decrease HPV infectivity and inhibit the expression of viral oncogenes (E6, E7) on mRNA and protein levels. However, the crude latex and its fractions do not affect the stability of structural proteins in HPV pseudovirions and they do not inhibit the virus from attaching to the cell surface. In addition, the protein fraction causes increased TNFα secretion, which may indicate the induction of an inflammatory response. These findings indicate that the antiviral properties of C. majus latex arise both from alkaloids and proteins contained therein, acting on different stages of the viral replication cycle.

Dissolving Microneedle Delivery of a Prophylactic HPV Vaccine.

Prophylactic vaccines capable of preventing human papillomavirus (HPV) infections are still inaccessible to a vast majority of the global population due to their high cost and challenges related to multiple administrations performed in a medical setting. In an effort to improve distribution and administration, we have developed dissolvable microneedles loaded with a thermally stable HPV vaccine candidate consisting of Qβ virus-like particles (VLPs) displaying a highly conserved epitope from the L2 protein of HPV (Qβ-HPV). Polymeric microneedle delivery of Qβ-HPV produces similar amounts of anti-HPV16 L2 IgG antibodies compared to traditional subcutaneous injection while delivering a much smaller amount of intradermal dose. However, a dose sparing effect was found. Furthermore, immunization yielded neutralizing antibody responses in a HPV pseudovirus assay. The vaccine candidate was confirmed to be stable at room temperature after storage for several months, potentially mitigating many of the challenges associated with cold-chain distribution. The ease of self-administration and minimal invasiveness of such microneedle patch vaccines may enable wide-scale distribution of the HPV vaccine and lead to higher patient compliance. The Qβ VLP and its delivery technology is a plug-and-play system that could serve as a universal platform with a broad range of applications. Qβ VLPs may be stockpiled for conjugation to a wide range of epitopes, which are then packaged and delivered directly to the patient via noninvasive microneedle patches. Such a system paves the way for rapid distribution and self-administration of vaccines.

Integrin alpha 6 as a stemness driver is a novel promising target for HPV (+) head and neck squamous cell carcinoma

Although the incidence rates of head and neck squamous cell carcinoma (HNSCC) associated with human papilloma virus (HPV) infection have recently been on the rise, the underlying mechanism of its tumorigenesis remains largely unknown. Here, we investigated whether HNSCC cells with high expression of integrin alpha 6 (ITGα6), one of the HPV receptors, have a preference during HPV infection. In addition, we examined the gain or loss of function of the ITGα6 gene in HPV + ve HNSCC cells, as well as its prognostic value in patients with HNSCC. HPV pseudovirus was found to be more infective, with HNSCC cells featuring an overexpressed ITGα6 gene compared to the control cells. Overexpression and suppression of ITGα6 respectively increases and decreases stemness phenotypes of HPV + ve HNSCC cells. Furthermore, ITGα6 can regulate stemness by partially mediating AKT pathway in HPV + ve HNSCC cells. Finally, patients with HPV + ve HNSCC had a poor prognosis in cases of elevated ITGα6 expression; however, the expression levels of ITGα6 did not influence the survival rates of HPV-negative HNSCC patients. In conclusion, ITGα6 can serve as a potential therapeutic target for HPV + ve HNSCC cancer-like stem cells (CSCs).

Immunogenicity of Plant-Produced Human Papillomavirus (HPV) Virus-Like Particles (VLPs).

Cervical cancer is ranked fourth among the top cancers in women and is the second most common cancer in low- and middle-income regions, with ~570,000 new cases reported in 2018, which attributed to 84% of worldwide cervical cancer cases. Three commercially available prophylactic Human papillomavirus (HPV) vaccines are effective at preventing HPV infections. However, these vaccines are expensive due to their complex production systems, therefore limiting their use in developing countries. Recently, the use of plants to produce vaccines has emerged as a cost-effective alternative to conventionally used expression systems. Here, L1 proteins of eight high-risk (HPV 16, 18, 31, 33, 35, 45, 52, and 58) and two low risk (HPV 6 and 34) HPV types were successfully expressed in Nicotiana benthamiana, and transmission electron microscopy (TEM) analysis showed the presence of VLPs and/or capsomeres. Immunogenicity studies were conducted in mice utilizing HPV 35, 52, and 58 and showed that type-specific L1-specific antibodies were produced which were able to successfully neutralize homologous HPV pseudovirions in pseudovirion-based neutralization assays (PBNAs). This work demonstrated the potential for using plant-based transient expression systems to produce affordable and immunogenic HPV vaccines, particularly for developing countries.

