RESUMEN
Recent insight into the mechanisms of induction of tissue-resident memory (TRM) CD8+ T cells (CD8+ TRM) enables the development of novel vaccine strategies against sexually transmitted infections. To maximize both systemic and genital intraepithelial CD8+ T cells against vaccine Ags, we assessed combinations of i.m. and intravaginal routes in heterologous prime-boost immunization regimens with unrelated viral vectors. Only i.m. prime followed by intravaginal boost induced concomitant strong systemic and intraepithelial genital-resident CD8+ T cell responses. Intravaginal boost with vectors expressing vaccine Ags was far superior to intravaginal instillation of CXCR3 chemokine receptor ligands or TLR 3, 7, and 9 agonists to recruit and increase the pool of cervicovaginal CD8+ TRM Transient Ag presentation increased trafficking of cognate and bystander circulating activated, but not naive, CD8+ T cells into the genital tract and induced in situ proliferation and differentiation of cognate CD8+ TRM Secondary genital CD8+ TRM were induced in the absence of CD4+ T cell help and shared a similar TCR repertoire with systemic CD8+ T cells. This prime-pull-amplify approach elicited systemic and genital CD8+ T cell responses against high-risk human papillomavirus type 16 E7 oncoprotein and conferred CD8-mediated protection to a vaccinia virus genital challenge. These results underscore the importance of the delivery route of nonreplicating vectors in prime-boost immunization to shape the tissue distribution of CD8+ T cell responses. In this context, the importance of local Ag presentation to elicit genital CD8+ TRM provides a rationale to develop novel vaccines against sexually transmitted infections and to treat human papillomavirus neoplasia.
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Linfocitos T CD8-positivos/inmunología , Papillomavirus Humano 16/inmunología , Vacunas contra Papillomavirus/inmunología , Animales , Células HEK293 , Humanos , Ratones , Ratones Congénicos , Ratones Endogámicos C57BL , Vacunas contra Papillomavirus/genética , VacunaciónRESUMEN
Infectious human papillomavirus 16 (HPV16) L1/L2 pseudovirions were found to remain largely intact during vesicular transport to the nucleus. By electron microscopy, capsids with a diameter of 50 nm were clearly visible within small vesicles attached to mitotic chromosomes and to a lesser extent within interphase nuclei, implying nuclear disassembly. By confocal analysis, it was determined that nuclear entry of assembled L1 is dependent upon the presence of the minor capsid protein, L2, but independent of encapsidated DNA. We also demonstrate that L1 nuclear localization and mitotic chromosome association can occur in vivo in the murine cervicovaginal challenge model of HPV16 infection. These findings challenge the prevailing concepts of PV uncoating and disassembly. More generally, they document that a largely intact viral capsid can enter the nucleus within a transport vesicle, establishing a novel mechanism by which a virus accesses the nuclear cellular machinery.IMPORTANCE Papillomaviruses (PVs) comprise a large family of nonenveloped DNA viruses that include HPV16, among other oncogenic types, the causative agents of cervical cancer. Delivery of the viral DNA into the host cell nucleus is necessary for establishment of infection. This was thought to occur via a subviral complex following uncoating of the larger viral capsid. In this study, we demonstrate that little disassembly of the PV capsid occurs prior to nuclear delivery. These surprising data reveal a previously unrecognized viral strategy to access the nuclear replication machinery. Understanding viral entry mechanisms not only increases our appreciation of basic cell biological pathways but also may lead to more effective antiviral interventions.
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Proteínas de la Cápside/metabolismo , Núcleo Celular/virología , Papillomavirus Humano 16/fisiología , Proteínas Oncogénicas Virales/metabolismo , Internalización del Virus , Animales , Cápside/metabolismo , Cápside/ultraestructura , Línea Celular , Modelos Animales de Enfermedad , Papillomavirus Humano 16/ultraestructura , Humanos , Microscopía Electrónica , Infecciones por Papillomavirus/patología , Infecciones por Papillomavirus/virologíaRESUMEN
UNLABELLED: We have established a cell-free in vitro system to study human papillomavirus type 16 (HPV16) assembly, a poorly understood process. L1/L2 capsomers, obtained from the disassembly of virus-like particles (VLPs), were incubated with nuclear extracts to provide access to the range of cellular proteins that would be available during assembly within the host cell. Incorporation of a reporter plasmid "pseudogenome" was dependent on the presence of both nuclear extract and ATP. Unexpectedly, L1/L2 VLPs that were not disassembled prior to incubation with a reassembly mixture containing nuclear extract also encapsidated a reporter plasmid. As with HPV pseudoviruses (PsV) generated intracellularly, infection by cell-free particles assembled in vitro required the presence of L2 and was susceptible to the same biochemical inhibitors, implying the cell-free assembled particles use the infectious pathway previously described for HPV16 produced in cell culture. Using biochemical and electron microscopy analyses, we observed that, in the presence of nuclear extract, intact VLPs partially disassemble, providing a mechanistic explanation to how the exogenous plasmid was packaged by these particles. Further, we provide evidence that capsids containing an <8-kb pseudogenome are resistant to the disassembly/reassembly reaction. Our results suggest a novel size discrimination mechanism for papillomavirus genome packaging in which particles undergo iterative rounds of disassembly/reassembly, seemingly sampling DNA until a suitably sized DNA is encountered, resulting in the formation of a stable virion structure. IMPORTANCE: Little is known about papillomavirus assembly biology due to the difficulties in propagating virus in vitro. The cell-free assembly method established in this paper reveals a new mechanism for viral genome packaging and will provide a tractable system for further dissecting papillomavirus assembly. The knowledge gained will increase our understanding of virus-host interactions, help to identify new targets for antiviral therapy, and allow for the development of new gene delivery systems based on in vitro-generated papillomavirus vectors.
