RESUMEN
Cows produce antibodies with a disulfide-bonded antigen-binding domain embedded within ultralong heavy chain third complementarity determining regions. This "knob" domain is analogous to natural cysteine-rich peptides such as knottins in that it is small and stable but can accommodate diverse loops and disulfide bonding patterns. We immunized cattle with SARS-CoV-2 spike and found ultralong CDR H3 antibodies that could neutralize several viral variants at picomolar IC50 potencies in vitro and could protect from disease in vivo. The independent CDR H3 peptide knobs were expressed and maintained the properties of the parent antibodies. The knob interaction with SARS-CoV-2 spike was revealed by electron microscopy, X-ray crystallography, NMR spectroscopy, and mass spectrometry and established ultralong CDR H3-derived knobs as the smallest known recombinant independent antigen-binding fragment. Unlike other vertebrate antibody fragments, these knobs are not reliant on the immunoglobulin domain and have potential as a new class of therapeutics.
Asunto(s)
COVID-19 , SARS-CoV-2 , Femenino , Animales , Bovinos , Anticuerpos , Fragmentos Fab de Inmunoglobulinas/genética , DisulfurosRESUMEN
Immunosuppression increases the risk of cancers that are associated with viral infection1. In particular, the risk of squamous cell carcinoma of the skin-which has been associated with beta human papillomavirus (ß-HPV) infection-is increased by more than 100-fold in immunosuppressed patients2-4. Previous studies have not established a causative role for HPVs in driving the development of skin cancer. Here we show that T cell immunity against commensal papillomaviruses suppresses skin cancer in immunocompetent hosts, and the loss of this immunity-rather than the oncogenic effect of HPVs-causes the markedly increased risk of skin cancer in immunosuppressed patients. To investigate the effects of papillomavirus on carcinogen-driven skin cancer, we colonized several strains of immunocompetent mice with mouse papillomavirus type 1 (MmuPV1)5. Mice with natural immunity against MmuPV1 after colonization and acquired immunity through the transfer of T cells from immune mice or by MmuPV1 vaccination were protected against skin carcinogenesis induced by chemicals or by ultraviolet radiation in a manner dependent on CD8+ T cells. RNA and DNA in situ hybridization probes for 25 commensal ß-HPVs revealed a significant reduction in viral activity and load in human skin cancer compared with the adjacent healthy skin, suggesting a strong immune selection against virus-positive malignant cells. Consistently, E7 peptides from ß-HPVs activated CD8+ T cells from unaffected human skin. Our findings reveal a beneficial role for commensal viruses and establish a foundation for immune-based approaches that could block the development of skin cancer by boosting immunity against the commensal HPVs present in all of our skin.
Asunto(s)
Carcinoma de Células Escamosas/inmunología , Carcinoma de Células Escamosas/prevención & control , Papillomaviridae/inmunología , Infecciones por Papillomavirus/inmunología , Infecciones por Papillomavirus/virología , Neoplasias Cutáneas/prevención & control , Neoplasias Cutáneas/virología , Simbiosis , Anciano , Anciano de 80 o más Años , Animales , Linfocitos T CD8-positivos/inmunología , Carcinogénesis/efectos de la radiación , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/virología , Femenino , Humanos , Huésped Inmunocomprometido/inmunología , Masculino , Ratones , Persona de Mediana Edad , Oncogenes , Papillomaviridae/genética , Papillomaviridae/patogenicidad , ARN Viral/análisis , ARN Viral/genética , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/patología , Rayos UltravioletaRESUMEN
The Antillean manatee Trichechus manatus manatus is an Endangered species living along the Atlantic coasts of the Americas from Florida (USA), throughout the Caribbean, to Brazil. In July 2020, a manatee with multiple wounds due to boat-inflicted trauma was rescued from the coast east of Cayo Mata, Salinas, Puerto Rico. This manatee had neutropenia, leukopenia, and monocytosis associated with immunosuppression and nutritional deficiency anemia, as well as bacteria and fungi within the lesions. The manatee had genital lesions which included papules and linear plaques, microscopically characterized by mucosal hyperplasia with cytopathic changes typical of papillomavirus infection. Superficial epithelial cells had strong nuclear immunolabeling when examined using a monoclonal antibody specific to papillomavirus. The sequencing data of PCR products with papillomavirus-specific degenerative primers indicated that these lesions contained a novel manatee papillomavirus (Trichechus manatus papillomavirus, TmPV). The genomic DNA was amplified using a rolling circle amplification, and fully sequenced to be 7586 bp (GenBank accession no. OK073977). Other TmPVs were previously isolated from Florida manatees T. manatus latirostris. This novel virus was designated TmPV type 5 (TmPV5) based on its genomic characterization and sequence comparison. The TmPV5 genome shared 50.7, 48.9, 69.4, and 62.1% similarities with TmPV1, TmPV2, TmPV3, and TmPV4, respectively. TmPV5 is classified in the genus Rhopapillomavirus together with other manatee papillomaviruses. After 2.5 mo of veterinary treatment and rehabilitation, the manatee recovered and was released. This is the first report of papillomatosis in a free-ranging Antillean manatee.