Human Papillomavirus Seroprevalence and Seroconversion Among Men Living With HIV: Cohort Study in South Africa.

Men living with HIV (MLHIV) have a high burden of human papillomavirus (HPV)-related cancer. Understanding serological dynamics of HPV in men can guide decisions on introducing HPV vaccination and monitoring impact. We determined HPV seroprevalence and evaluated factors associated with HPV seroconversion among MLHIV in Johannesburg, South Africa. We enrolled 304 sexually active MLHIV 18 years and older and collected sociobehavioral data, blood samples (CD4 counts, HIV-1 plasma viral load, and HPV serology), and genital and anal swabs [HPV DNA and HPV viral load (VL)] at enrollment and 6-monthly for up to 18 months. Antibodies to 15 HPV types were measured using HPV pseudovirions. Generalized estimating equations were used to evaluate correlates of HPV seroconversion. Median age at enrollment was 38 years (IQR: 22-59), 25% reported >1 sexual partner in the past 3 months, and 5% reported ever having sex with other men. Most participants (65%) were on antiretroviral therapy (ART), with median CD4 count of 445 cells/µL (IQR: 328-567). Seroprevalence for any HPV type was 66% (199/303). Baseline seropositivity for any bivalent (16/18), quadrivalent (6/11/16/18), and nonavalent (6/11/16/18/31/33/45/52/58) vaccine types was 19%, 37%, and 60%, respectively. At 18 months, type-specific seroconversion among 59 men whose genital samples were HPV DNA positive but seronegative for the same type at enrollment was 22% (13/59). Type-specific seroconversion was higher among men with detectable HIV plasma viral load (adjusted odds ratio = 2.78, 95% CI: 1.12 to 6.77) and high HPV VL (adjusted odds ratio = 3.32, 95% CI: 1.42 to 7.74). Seropositivity and exposure to nonavalent HPV types were high among MLHIV. HPV vaccination of boys before they become sexually active could reduce the burden of HPV infection among this at-risk population.

Cell-penetrating peptide inhibits retromer-mediated human papillomavirus trafficking during virus entry

Virus replication requires critical interactions between viral proteins and cellular proteins that mediate many aspects of infection, including the transport of viral genomes to the site of replication. In human papillomavirus (HPV) infection, the cellular protein complex known as retromer binds to the L2 capsid protein and sorts incoming virions into the retrograde transport pathway for trafficking to the nucleus. Here, we show that short synthetic peptides containing the HPV16 L2 retromer-binding site and a cell-penetrating sequence enter cells, sequester retromer from the incoming HPV pseudovirus, and inhibit HPV exit from the endosome, resulting in loss of viral components from cells and in a profound, dose-dependent block to infection. The peptide also inhibits cervicovaginal HPV16 pseudovirus infection in a mouse model. These results confirm the retromer-mediated model of retrograde HPV entry and validate intracellular virus trafficking as an antiviral target. More generally, inhibiting virus replication with agents that can enter cells and disrupt essential protein-protein interactions may be applicable in broad outline to many viruses.

The Underlying Mechanism of 3-Hydroxyphthalic Anhydride-Modified Bovine Beta-Lactoglobulin to Block Human Papillomavirus Entry Into the Host Cell.

We have previously demonstrated that 3-hydroxyphthalic anhydride (3HP)-modified bovine beta-lactoglobulin (3HP-β-LG) is highly effective in inhibiting entry of pseudovirus (PsV) of high- and low-risk human papillomavirus (HPV) into the target cell. Intravaginally applied 3HP-β-LG-containing vaginal gel could significantly inhibit HPV infection and reduce viral load in the cervical region. However, we still do not understand the underlying molecular mechanism by which 3HP-β-LG is able to inhibit HPV infection. Here, though, we showed that 3HP-β-LG did not inactivate HPV PsV, but rather blocked entry of HPV PsV into the target cell via its interaction with virus, not cell. It bound to the positively charged region in the HPV L1 protein, suggesting that 3HP-β-LG binds to HPV L1 protein through the interaction between the negatively charged region in 3HP-β-LG and the positively charged region in HPV L1 protein, thus competitively blocking the binding of HPV to the receptor on the basement membrane in vaginal mucosa. Although 3HP-modified chicken ovalbumin (3HP-OVA) also carries high net negative charges, it exhibited no anti-HPV activity, suggesting that the interaction between 3HP-modified protein and HPV L1 protein relies on both electrostatic and matchable conformation of the binding sites in both proteins. When topically applied, 3HP-β-LG did not enter the host cell or blood circulation. These findings suggest that 3HP-β-LG targets HPV L1 protein and blocks HPV entry into the host cell, thus being safe and effective for topical application in the treatment of HPV infection.