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Proteínas de la Cápside/metabolismo , ADN Viral/metabolismo , Genoma Viral , Papillomavirus Humano 16/fisiología , Proteínas Oncogénicas Virales/metabolismo , Ensamble de Virus , Genes Reporteros , PlásmidosRESUMEN
UNLABELLED: No herpes simplex virus 2 (HSV-2) vaccine has been licensed for use in humans. HSV-2 glycoproteins B (gB) and D (gD) are targets of neutralizing antibodies and T cells, but clinical trials involving intramuscular (i.m.) injection of HSV-2 gB and gD in adjuvants have not been effective. Here we evaluated intravaginal (ivag) genetic immunization of C57BL/6 mice with a replication-defective human papillomavirus pseudovirus (HPV PsV) expressing HSV-2 gB (HPV-gB) or gD (HPV-gD) constructs to target different subcellular compartments. HPV PsV expressing a secreted ectodomain of gB (gBsec) or gD (gDsec), but not PsV expressing a cytoplasmic or membrane-bound form, induced circulating and intravaginal-tissue-resident memory CD8(+) T cells that were able to secrete gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) as well as moderate levels of serum HSV neutralizing antibodies. Combined immunization with HPV-gBsec and HPV-gDsec (HPV-gBsec/gDsec) vaccines conferred longer survival after vaginal challenge with HSV-2 than immunization with HPV-gBsec or HPV-gDsec alone. HPV-gBsec/gDsec ivag vaccination was associated with a reduced severity of genital lesions and lower levels of viral shedding in the genital tract after HSV-2 challenge. In contrast, intramuscular vaccination with a soluble truncated gD protein (gD2t) in alum and monophosphoryl lipid A (MPL) elicited high neutralizing antibody titers and improved survival but did not reduce genital lesions and viral shedding. Vaccination combining ivag HPV-gBsec/gDsec and i.m. gD2t-alum-MPL improved survival and reduced genital lesions and viral shedding. Finally, high levels of circulating HSV-2-specific CD8(+) T cells, but not serum antibodies, correlated with reduced viral shedding. Taken together, our data underscore the potential of HPV PsV as a platform for a topical mucosal vaccine to control local manifestations of primary HSV-2 infection. IMPORTANCE: Genital herpes is a highly prevalent chronic disease caused by HSV infection. To date, there is no licensed vaccine against HSV infection. This study describes intravaginal vaccination with a nonreplicating HPV-based vector expressing HSV glycoprotein antigens. The data presented in this study underscore the potential of HPV-based vectors as a platform for the induction of genital-tissue-resident memory T cell responses and the control of local manifestations of primary HSV infection.
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Linfocitos T CD8-positivos/inmunología , Herpes Genital/prevención & control , Vacunas contra Herpesvirus/inmunología , Papillomaviridae/genética , Proteínas del Envoltorio Viral/inmunología , Esparcimiento de Virus , Administración Intravaginal , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Modelos Animales de Enfermedad , Femenino , Vectores Genéticos , Herpes Genital/inmunología , Herpes Genital/patología , Herpesvirus Humano 2/genética , Herpesvirus Humano 2/inmunología , Vacunas contra Herpesvirus/administración & dosificación , Vacunas contra Herpesvirus/genética , Memoria Inmunológica , Inyecciones Intramusculares , Interferón gamma/metabolismo , Ratones Endogámicos C57BL , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Análisis de Supervivencia , Factor de Necrosis Tumoral alfa/metabolismo , Vacunación/métodos , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Proteínas del Envoltorio Viral/genéticaRESUMEN
Full-length genomic DNA of the recently identified laboratory mouse papillomavirus 1 (MusPV1) was synthesized in vitro and was used to establish and characterize a mouse model of papillomavirus pathobiology. MusPV1 DNA, whether naked or encapsidated by MusPV1 or human papillomavirus 16 (HPV 16) capsids, efficiently induced the outgrowth of papillomas as early as 3 weeks after application to abraded skin on the muzzles and tails of athymic NCr nude mice. High concentrations of virions were extracted from homogenized papillomatous tissues and were serially passaged for >10 generations. Neutralization by L1 antisera confirmed that infectious transmission was capsid mediated. Unexpectedly, the skin of the murine back was much less susceptible to virion-induced papillomas than the muzzle or tail. Although reporter pseudovirions readily transduced the skin of the back, infection with native MusPV1 resulted in less viral genome amplification and gene expression on the back, including reduced expression of the L1 protein and very low expression of the L2 protein, results that imply skin region-specific control of postentry aspects of the viral life cycle. Unexpectedly, L1 protein on the back was predominantly cytoplasmic, while on the tail the abundant L1 was cytoplasmic in the lower epithelial layers and nuclear in the upper layers. Nuclear localization of L1 occurred only in cells that coexpressed the minor capsid protein, L2. The pattern of L1 protein staining in the infected epithelium suggests that L1 expression occurs earlier in the MusPV1 life cycle than in the life cycle of high-risk HPV and that virion assembly is regulated by a previously undescribed mechanism.