Asunto(s)
Papiloma , Trichechus manatus , Animales , Genitales , Papiloma/veterinaria , Papillomaviridae/genética , Puerto RicoRESUMEN
BACKGROUND: Human papillomavirus (HPV) etiology has become evident in head and neck cancers (HNCs) and HPV positivity showed a strong association with its malignant progression. Since aberrant DNA methylation is known to drive carcinogenesis and progression in HNCs, we investigated to determine target gene(s) associated with this modification. METHODS: We characterized epigenetic changes in tumor-related genes (TRGs) that are known to be associated with HNC development and its progression. RESULTS: The expression levels of 42 candidate HNC-associated genes were analyzed. Of these, 7 TGRs (CHFR, RARß, GRB7, EREG, RUNX2, RUNX3, and SMG-1) showed decreased expressions in HPV-positive (+) HNC cells compared with HPV-negative (-) HNC cells. When gene expression levels were compared corresponding to the DNA methylation conditions, GRB7 and EREG showed significant differential expression between HPV+ and HPV- cells, which suggested these genes as primary targets of epigenetic regulation in HPV-induced carcinogenesis. Furthermore, treatment with a demethylation agent, 5-aza-2'-deoxycytidine (5-aza-dc), caused restoration of EREG expression and was associated with hypomethylation of its promoter in HPV+ cells, while no changes was noted in HPV- cells. EREG promoter hypermethylation in HPV+ cells was confirmed using methylation-specific PCR (MS-PCR). CONCLUSION: We conclude that EREG is the target of epigenetic regulation in HPV+ HNCs and its suppressed expression through promoter hypermethylation is associated with the development of HPV-associated HNCs.
Asunto(s)
Alphapapillomavirus/genética , Epigénesis Genética , Epirregulina/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Alphapapillomavirus/patogenicidad , Azacitidina , Carcinogénesis/genética , Línea Celular Tumoral , ADN (Citosina-5-)-Metiltransferasa 1/genética , ADN (Citosina-5-)-Metiltransferasas/genética , Metilación de ADN/genética , ADN Metiltransferasa 3A , Decitabina , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Proteínas de Neoplasias/genética , Regiones Promotoras Genéticas/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Carcinoma de Células Escamosas de Cabeza y Cuello/virologíaRESUMEN
Previous studies of naturally occurring mouse papillomavirus (PV) MmuPV1-induced tumors in B6.Cg-Foxn1nu/nu mice suggest that T cell deficiency is necessary and sufficient for the development of such tumors. To confirm this, MmuPV1-induced tumors were transplanted from T cell-deficient mice into immunocompetent congenic mice. Consequently, the tumors regressed and eventually disappeared. The elimination of MmuPV1-infected skin/tumors in immunocompetent mice was consistent with the induction of antitumor T cell immunity. This was confirmed by adoptive cell experiments using hyperimmune splenocytes collected from graft-recipient mice. In the present study, such splenocytes were injected into T cell-deficient mice infected with MmuPV1, and they eliminated both early-stage and fully formed tumors. We clearly show that anti-tumor T cell immunity activated during tumor regression in immunocompetent mice effectively eliminates tumors developing in T cell-deficient congenic mice. The results corroborate the notion that PV-induced tumors are strongly linked to the immune status of the host, and that PV antigens are major anti-tumor antigens. Successful anti-PV T cell responses should, therefore, lead to effective anti-tumor immune therapy in human PV-infected patients.