Abstract 2540: Mitotic Golgi vesiculation enables human papillomavirus to evade cGAS/STING

Abstract Oncogenic human papillomaviruses (HPVs) infect and replicate in differentiating mucosal epithelium, causing 5% of cancers worldwide and essentially all cervical cancers. During infection, HPV must traffic viral genome (vDNA) to the nuclei of basal keratinocytes. Minor capsid protein L2 facilitates intracellular transport of vDNA to the Golgi; upon mitosis, the L2-vDNA complex penetrates limiting membranes and localizes to mitotic chromosomes, ensuring infection of both daughter cells. Cytosolic DNA sensor cGAS recognizes cytosolic dsDNA and produces second messenger cGAMP; cGAMP causes activation and relocalization of ER-resident STING to the Golgi, where it recruits TBK1 to phosphorylate and activate IRF3, initiating a type-I IFN response. The cGAS/STING pathway is assumed, though never demonstrated, to be inactive during mitosis to avoid detecting self-DNA. Since the Golgi is a platform for STING/TBK1/IRF3 recruitment and activation, we hypothesize natural Golgi dispersal deactivates cGAS/STING during mitosis. Further, we hypothesize HPV has evolved to traffic to and translocate from the mitotic Golgi as an immunoevasive tactic to avoid detection by cGAS/STING during mitosis. HaCaTs, a human keratinocyte line, were transfected with DNA or infected with HPV pseudovirions and analyzed for cGAS/STING activation. DNA transfection resulted in IRF3 phosphorylation and nuclear translocation, and STING translocation to the Golgi, indicating cGAS/STING activity. Strikingly, chemical disruption of the Golgi ribbon potently blocked IRF3 activation in response to foreign DNA, suggesting Golgi morphology might modulate cGAS/STING activity during mitosis. In accordance, DNA-dependent IRF3 phosphorylation was transiently reduced in synchronized mitotic cells, but chemical impairment of mitotic Golgi vesiculation enabled cGAS/STING activation, even without foreign DNA transfection. Further, HPV infection resulted in minimal IRF3 phosphorylation, indicating HPV can efficiently evade detection during initial infection. To determine if HPV’s unique trafficking enables evasion, we used cationic liposomes to permit premature virion translocation across limiting membranes. Such treatment renders a non-infectious, translocation-defective mutant HPV infectious, yet susceptible to cGAS/STING sensing. Overall, the cGAS/STING pathway may be inactivated by mitotic Golgi dispersal, allowing HPV to evade detection during mitosis. Citation Format: Brittany L. Forte, Shauna M. Bratton, Samuel K. Campos. Mitotic Golgi vesiculation enables human papillomavirus to evade cGAS/STING [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2540.

Establishment and evaluation of a triple-color human papillomavirus pseudovirion neutralization assay