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Proteínas de la Cápside/metabolismo , Regulación Viral de la Expresión Génica , Papillomaviridae/metabolismo , Infecciones por Papillomavirus/veterinaria , Enfermedades de los Roedores/virología , Animales , Proteínas de la Cápside/genética , Núcleo Celular/virología , Citoplasma/virología , Femenino , Ratones/virología , Ratones Desnudos , Papillomaviridae/genética , Infecciones por Papillomavirus/virología , Transporte de Proteínas , Conejos , Ratas , Ratas Sprague-DawleyRESUMEN
In 2019, there were about 100,000 kidney transplants globally, with more than a quarter of them performed in the United States. Unfortunately, some engrafted organs are lost to polyomavirus-associated nephropathy (PyVAN) caused by BK and JC viruses (BKPyV and JCPyV). Both viruses cause brain disease and possibly bladder cancer in immunosuppressed individuals. Transplant patients are routinely monitored for BKPyV viremia, which is an accepted hallmark of nascent nephropathy. If viremia is detected, a reduction in immunosuppressive therapy is standard care, but the intervention comes with increased risk of immune rejection of the engrafted organ. Recent reports have suggested that transplant recipients with high levels of polyomavirus-neutralizing antibodies are protected against PyVAN. Virus-like particle (VLP) vaccines, similar to approved human papillomavirus vaccines, have an excellent safety record and are known to induce high levels of neutralizing antibodies and long-lasting protection from infection. In this study, we demonstrate that VLPs representing BKPyV genotypes I, II, and IV, as well as JCPyV genotype 2 produced in insect cells elicit robust antibody titers. In rhesus macaques, all monkeys developed neutralizing antibody titers above a previously proposed protective threshold of 10,000. A second inoculation, administered 19 weeks after priming, boosted titers to a plateau of ≥ 25,000 that was maintained for almost two years. No vaccine-related adverse events were observed in any macaques. A multivalent BK/JC VLP immunogen did not show inferiority compared to the single-genotype VLP immunogens. Considering these encouraging results, we believe a clinical trial administering the multivalent VLP vaccine in patients waiting to receive a kidney transplant is warranted to evaluate its ability to reduce or eliminate PyVAN.
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Virus BK , Enfermedades Renales , Infecciones por Polyomavirus , Poliomavirus , Infecciones Tumorales por Virus , Vacunas de Partículas Similares a Virus , Animales , Humanos , Macaca mulatta , Viremia/prevención & control , Anticuerpos NeutralizantesRESUMEN
We have recently reported that the sublingual (s.l.) mucosa is an efficient site for inducing systemic and mucosal immune responses. In this study, the potential of s.l. immunization to induce remote Ab responses and CD8(+) cytotoxic responses in the female genital tract was examined in mice by using a nonreplicating Ag, OVA, and cholera toxin (CT) as an adjuvant. Sublingual administration of OVA and CT induced Ag-specific IgA and IgG Abs in blood and in cervicovaginal secretions. These responses were associated with large numbers of IgA Ab-secreting cells (ASCs) in the genital mucosa. Genital ASC responses were similar in magnitude and isotype distribution after s.l., intranasal, or vaginal immunization and were superior to those seen after intragastric immunization. Genital, but not blood or spleen, IgA ASC responses were inhibited by treatment with anti-CCL28 Abs, suggesting that the chemokine CCL28 plays a major role in the migration of IgA ASC progenitors to the reproductive tract mucosa. Furthermore, s.l. immunization with OVA induced OVA-specific effector CD8(+) cytolytic T cells in the genital mucosa, and these responses required coadministration of the CT adjuvant. Furthermore, s.l. administration of human papillomavirus virus-like particles with or without the CT adjuvant conferred protection against genital challenge with human papillomavirus pseudovirions. Taken together, these findings underscore the potential of s.l. immunization as an efficient vaccination strategy for inducing genital immune responses and should impact on the development of vaccines against sexually transmitted diseases.