Asunto(s)
Modelos Animales de Enfermedad , Inmunidad Celular/inmunología , Papillomaviridae/inmunología , Infecciones por Papillomavirus/complicaciones , Neoplasias Cutáneas/prevención & control , Linfocitos T/inmunología , Animales , Femenino , Ratones , Ratones Congénicos , Ratones Endogámicos C57BL , Ratones Desnudos , Infecciones por Papillomavirus/virología , Neoplasias Cutáneas/etiología , Neoplasias Cutáneas/patologíaRESUMEN
Infection by mouse papillomavirus (PV), MmuPV1, of T cell-deficient, B6.Cg-Foxn1(nu)/J nude mice revealed that four, distinct squamous papilloma phenotypes developed simultaneously after infection of experimental mice. Papillomas appeared on the muzzle, vagina, and tail at or about day 42days post-inoculation. The dorsal skin developed papillomas and hair follicle tumors (trichoblastomas) as early as 26days after infection. Passive transfer of hyperimmune sera from normal congenic mice immunized with MmuPV1 virus-like particles (VLPs) to T cell-deficient strains of mice prevented infection by virions of experimental mice. This study provides further evidence that T cell deficiency is critical for tumor formation by MmuPV1 infection.
Asunto(s)
Papiloma/virología , Infecciones por Papillomavirus/virología , Neoplasias Cutáneas/virología , Linfocitos T/virología , Virión/metabolismo , Animales , Modelos Animales de Enfermedad , Ratones Congénicos , Ratones Desnudos , Ratones Transgénicos , Neoplasias Cutáneas/patología , Linfocitos T/inmunologíaRESUMEN
We isolated two new manatee papillomavirus (PV) types, TmPV3 and TmPV4, from a Florida manatee (Trichechus manatus latirostris). Two PV types were previously isolated from this species. TmPV1 is widely dispersed amongst manatees and a close-to-root PV; not much is known about TmPV2. The genomes of TmPV3 and TmPV4 were 7622 and 7771 bp in size, respectively. Both PVs had a genomic organization characteristic of all PVs, with one non-coding region and seven ORFs, including the E7 ORF that is absent in other cetacean PVs. Although these PVs were isolated from separate genital lesions of the same manatee, an enlarged E2/E4 ORF was found only in the TmPV4 genome. The full genome and L1 sequence similarities between TmPV3 and TmPV4 were 63.2 and 70.3 %, respectively. These genomes shared only 49.1 and 50.2 % similarity with TmPV1. The pairwise alignment of L1 nucleotide sequences indicated that the two new PVs nested in a monophyletic group of the genus Rhopapillomavirus, together with the cutaneotropic TmPV1 and TmPV2.
Asunto(s)
Clonación Molecular , Genoma Viral , Papillomaviridae/genética , Infecciones por Papillomavirus/veterinaria , Trichechus manatus/virología , Animales , ADN Viral/genética , Papillomaviridae/clasificación , Papillomaviridae/aislamiento & purificación , Infecciones por Papillomavirus/virología , FilogeniaRESUMEN
BACKGROUND: Current vaccines against Human Papillomavirus (HPV) are highly effective and based on recombinant virus-like particles (VLPs) of the major capsid protein L1. Since these vaccines are HPV type-specific and expensive for global implementation, an alternative, broader-spectrum immunogen would be the N-terminus of the minor capsid protein L2 that induces low titered broadly cross-neutralizing antibodies. Here we analyzed the reactivity of different synthetic L2 peptides containing N-terminus amino acids 17-36 in order to test their antigenicity. METHODS: Different synthetic peptides were designed to target the 17-36 amino acid sequences, present in highly antigenic amino-terminus of L2 protein. Six different peptides including Cys22-Cys28 disulfide bonded cyclized L2 peptide were examined for their antigenicity against mouse monoclonal antibody RG-1 and rabbit polyclonal antisera to HPV L2 by enzyme-linked immunosorbent assay (ELISA). RESULTS: Here we report that the cyclized form of synthetic L2 peptide, which is formed through Cys22-Cys28 disulfide bridges, has the highest reactivity to antibodies than other synthetic L2 peptides. CONCLUSION: A cyclized L2 peptide has potential to be an excellent candidate to formulate a low-cost, broadly protective pan-oncogenic HPV vaccine.