Objective: To establish a triple-color pseudovirion-based neutralization assay (PBNA) and evaluate its capability of detecting immunogenicity of the sera generated by the immunization of HPV 9-valent vaccine. Methods: HPV pseudovirus (PsVs) 6/11/16/18/31/33/45/52/58 with the encapsidated fluorescence expressing red fluorescent plasmid N31-MCHREEY, green fluorescent N31-EGFP or blue fluorescent N31-mTagBFP were generated. The concentration of HPV PsVs and the infection titers of HPV PsVs were detected by double-antibody sandwich ELISA and TCID(50), respectively. The single- and triple color HPV 16/33/45 PsVs were used to detect the neutralization titers of mice sera immunized with HPV 9-valent vaccine and confirmed the accuracy and specificity of the triple-color PBNAs. Then, the single- and triple color HPV 6/11/18/31/33/45/52/58 PsVs were employed to detect the neutralization titers of cynomolgus macaques sera immunized with HPV 9-valent vaccine and determined whether the triple-color PBNAs could be applied to evaluate the immunogenicity of the sera generated by the immunization of HPV9-valent vaccine. Results: The concentration of HPV16 PsVs encapsulating green, red or blue fluorescent plasmid was 5.0 to 6.0 μg/ml and HPV6/11/18/31/33/45/52/59 triple-color HPV PsVs was about 1.0 to 3.0 μg/ml. 9 types HPV PsVs containing EGFP, Mcherry or mTagBFP reporter plasmid were obtained and the concentration can meet the need of neutralization detection. 9 types single-color fluorescent HPV PsVs had similar infectivity against 293FT cells with the infection titer values between 1×10(4) and 1×10(5). The results of PBNAs showed that there was no significant difference in the anti-HPV neutralization titers of mice sera induced by HPV 9-valent vaccine between single-color and triple-color HPV16/33/45 PsVs (P>0.05). Similarly, there was also no significant difference in the anti-HPV neutralization titers of cynomolgus macaques sera induced by HPV 9-valent vaccine between single-color and triple-color HPV6/11/18/31/33/45/52/58 PsVs (P>0.05). Conclusion: We successfully established the triple-color PBNAs and verified the accuracy and specificity of triple-color PBNAs consistent with single-color PBNAs. The triple-color PBNAs can be applied to evaluate the immunogenicity of HPV 9-valent vaccine's immune serum.

Griffithsin carrageenan fast dissolving inserts prevent SHIV HSV-2 and HPV infections in vivo

Human immunodeficiency virus (HIV) pre-exposure prophylaxis (PrEP) strategies with proven in vivo efficacy rely on antiretroviral drugs, creating the potential for drug resistance and complicated treatment options in individuals who become infected. Moreover, on-demand products are currently missing from the PrEP development portfolio. Griffithsin (GRFT) is a non-antiretroviral HIV entry inhibitor derived from red algae with an excellent safety profile and potent activity in vitro. When combined with carrageenan (CG), GRFT has strong activity against herpes simplex virus-2 (HSV-2) and human papillomavirus (HPV) in vitro and in vivo. Here, we report that GRFT/CG in a freeze-dried fast dissolving insert (FDI) formulation for on-demand use protects rhesus macaques from a high dose vaginal SHIV SF162P3 challenge 4 h after FDI insertion. Furthermore, the GRFT/CG FDI also protects mice vaginally against HSV-2 and HPV pseudovirus. As a safe, potent, broad-spectrum, on-demand non-antiretroviral product, the GRFT/CG FDI warrants clinical development.

Abstract LB-333: PARP-1 supports HPV infection and HPV-associated head and neck cancer

Abstract Background: As the incidence of human papillomavirus (HPV)-related head and neck squamous cell carcinoma (HNSCC) continues to rise, mechanistic knowledge of HPV16-driven HNSCC remains incomplete. HPV(+) and HPV(-) HNSCCs are molecularly distinct, with altered signaling pathways that have been characterized by proteomic technologies. We recently reported that poly(ADP-ribose)polymerase-1 (PARP-1) is overexpressed in HPV(+) compared to HPV(-) HNSCC. PARP-1 is a multi-functional protein with the ability to produce polymers of ADP-ribose attached to proteins and with major known roles in maintaining genomic integrity, DNA damage repair, transcription, DNA replication, and cell cycle regulation. However, PARP-1 functions in HPV infection and in head and neck cancer have not been fully uncovered. Here, we examine the role of PARP-1 in HPV infection, the interactions of HPV with cellular proteins, and the therapeutic implications of PARP-1 inhibition in HPV(+) HNSCC. Methods: PARP activity was assessed by immunoblotting for levels of poly(ADP-ribose) and by enzymatic PARP-1 ELISA. Expression of HPV genes was measured by RT-PCR. Murine kidney epithelial cells were established from PARP-1 wild-type and knockout mice, and the absence of PARP-1 expression was confirmed by RT-PCR and immunoblotting. Infection with HPV pseudovirus (PsV) containing a GFP expression plasmid was detected by L1 immunofluorescence staining and GFP detection. Survival studies were done in vitro and in vivo with PARP inhibitors veliparib and olaparib. Results: In addition to being overexpressed, we found that PARP-1 was highly enzymatically active in HPV(+) HNSCC as compared to HPV(-) tumors. HPV16 PsV and major capsid protein L1 activated PARP-1 in vitro and in cells. Depletion of PARP-1 and chemical PARP inhibition repressed HPV16 cellular entry, and infection of cells with HPV PsV increased sensitivity of HPV(-) cells to PARP inhibition. Conclusions: We identified a functional role of PARP-1 in initial HPV infection and in HPV-associated cancer. PARP-1 is activated by HPV major capsid protein L1, and HPV(+) HNSCCs maintain PARP-1 enzymatic activity. On top of its role in initial HPV infection, PARP-1 also appears to be important for survival of HPV-infected cells, as inhibition of PARP-1 sensitized cells to HPV PsV infection. These findings suggest that PARP inhibition may hold significant therapeutic potential in the treatment of HPV-associated HNSCC, with our work resulting in the initiation of a window clinical trial ongoing at the Yale Cancer Center. Citation Format: Cassie Pan, Asel Biktasova, Michael Hajek, Andrew Sewell, Tejas Sathe, Gary Bellinger, Wendell Yarbrough, Natalia Issaeva. PARP-1 supports HPV infection and HPV-associated head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-333.