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Anticuerpos Antibacterianos/biosíntesis , Células Productoras de Anticuerpos/inmunología , Toxina del Cólera/inmunología , Papillomavirus Humano 16/inmunología , Ovalbúmina/inmunología , Infecciones por Papillomavirus/prevención & control , Linfocitos T Citotóxicos/inmunología , Neoplasias del Cuello Uterino/prevención & control , Adyuvantes Inmunológicos/administración & dosificación , Administración Sublingual , Animales , Anticuerpos Antivirales/biosíntesis , Células Productoras de Anticuerpos/citología , Células Productoras de Anticuerpos/virología , Diferenciación Celular/inmunología , Células Cultivadas , Toxina del Cólera/administración & dosificación , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Membrana Mucosa/inmunología , Membrana Mucosa/virología , Ovalbúmina/administración & dosificación , Infecciones por Papillomavirus/inmunología , Infecciones por Papillomavirus/virología , Linfocitos T Citotóxicos/virología , Neoplasias del Cuello Uterino/inmunología , Neoplasias del Cuello Uterino/virología , Virión/inmunologíaRESUMEN
Although millions of distinct virus species likely exist, only approximately 9000 are catalogued in GenBank's RefSeq database. We selectively enriched for the genomes of circular DNA viruses in over 70 animal samples, ranging from nematodes to human tissue specimens. A bioinformatics pipeline, Cenote-Taker, was developed to automatically annotate over 2500 complete genomes in a GenBank-compliant format. The new genomes belong to dozens of established and emerging viral families. Some appear to be the result of previously undescribed recombination events between ssDNA and ssRNA viruses. In addition, hundreds of circular DNA elements that do not encode any discernable similarities to previously characterized sequences were identified. To characterize these 'dark matter' sequences, we used an artificial neural network to identify candidate viral capsid proteins, several of which formed virus-like particles when expressed in culture. These data further the understanding of viral sequence diversity and allow for high throughput documentation of the virosphere.
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Virus ADN , ADN Circular/genética , Animales , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Infecciones por Virus ADN/virología , Virus ADN/clasificación , Virus ADN/genética , ADN Viral/genética , Genoma Viral/genética , Humanos , Anotación de Secuencia Molecular , Programas InformáticosRESUMEN
Papillomavirus capsids can package a wide variety of nonviral DNA plasmids and deliver the packaged genetic material to cells, making them attractive candidates for targeted gene delivery vehicles. However, the papillomavirus vectors generated by current methods are unlikely to be suitable for clinical applications. We have developed a chemically defined, cell-free, papillomavirus-based vector production system that allows the incorporation of purified plasmid DNA (pseudogenome) into high-titer papillomavirus L1/L2 capsids. We investigated the incorporation of several DNA forms into a variety of different papillomavirus types, including human and animal types. Our results show that papillomavirus capsids can package and transduce linear or circular DNA under defined conditions. Packaging and transduction efficiencies were surprisingly variable across capsid types, DNA forms, and assembly reaction conditions. The pseudoviruses produced by these methods are sensitive to the same entry inhibitors as cell-derived pseudovirions, including neutralizing antibodies and heparin. The papillomavirus vector production systems developed in this study generated as high as 1011 infectious units/mg of L1. The pseudoviruses were infectious both in vitro and in vivo and should be compatible with good manufacturing practice (GMP) requirements.
RESUMEN
Human papillomaviruses (HPVs) cause approximately 5% of cancer cases worldwide. Fortunately, three prophylactic vaccines have been approved to protect against HPV infections. Gardasil-9, the most recent HPV vaccine, is predicted to offer protection against the HPV types that cause â¼90% of cervical cancer, 86% of HPV-associated penile cancers, and â¼93% of HPV-associated head & neck cancers. As an alternative to Gardasil-9, we developed and tested a novel candidate vaccine targeting conserved epitopes in the HPV minor capsid protein, L2. We displayed a tandem HPV31/16L2 peptide (amino acid 17-31) or consensus peptides from HPV L2 (amino acid 69-86 or 108-122) on the surface of bacteriophage MS2 virus-like particles (VLPs). Mice immunized with the MS2 VLPs displaying the tandem peptide or immunized with a mixture of VLPs (displaying the tandem peptide and consensus peptide 69-86) elicited high titer antibodies against individual L2 epitopes. Moreover, vaccinated mice were protected from cervicovaginal infection with HPV pseudoviruses 16, 31, 45, 58 and sera from immunized mice neutralized HPV pseudoviruses 18 and 33 at levels similar to mice immunized with Gardasil-9. These results suggest that immunization with a tandem, L2 peptide or a low valency mixture of L2 peptide-displaying VLPs can provide broad protection against multiple HPV types.