Asunto(s)
Proteínas de la Cápside/inmunología , Disulfuros/química , Epítopos/química , Epítopos/inmunología , Proteínas Oncogénicas Virales/inmunología , Infecciones por Papillomavirus/prevención & control , Fragmentos de Péptidos/farmacología , Vacunas Sintéticas/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Disulfuros/inmunología , Ensayo de Inmunoadsorción Enzimática , Humanos , Ratones , Datos de Secuencia Molecular , Papillomaviridae/inmunología , Infecciones por Papillomavirus/inmunología , Infecciones por Papillomavirus/virología , Vacunas contra Papillomavirus/inmunología , Vacunas contra Papillomavirus/uso terapéutico , Fragmentos de Péptidos/inmunología , Conejos , Homología de Secuencia de AminoácidoRESUMEN
BACKGROUND: Human papillomavirus (HPV)-positive oropharyngeal cancer is associated with improved survival and treatment response as compared to HPV-negative cancers. P16 overexpression is widely accepted as a surrogate marker for HPV positivity. METHODS: A total of 92 serum samples from 75 head and neck squamous cell carcinoma (HNSCC) patients were examined for HPV16 and 18 E7 antibodies by ELISA. Available tissue was tested for HPV-DNA by PCR, and p16 immunohistochemistry was obtained from a deidentified database. RESULTS: Of 75 HNSCC patients, 25 were HPV E7 seropositive. Seropositivity was strongly associated with cancers of the oropharynx, and correlated with positive p16 immunohistochemistry (IHC) and HPV-DNA. Post-treatment serum was available in a limited subset of patients, revealing a decrease in antibody titers following response to treatment. CONCLUSIONS: HPV E7 seropositivity correlated with positive tumor HPV-DNA and p16 expression, and was strongly associated with cancers of the oropharynx. E7 serology warrants further study as a potential biomarker in HPV-positive HNSCC.
Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Neoplasias de Cabeza y Cuello/metabolismo , Proteínas E7 de Papillomavirus/sangre , Infecciones por Papillomavirus/virología , Adulto , Anciano , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/virología , ADN Viral/genética , Ensayo de Inmunoadsorción Enzimática , Femenino , Neoplasias de Cabeza y Cuello/patología , Neoplasias de Cabeza y Cuello/virología , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/aislamiento & purificación , Humanos , Técnicas para Inmunoenzimas , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Infecciones por Papillomavirus/metabolismo , Infecciones por Papillomavirus/patología , Reacción en Cadena de la Polimerasa , Pronóstico , Estudios SeroepidemiológicosRESUMEN
Correctly folded virus-like particles (VLPs) of papillomavirus (PV) display conformationally dependent epitopes that are type specific, maintained on authentic virions, and induce neutralizing antibodies. Alignment of the L1 amino acid (aa) sequences of 84 PVs revealed that the lengths of their N-termini are diverse and that multiple, possible initiation methionine (met) codons exist. The L1 gene of MusPV (MmuPV1), that naturally infects immunodeficient laboratory mouse strain (NMRI-Foxn1(nu)/Foxn1(nu)), has four met codons at the 1st, 2nd, 28th, and 30th aas from its N-terminus. Of these, the 3rd and 4th mets, that are at the 28th and 30th aa position from the N-termius, respectively, are located at the position where most PVs have their first met. These two mets, located at the 9th and 11th from the YLPP conserved aas of most PVs, should be considered as consensus initiation codons of PV L1s. Three L1 proteins of MusPV, starting from the 2nd, 3rd, and 4th mets, were expressed using a baculovirus expression system and characterized for their ability to self-assemble into VLPs. While MusPV L1 proteins starting from the 2nd met expressed an L1 protein that did not fold into VLPs, the L1s starting from the 3rd and 4th mets generated correct VLPs in abundant quantities. We now conclude that the highest quantity and best quality VLPs are made from the consensus L1 met of MusPV.
Asunto(s)
Aminoácidos/genética , Proteínas de la Cápside/genética , Epítopos/genética , Papillomaviridae/genética , Animales , Anticuerpos Antivirales/genética , Ratones , Conformación Proteica , Alineación de Secuencia , Virión/genéticaRESUMEN
Chikungunya fever virus (CHIKV) is a mosquito-borne alphavirus that causes wide-spread human infections and epidemics in Asia, Africa and recently, in the Americas. CHIKV is considered a priority pathogen by CEPI and WHO. Despite recent approval of a live-attenuated CHIKV vaccine, development of additional vaccines is warranted due to the worldwide outbreaks of CHIKV. Previously, we developed immunization DNA (iDNA) plasmid capable of launching live-attenuated CHIKV vaccine in vivo. Here we report the use of CHIKV iDNA plasmid to prepare a novel, live-attenuated CHIKV vaccine V5040 with rearranged RNA genome. In V5040, genomic RNA was rearranged to encode capsid gene downstream from the glycoprotein genes. Attenuated mutations derived from experimental CHIKV 181/25 vaccine were also engineered into E2 gene of V5040. The DNA copy of rearranged CHIKV genomic RNA with attenuated mutations was cloned into iDNA plasmid pMG5040 downstream from the CMV promoter. After transfection in vitro, pMG5040 launched replication of V5040 virus with rearranged genome and attenuating E2 mutations. Furthermore, V5040 virus was evaluated in experimental murine models for general safety and immunogenicity. Vaccination with V5040 virus subcutaneously resulted in elicitation of CHIKV-specific, virus-neutralizing antibodies. The results warrant further evaluation of V5040 virus with rearranged genome as a novel live-attenuated vaccine for CHIKV.