Displaying 31RG-1 peptide on the surface of HPV16 L1 by use of a human papillomavirus chimeric virus-like particle induces cross-neutralizing antibody responses in mice

ABSTRACTCurrent available human papillomavirus (HPV) vaccines are based on the major capsid protein L1 virus-like particles (VLPs), which mainly induce type-specific neutralizing antibodies against vaccine types. Continuing to add more types of VLPs in a vaccine raises the complexity and cost of production which remains the principal impediment to achieve broad implementation of HPV vaccines, particularly in developing regions. In this study, we constructed 16L1-31L2 chimeric VLP (cVLP) by displaying HPV31 L2 aa.17-38 on the h4 coil surface region of HPV16 L1, and assessed its immunogenicity in mouse model. We found that the cVLP adjuvanted with alum plus monophosphoryl lipid A could induce cross-neutralizing antibody responses against 16 out of 17 tested HPV pseudoviruses, and the titer against HPV16 was as high as that was induced by HPV16 L1VLP (titer > 105), more importantly, titers over 103 were observed against two HR-HPVs including HPV31 (titer, 2,200) and −59 (titer, 1,013), among which HPV59 was not covered by Gardasil-9, and medium or low titers of cross-neutralizing antibodies against other 13 tested HPV pseudoviruses were also observed. Our data demonstrate that 16L1-31L2 cVLP is a promising candidate for the formulation of broader spectrum HPV vaccines.

Prophylactic immunization with human papillomavirus vaccines induces oral immunity in mice.

Although it has been shown that prophylactic vaccination can induce genital immunity, there is inadequate information on human papillomavirus (HPV) vaccine-induced oral immunity, which is of particular interest due to HPV-associated oropharyngeal malignancies and recurrent respiratory papillomatosis. Therefore, we assessed the efficacy of various HPV vaccines against oral HPV pseudovirus (PsV) infection in mice. Preclinical scientific investigation. C57BL/6 mice were vaccinated three times at 2-week intervals with either Gardasil (Merck, Kenilworth, NJ) (50 µL intramuscular injection) or a candidate pan-HPV L2 vaccine with alum adjuvant (25 µg subcutaneous injection). Additional mice were immunized with passive transfer of either Gardasil (Merck) human antisera or nonimmunized sera (100 µL intraperitoneal injection). All vaccinated and naïve control mice were then challenged with HPV16 E6E7 luciferase PsV in the oral mucosa. Visualization of HPV PsV infection was monitored through in vivo luciferase imaging. Oral luciferase-expressing HPV16 PsV infection was not detected in Gardasil (Merck), L2 vaccine, and Gardasil (Merck) antisera-immunized mice, whereas robust luciferase expression was observed in all control mice. An in vitro neutralization assay from sera of Gardasil-vaccinated (Merck) mice confirmed that vaccine efficacy was due to neutralizing antibodies. Oral HPV16 PsV infection in mice was completely prevented with all methods of prophylactic HPV immunization. These findings provide preliminary evidence that human vaccines induce protection against oral HPV infection, which has significant public health implications for HPV-associated oropharyngeal malignancies. NA. Laryngoscope, 128:E16-E20, 2018.