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Proteínas de la Cápside/inmunología , Papillomaviridae/inmunología , Infecciones por Papillomavirus/prevención & control , Vacunas contra Papillomavirus/inmunología , Animales , Anticuerpos Antivirales/sangre , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Protección Cruzada , Modelos Animales de Enfermedad , Epítopos/química , Epítopos/genética , Epítopos/inmunología , Vacuna Tetravalente Recombinante contra el Virus del Papiloma Humano Tipos 6, 11 , 16, 18/inmunología , Humanos , Levivirus/genética , Levivirus/inmunología , Ratones , Ratones Endogámicos BALB C , Pruebas de Neutralización , Infecciones por Papillomavirus/inmunología , Vacunas contra Papillomavirus/genética , Vacunas contra Papillomavirus/normas , Péptidos/química , Péptidos/genética , Péptidos/inmunología , Vacunas de Partículas Similares a Virus/genética , Vacunas de Partículas Similares a Virus/inmunología , Vacunas de Partículas Similares a Virus/aislamiento & purificaciónRESUMEN
We report that during assembly of HPV16 pseudovirus (PsV) the minor capsid protein, L2, interacts with the host nucleolar protein nucleophosmin (NPM1/B23). Exogenously-expressed L2 colocalized with NPM1, a complex containing both proteins could be immunoprecipitated, and L2 could redirect to the nucleus NPM1 that was pharmacologically or genetically restricted to the cytoplasm. Coexpression of the major capsid protein, L1, prevented both the colocalization and the biochemical association, and L1 pentamers could displace L2 from L2/NPM1 complexes attached to a nuclear matrix. HPV16 PsV that was produced in a cell line with reduced NPM1 levels had significantly lower infectivity compared to PsV produced in the parental cell line, although the PsV preparations had comparable L1 and L2 ratios and levels of encapsidated DNA. The PsV produced in NPM1-deficient cells showed increased trypsin sensitivity and exhibited decreased L2 levels during endocytosis. These results suggest a critical role for NPM1 in establishing the correct interactions between L2 and L1 during HPV capsid assembly. A decrease in cellular levels of NPM1 results in the formation of seemingly normal, but unstable, capsids that result in a premature loss of L2, thus inhibiting successful infection. No role for NPM1 in HPV infectious entry was found.
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Human papilloma virus-like particles (HPV VLP) serve as the basis of the current licensed vaccines for HPV. We have previously shown that encapsidation of DNA expressing the model antigen M/M2 from respiratory syncytial virus (RSV) in HPV pseudovirions (PsV) is immunogenic when delivered intravaginally. Because the HPV capsids confer tropism for basal epithelium, they represent attractive carriers for vaccination targeted to the skin using microneedles. In this study we asked: 1) whether HPV16 VLP administered by microneedles could induce protective immune responses to HPV16 and 2) whether HPV16 PsV-encapsidated plasmids delivered by microneedles could elicit immune responses to both HPV and the antigen delivered by the transgene. Mice immunized with HPV16 VLP coated microneedles generated robust neutralizing antibody responses and were protected from HPV16 challenge. Microneedle arrays coated with HPV16-M/M2 or HPV16-F protein (genes of RSV) were then tested and dose-dependent HPV and F-specific antibody responses were detected post-immunization, and M/M2-specific T-cell responses were detected post RSV challenge, respectively. HPV16 PsV-F immunized mice were fully protected from challenge with HPV16 PsV and had reduced RSV viral load in lung and nose upon intranasal RSV challenge. In summary, HPV16 PsV-encapsidated DNA delivered by microneedles induced neutralizing antibody responses against HPV and primed for antibody and T-cell responses to RSV antigens encoded by the encapsidated plasmids. Although the immunogenicity of the DNA component was just above the dose response threshold, the HPV-specific immunity was robust. Taken together, these data suggest microneedle delivery of lyophilized HPV PsV could provide a practical, thermostable combined vaccine approach that could be developed for clinical evaluation.
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Infecciones por Papillomavirus/prevención & control , Vacunas contra Papillomavirus/inmunología , Plásmidos/inmunología , Piel/inmunología , Neoplasias del Cuello Uterino/prevención & control , Vacunación , Administración Cutánea , Animales , Anticuerpos Neutralizantes/biosíntesis , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/biosíntesis , Anticuerpos Antivirales/inmunología , ADN Viral/genética , ADN Viral/inmunología , Femenino , Expresión Génica , Genes Reporteros , Papillomavirus Humano 16/efectos de los fármacos , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/inmunología , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Ratones , Microinyecciones , Agujas , Infecciones por Papillomavirus/inmunología , Infecciones por Papillomavirus/virología , Vacunas contra Papillomavirus/administración & dosificación , Vacunas contra Papillomavirus/genética , Plásmidos/administración & dosificación , Plásmidos/genética , Virus Sincitiales Respiratorios/genética , Virus Sincitiales Respiratorios/inmunología , Transgenes , Neoplasias del Cuello Uterino/inmunología , Neoplasias del Cuello Uterino/virología , Vacunas de Partículas Similares a Virus/administración & dosificación , Vacunas de Partículas Similares a Virus/genética , Vacunas de Partículas Similares a Virus/inmunología , Proteínas Virales de Fusión/administración & dosificación , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/inmunología , Proteínas de la Matriz Viral/administración & dosificación , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/inmunologíaRESUMEN
Two HPV virus-like particle (VLP) vaccines, HPV-16/18 (GlaxoSmithKline, Cervarix®) and HPV-6/11/16/18 (Merck, Gardasil®), are currently licensed in the United States. Given the similar antigenic content but different adjuvant formulations in the 2 vaccines, they provide an efficient method for evaluating adjuvants and comparing the kinetics of the innate and adaptive immune responses. We randomized women to receive either Cervarix® or Gardasil®, followed 6 month vaccination delivery schedules per manufacturer's recommendations, and analyzed the humoral immune response, T cell response, and circulating plasma cytokine levels in response to vaccination. Cervarix® recipients had higher anti-HPV-16 antibody and neutralization titers at month 7, and elevated anti-HPV-18 antibody and neutralization titers at months 7 and 12. Antibody avidity was similar for the 2 vaccines. HPV-31 was the only phylogenetically related non-vaccine HPV type, for which there is evidence of cross-protection, to be cross-neutralized and only in response to Cervarix®. Comparing CD4+ T cell cytokine responses at month 12, there was a trend of increased levels of IL-2 and TNF-α in the Cervarix® groups versus the Gardasil® groups that was consistent across all 4 tested HPV types (16/18/33/45). Elevated levels of circulating plasma cytokine/chemokines were observed post first vaccination in Gardasil® recipients and proinflammatory cytokines were elevated following 1st and 3rd Cervarix® vaccinations. Cervarix® and Gardasil® are both highly immunogenic vaccines. Higher antibody levels and CD4 T cell responses were achieved with Cervarix® after 3 doses, although similar affinity maturation was measured for the 2 vaccines. The clinical implications of the differences in immune responses are unknown.