Asunto(s)
Anticuerpos Antivirales , Fiebre Chikungunya , Virus Chikungunya , Genoma Viral , Vacunas Atenuadas , Vacunas Virales , Replicación Viral , Animales , Vacunas Atenuadas/inmunología , Vacunas Atenuadas/genética , Vacunas Atenuadas/administración & dosificación , Ratones , Virus Chikungunya/genética , Virus Chikungunya/inmunología , Vacunas Virales/inmunología , Vacunas Virales/genética , Vacunas Virales/administración & dosificación , Fiebre Chikungunya/prevención & control , Fiebre Chikungunya/inmunología , Fiebre Chikungunya/virología , Anticuerpos Antivirales/sangre , Femenino , Humanos , Chlorocebus aethiops , Anticuerpos Neutralizantes/sangre , Células Vero , Ratones Endogámicos BALB CRESUMEN
Mouse parvoviruses (MPVs) are small, single-stranded, 5 kb DNA viruses that are subclinical and endemic in many laboratory mouse colonies. MPVs cause more distinctive deleterious effects in immune-compromised or genetically-engineered mice than immuno-competent mice. At the University of Louisville (U of L), there was an unexpected increase of MPV sero-positivity for MPV infections in mouse colonies between January 2006 and February 2007, resulting in strategic husbandry changes aimed at controlling MPV spread throughout the animal facility. To investigate these MPVs, VP2 genes of seven MPVs were cloned and sequenced from eight documented incidences by PCR technology. The mutations in these VP2 genes were compared to those found at the Genbank database (NCBI; http://www.ncbi.nlm.nih.gov) and an intra-institutional phylogenetic tree for MPV infections at U of L was constructed. We discovered that the seven MPV isolates were different from those in Genbank and were not identical to each other. These MPVs were designated MPV-UL1 to 7; none of them were minute virus of mice (MVMs). Four isolates could be classified as MPV1, one was classified as MPV2, and two were defined as novel types with less than 96% and 94% homology with existing MPV types. Considering that all seven isolates had mutations in their VP2 genes and no mutations were observed in VP2 genes of MPV during a four-month time period of incubation, we concluded that all seven MPVs isolated at U of L between 2006 and 2007 probably originated from different sources. Serological survey for MPV infections verified that each MPV outbreak was controlled without further contamination within the institution.
Asunto(s)
Infecciones por Parvoviridae/virología , Parvovirus/genética , Filogenia , Enfermedades de los Roedores/virología , Animales , Proteínas de la Cápside/genética , Ratones/virología , Virus Diminuto del Ratón/genética , Infecciones por Parvoviridae/epidemiología , Infecciones por Parvoviridae/veterinaria , Parvovirus/aislamiento & purificación , Enfermedades de los Roedores/epidemiología , Homología de Secuencia de AminoácidoRESUMEN
Cutaneous squamous cell carcinoma (cSCC) is the second most common cancer, with increased incidence in immunosuppressed patients. ß-Human papillomavirus has been proposed as a contributor to cSCC risk partly on the basis of increased ß-human papillomavirus viral load and seropositivity observed among patients with cSCC. Experimental data in mice colonized with mouse papillomavirus type 1 suggest that T cell immunity against ß-human papillomavirus suppresses skin cancer in immunocompetent hosts, and the loss of this immunity leads to the increased risk of cSCC. In this study, we show that CD8+ T cell depletion in mouse papillomavirus type 1âcolonized mice that underwent skin carcinogenesis protocol led to increased viral load in the skin and seropositivity for antiâmouse papillomavirus type 1 antibodies. These findings provide evidence that compromised T cell immunity can be the link that connects increased ß-human papillomavirus detection to cSCC risk.