Characterization of serum antibodies from women immunized with Gardasil: A study of HPV-18 infection of primary human keratinocytes

The prevalent human papillomaviruses (HPVs) infect human epithelial tissues. Infections by the mucosotropic HPV genotypes cause hyperproliferative ano-genital lesions. Persistent infections by high-risk (HR) HPVs such as HPV-16, HPV-18 and related types can progress to high grade intraepithelial neoplasias and cancers. Prophylactic HPV vaccines are based on DNA-free virus-like particles (VLPs) composed of the major capsid protein L1 of HPV-16, -18, -6 and -11 (Gardasil) or HPV-16 and -18 (Cervarix). Sera from vaccinated animals effectively prevent HPV pseudovirions to infect cell lines and mouse cervical epithelia. Both vaccines have proven to be highly protective in people. HPV pseudovirions are assembled in HEK293TT cells from matched L1 and L2 capsid proteins to encapsidate a reporter gene. Pseudovirions and genuine virions have structural differences and they infect cell lines or primary human keratinocytes (PHKs) with different efficiencies. In this study, we show that sera and isolated IgG from women immunized with Gardasil prevent authentic HPV-18 virions from infecting PHKs, whereas non-immune sera and purified IgG thereof are uniformly ineffective. Using early passage PHKs, neutralization is achieved only if immune sera are added within 2–4h of infection. We attribute the timing effect to a conformational change in HPV virions, thought to occur upon initial binding to heparan sulfate proteoglycans (HSPG) on the cell surface. This interpretation is consistent with the inability of immune IgG bound to or taken up by PHKs to neutralize the virus. Interestingly, the window of neutralization increases to 12–16h in slow growing, late passage PHKs, suggestive of altered cell surface molecules. In vivo, this window might be further lengthened by the time required to activate the normally quiescent basal cells to become susceptible to infection. Our observations help explain the high efficacy of HPV vaccines.

Production of Human papillomavirus pseudovirions in plants and their use in pseudovirion-based neutralisation assays in mammalian cells.

Human papillomaviruses (HPV) cause cervical cancer and have recently also been implicated in mouth, laryngeal and anogenital cancers. There are three commercially available prophylactic vaccines that show good efficacy; however, efforts to develop second-generation vaccines that are more affordable, stable and elicit a wider spectrum of cross-neutralising immunity are still ongoing. Testing antisera elicited by current and candidate HPV vaccines for neutralizing antibodies is done using a HPV pseudovirion (PsV)-based neutralisation assay (PBNA). PsVs are produced by transfection of mammalian cell cultures with plasmids expressing L1 and L2 capsid proteins, and a reporter gene plasmid, a highly expensive process. We investigated making HPV-16 PsVs in plants, in order to develop a cheaper alternative. The secreted embryonic alkaline phosphatase (SEAP) reporter gene and promoter were cloned into a geminivirus-derived plant expression vector, in order to produce circular dsDNA replicons. This was co-introduced into Nicotiana benthamiana plants with vectors expressing L1 and L2 via agroinfiltration, and presumptive PsVs were purified. The PsVs contained DNA, and could be successfully used for PBNA with anti-HPV antibodies. This is the first demonstration of the production of mammalian pseudovirions in plants, and the first demonstration of the potential of plants to make DNA vaccines.

Human papillomavirus vaccination induces neutralising antibodies in oral mucosal fluids.

Background:Mucosal human papillomaviruses (HPV) are a major cause of cancers and papillomas of the anogenital and oropharyngeal tract. HPV-vaccination elicits neutralising antibodies in sera and cervicovaginal secretions and protects uninfected individuals from persistent anogenital infection and associated diseases caused by the vaccine-targeted HPV types. Whether immunisation can prevent oropharyngeal infection and diseases and whether neutralising antibodies represent the correlate of protection, is still unclear.Methods:We determined IgG and neutralising antibodies against low-risk HPV6 and high-risk HPV16/18 in sera and oral fluids from healthy females (n=20) before and after quadrivalent HPV-vaccination and compared the results with non-vaccinated controls.Results:HPV-vaccination induced type-specific antibodies in sera and oral fluids of the vaccinees. Importantly, the antibodies in oral fluids were capable of neutralising HPV pseudovirions in vitro, indicating protection from infection. The increased neutralising antibody levels against HPV16/18 in sera and oral fluids post-vaccination correlated significantly within an individual.Conclusions:We provide experimental proof that HPV-vaccination elicits neutralising antibodies to the vaccine-targeted types in oral fluids. Hence, immunisation may confer direct protection against type-specific HPV infection and associated diseases of the oropharyngeal tract. Measurement of antibodies in oral fluids represents a suitable tool to assess vaccine-induced protection within the mucosal milieu of the orophayrynx.