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Inmunidad Adaptativa , Vacunas contra Papillomavirus/inmunología , Adolescente , Adulto , Anticuerpos Antivirales/sangre , Linfocitos T CD4-Positivos/inmunología , Quimiocinas/sangre , Citocinas/sangre , Femenino , Vacuna Tetravalente Recombinante contra el Virus del Papiloma Humano Tipos 6, 11 , 16, 18 , Humanos , Inmunidad Innata , Adulto JovenRESUMEN
Papillomavirus L2-based vaccines have generally induced low-level or undetectable neutralizing antibodies in standard in vitro assays yet typically protect well against in vivo experimental challenge in animal models. Herein we document that mice vaccinated with an L2 vaccine comprising a fusion protein of the L2 amino acids 11 to 88 of human papillomavirus type 16 (HPV16), HPV18, HPV1, HPV5, and HPV6 were uniformly protected from cervicovaginal challenge with HPV16 pseudovirus, but neutralizing antibodies against HPV16, -31, -33, -45, or -58 were rarely detected in their sera using a standard in vitro neutralization assay. To address this discrepancy, we developed a neutralization assay based on an in vitro infectivity mechanism that more closely mimics the in vivo infectious process, specifically by spaciotemporally separating primary and secondary receptor engagement and correspondingly by altering the timing of exposure of the dominant L2 cross-neutralizing epitopes to the antibodies. With the new assay, titers in the 100 to 10,000 range were measured for most sera, whereas undetectable neutralizing activities were observed with the standard assay. In vitro neutralizing titers measured in the serum of mice after passive transfer of rabbit L2 immune serum correlated with protection from cervicovaginal challenge of the mice. This "L2-based" in vitro neutralization assay should prove useful in critically evaluating the immunogenicity of L2 vaccine candidates in preclinical studies and future clinical trials.
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Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Proteínas de la Cápside/inmunología , Pruebas de Neutralización/métodos , Proteínas Oncogénicas Virales/inmunología , Vacunas contra Papillomavirus/inmunología , Animales , Modelos Animales de Enfermedad , Femenino , Inmunización Pasiva , Ratones , Ratones Endogámicos BALB C , Infecciones por Papillomavirus/prevención & control , Conejos , Sensibilidad y EspecificidadRESUMEN
The induction of persistent intraepithelial CD8+ T cell responses may be key to the development of vaccines against mucosally transmitted pathogens, particularly for sexually transmitted diseases. Here we investigated CD8+ T cell responses in the female mouse cervicovaginal mucosa after intravaginal immunization with human papillomavirus vectors (HPV pseudoviruses) that transiently expressed a model antigen, respiratory syncytial virus (RSV) M/M2, in cervicovaginal keratinocytes. An HPV intravaginal prime/boost with different HPV serotypes induced 10-fold more cervicovaginal antigen-specific CD8+ T cells than priming alone. Antigen-specific T cell numbers decreased only 2-fold after 6 months. Most genital antigen-specific CD8+ T cells were intra- or subepithelial, expressed αE-integrin CD103, produced IFN-γ and TNF-α, and displayed in vivo cytotoxicity. Using a sphingosine-1-phosphate analog (FTY720), we found that the primed CD8+ T cells proliferated in the cervicovaginal mucosa upon HPV intravaginal boost. Intravaginal HPV prime/boost reduced cervicovaginal viral titers 1,000-fold after intravaginal challenge with vaccinia virus expressing the CD8 epitope M2. In contrast, intramuscular prime/boost with an adenovirus type 5 vector induced a higher level of systemic CD8+ T cells but failed to induce intraepithelial CD103+CD8+ T cells or protect against recombinant vaccinia vaginal challenge. Thus, HPV vectors are attractive gene-delivery platforms for inducing durable intraepithelial cervicovaginal CD8+ T cell responses by promoting local proliferation and retention of primed antigen-specific CD8+ T cells.
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Linfocitos T CD8-positivos/inmunología , Papillomavirus Humano 16/genética , Vacunas contra Papillomavirus/administración & dosificación , Vacunación , Administración Intravaginal , Animales , Antígenos Virales/biosíntesis , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/fisiología , Proliferación Celular , Citotoxicidad Inmunológica , Femenino , Genes Reporteros , Vectores Genéticos , Células HEK293 , Papillomavirus Humano 16/inmunología , Humanos , Inmunización Secundaria , Memoria Inmunológica , Interferón gamma/metabolismo , Luciferasas de Luciérnaga/biosíntesis , Luciferasas de Luciérnaga/genética , Ganglios Linfáticos/inmunología , Ratones , Ratones Endogámicos BALB C , Membrana Mucosa/inmunología , Membrana Mucosa/virología , Vacunas contra Papillomavirus/genética , Infecciones por Virus Sincitial Respiratorio/prevención & control , Virus Sincitiales Respiratorios/inmunología , Bazo/inmunología , Estadísticas no Paramétricas , Factor de Necrosis Tumoral alfa/metabolismo , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Virus Vaccinia/genética , Virus Vaccinia/inmunología , Vagina/inmunología , Vagina/virologíaRESUMEN
The major capsid protein (L1) of human papillomaviruses (HPV) expressed in heterologous systems assembles into virus-like particles (VLPs). We report cloning and expression of codon optimized HPV L1 genes of the two high-risk HPV types 16 and 18 in methylotropic yeast, Pichia pastoris. The VLPs produced in P. pastoris were subjected to three step purification method involving density gradient centrifugations and size exclusion chromatography. The enriched VLPs were characterized using conformation-specific monoclonal antibodies in ELISA and by transmission electron microscopy. Mice immunized with a bivalent HPV16 and HPV18 VLPs developed high serum antibody titers to both HPV types that persisted for 190 days post vaccination. Serum of mice immunized with the HPV-VLP preparations could neutralize homologous pseudoviruses in an in vitro assays. Our results demonstrate that the L1 proteins expressed in P. pastoris fold properly as evidenced by assembly into VLPs and induction of type-specific neutralizing antibody response in mice. This work constitutes a step towards developing an alternate production platform for generating an affordable HPV vaccine to meet the needs of developing countries.
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Proteínas de la Cápside , Papillomavirus Humano 16/genética , Papillomavirus Humano 18/genética , Proteínas Oncogénicas Virales , Vacunas contra Papillomavirus/inmunología , Pichia/genética , Vacunas de Partículas Similares a Virus/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Clonación Molecular , Células HEK293 , Papillomavirus Humano 16/inmunología , Papillomavirus Humano 18/inmunología , Humanos , Ratones , Ratones Endogámicos BALB C , Proteínas Oncogénicas Virales/química , Proteínas Oncogénicas Virales/genética , Proteínas Oncogénicas Virales/inmunología , Infecciones por Papillomavirus/prevención & controlRESUMEN
The mechanisms of human papillomavirus (HPV) neutralization by antibodies are incompletely understood. We have used HPV16 pseudovirus infection of HaCaT cells to analyze how several neutralizing monoclonal antibodies (MAbs) generated against HPV16 L1 interfere with the process of keratinocyte infection. HPV16 capsids normally bind to both the cell surface and extracellular matrix (ECM) of HaCaT cells. Surprisingly, two strongly neutralizing MAbs, V5 and E70, did not prevent attachment of capsids to the cell surface. However, they did block association with the ECM and prevented internalization of cell surface-bound capsids. In contrast, MAb U4 prevented binding to the cell surface but not to the ECM. The epitope recognized by U4 was inaccessible when virions were bound to the cell surface but became accessible after endocytosis, presumably coinciding with receptor detachment. Treatment of capsids with heparin, which is known to interfere with binding to cell surface heparan sulfate proteoglycans (HSPGs), also resulted in HPV16 localization to the ECM. These results suggest that the U4 epitope on the intercapsomeric C-terminal arm is likely to encompass the critical HSPG interaction residues for HPV16, while the V5 and E70 epitopes at the apex of the capsomer overlap the ECM-binding sites. We conclude that neutralizing antibodies can inhibit HPV infection by multiple distinct mechanisms, and understanding these mechanisms can add insight to the HPV entry processes.
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Anticuerpos Monoclonales/farmacología , Anticuerpos Antivirales/farmacología , Membrana Celular/virología , Matriz Extracelular/virología , Papillomavirus Humano 16/efectos de los fármacos , Virión/inmunología , Cápside/metabolismo , Membrana Celular/metabolismo , Epítopos/inmunología , Matriz Extracelular/metabolismo , Proteoglicanos de Heparán Sulfato/metabolismo , Heparina/farmacología , Papillomavirus Humano 16/inmunología , Papillomavirus Humano 16/metabolismo , Humanos , Queratinocitos/virología , Pruebas de Neutralización , Virión/efectos de los fármacos , Virión/metabolismoRESUMEN
We identified the strategies to induce a CTL response to human papillomavirus (HPV) 16 E2 in HLA-A2 transgenic mice (AAD). A chimeric HPV16 virus-like particle (VLP) that includes full length HPV16 E7 and E2 (VLP-E7E2) was generated. The combination of E2 and E7 has the advantage that E2 is expressed in early dysplasia and neoplasia lesions, where E7 is expressed in more advance lesions. Since T cell response to E2 is less defined, we first evaluated the strategies to enhancing CD8(+) T cell responses to HPV E7, using different combinations of immune-modulators with VLP-E7E2. Data showed that the CTL response to E7 could be significantly enhanced by coinjection of GM-CSF and anti-CD40 antibodies with chimeric VLP-E7E2 without adjuvant. However, using the same combination, a low level of CD8(+) T cell response to E2 was detected. To enhance the CD8+ T cell response to E2, we analyzed T cell epitopes from E2 sequence. A heterogenous prime-boost with chimeric VLP-E7E2 and E2 peptides was performed. The data showed that the priming with chimeric VLP-E7E2, followed by boosting with E2 peptides, gave a better CTL response than 2 immunizations with E2 peptides. The enhanced immunity is due to the increase of CD11c(+) and CD11c(+) CD40(+) double positive dendritic cells in mice that received immune-modulators, GM-CSF and anti-CD40. Furthermore, the level of anti-L1 antibodies remains similar in mice immunized with chimeric VLP with/without immune-modulators. Thus, the data suggested that the chimeric VLP-E7E2 has a therapeutic potential for the treatment of HPV-associated CINs and cancer without diminishing VLPs potential as a prophylactic vaccine by inducing anti-L1 antibodies against free virus.
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Vacunas contra el Cáncer/farmacología , Proteínas de Unión al ADN/inmunología , Complejo de Antígeno L1 de Leucocito/efectos de los fármacos , Proteínas de Fusión Oncogénica/inmunología , Proteínas Oncogénicas Virales/inmunología , Linfocitos T Citotóxicos/inmunología , Proteínas Virales de Fusión/inmunología , Animales , Anticuerpos Antivirales/farmacología , Antígeno CD11c/inmunología , Antígenos CD40/inmunología , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/inmunología , Vacunas contra el Cáncer/uso terapéutico , Células Dendríticas/inmunología , Quimioterapia Combinada , Epítopos de Linfocito T/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Antígeno HLA-A2/genética , Humanos , Complejo de Antígeno L1 de Leucocito/inmunología , Ratones , Ratones Transgénicos , Proteínas E7 de PapillomavirusRESUMEN
The papillomavirus capsid is a nonenveloped icosahedral shell formed by the viral major structural protein, L1. It is known that disulfide bonds between neighboring L1 molecules help to stabilize the capsid. However, the kinetics of inter-L1 disulfide bond formation during particle morphogenesis have not previously been examined. We have recently described a system for producing high-titer papillomavirus-based gene transfer vectors (also known as pseudoviruses) in mammalian cells. Here we show that papillomavirus capsids produced using this system undergo a maturation process in which the formation of inter-L1 disulfide bonds drives condensation and stabilization of the capsid. Fully mature capsids exhibit improved regularity and resistance to proteolytic digestion. Although capsid maturation for other virus types has been reported to occur in seconds or minutes, papillomavirus capsid maturation requires overnight incubation. Maturation of the capsids of human papillomavirus types 16 and 18 proceeds through an ordered accumulation of dimeric and trimeric L1 species, whereas the capsid of bovine papillomavirus type 1 matures into more extensively cross-linked forms. The presence of encapsidated DNA or the minor capsid protein, L2, did not have major effects on the kinetics or extent of capsid maturation. Immature capsids and capsids formed from L1 mutants with impaired disulfide bond formation are infectious but physically fragile. Consequently, capsid maturation is essential for efficient purification of papillomavirus-based gene transfer vectors. Despite their obvious morphological differences, mature and immature capsids are similarly neutralizable by various L1- and L2-specific antibodies.
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Cápside/fisiología , Papillomaviridae/fisiología , Animales , Papillomavirus Bovino 1/genética , Papillomavirus Bovino 1/fisiología , Cápside/ultraestructura , Bovinos , Línea Celular , Cisteína/química , ADN Viral/análisis , Disulfuros/química , Humanos , Microscopía Electrónica , Mutagénesis Sitio-Dirigida , Pruebas de Neutralización , Oxidación-Reducción , Papillomaviridae/clasificación , Papillomaviridae/genética , Papillomaviridae/ultraestructura , Tripsina , Proteínas Estructurales Virales/genética , Proteínas Estructurales Virales/fisiología , Ensamble de VirusRESUMEN
Vaccination with papillomavirus L2 has been shown to induce neutralizing antibodies that protect against homologous type infection and cross-neutralize a limited number of genital HPVs. Surprisingly, we found that antibodies to bovine papillomavirus (BPV1) L2 amino acids 1-88 induced similar titers of neutralizing antibodies against Human papillomavirus (HPV)16 and 18 and BPV1 pseudoviruses and also neutralized HPV11 native virions. These antibodies also neutralized each of the other pseudovirus types tested, HPV31, HPV6 and Cottontail rabbit papillomavirus (CRPV) pseudoviruses, albeit with lower titers. HPV16, HPV18, HPV31, HPV6 and CRPV L2 anti-sera also displayed some cross-neutralization, but the titers were lower and did not encompass all pseudoviruses tested. This study demonstrates the presence of broadly cross-neutralizing epitopes at the N-terminus of L2 that are shared by cutaneous and mucosal types and by types that infect divergent species. BPV1 L2 was exceptionally effective at inducing cross-neutralizing antibodies to these shared epitopes.