RESUMEN
Chikungunya fever virus (CHIKV) is a mosquito-borne alphavirus that causes wide-spread human infections and epidemics in Asia, Africa and recently, in the Americas. There is no approved vaccine and CHIKV is considered a priority pathogen by CEPI and WHO. Previously, we developed immunization DNA (iDNA) plasmid capable of launching live-attenuated CHIKV vaccine in vivo . Here we report the use of CHIKV iDNA plasmid to prepare a novel, live-attenuated CHIKV vaccine V5040 with rearranged RNA genome for improved safety. In V5040, genomic RNA was rearranged to encode capsid gene downstream from the glycoprotein genes. To secure safety profile, attenuated mutations derived from experimental CHIKV 181/25 vaccine were also engineered into E2 gene of V5040. The DNA copy of rearranged CHIKV genomic RNA with attenuated mutations was cloned into iDNA plasmid pMG5040 downstream from the CMV promoter. After transfection in vitro, pMG5040 launched replication of V5040 virus with rearranged genome and attenuating E2 mutations. Furthermore, V5040 virus was evaluated in experimental murine models for safety and immunogenicity. Vaccination with V5040 virus subcutaneously resulted in elicitation of CHIKV-specific, virus-neutralizing antibodies. The results warrant further evaluation of V5040 virus with rearranged genome as a novel live-attenuated vaccine for CHIKV.
RESUMEN
MusPV, a novel papillomavirus (PV) that naturally infects laboratory mice, was isolated and characterized from a colony of NMRI-Foxn1(nu)/Foxn1(nu) (nude) mice in India. Because MusPV may have been missed during routine pathogen screening of mice in colonies worldwide, a variety of detection methods are described to detect MusPV. The clinical and histologic lesions of productive MusPV infections fit PV-associated features, including papillomas, koilocytes within the stratum granulosum of the hyperplastic/acanthotic papillomatous epithelium, and the presence of intranuclear virus particles in koilocytotic cells visualized by electron microscopy. Antiserum against disrupted PV virions, isolated from another species (canine), identified conserved viral antigens in productively infected cells by immunohistochemistry. A rolling circle technique was used to amplify viral circular DNAs followed by endonuclease restriction enzyme digestion to determine the correct size of PV DNA. Consensus PV degenerative primers, My09/11, commonly used to detect many different types of PVs by polymerase chain reaction (PCR), particularly mucosotropic HPVs, also identified MusPV and all rodent PVs tested. Since there was one nucleotide mismatch between the My09/11 primer set and the MusPV template, a new primer set, MusPV-My09/11, was designed to specifically detect MusPV in latent infections and spontaneous MusPV-induced papillomas. Southern blot analysis verified the presence of full size PV DNA in infected tissues. Virus-like particles (VLPs), generated from MusPV L1 genes, provided a substrate for serological testing of naturally and experimentally infected mice. In summary, a series of diagnostic assays were developed and validated to detect MusPV infection in skin tumors and serological response in laboratory mice.
Asunto(s)
Papiloma/veterinaria , Papillomaviridae/aislamiento & purificación , Infecciones por Papillomavirus/veterinaria , Enfermedades de los Roedores/diagnóstico , Enfermedades Cutáneas Virales/veterinaria , Animales , Animales de Laboratorio , Secuencia de Bases , Cartilla de ADN/química , ADN Viral/análisis , ADN Viral/genética , Femenino , Ratones , Ratones Endogámicos , Ratones Desnudos , Datos de Secuencia Molecular , Papiloma/diagnóstico , Papiloma/virología , Papillomaviridae/genética , Infecciones por Papillomavirus/diagnóstico , Infecciones por Papillomavirus/virología , Enfermedades de los Roedores/virología , Enfermedades Cutáneas Virales/diagnóstico , Enfermedades Cutáneas Virales/virologíaRESUMEN
High-risk human papillomavirus (HPV) is among the most common causes of head and neck cancer (HNC) with increasing incidence. HPV-associated HNC patients' clinical response to treatment varies drastically, which has made treatment de-escalation clinical trials challenging. To address the need for noninvasive biomarkers that differentiate patient outcomes, serum antibodies to E7 oncoprotein levels were evaluated in serial serum specimens from HPV-positive HNC patients (n = 48). We have found that increasing antibodies to E7 throughout treatment correlates with increased cancer recurrence or progression to mortality (p = .004) with 100% specificity as a predictive test.
RESUMEN
A papillomavirus (PV) that naturally infects laboratory mice will provide an extremely valuable tool for PV research. We describe here the isolation, cloning and molecular analysis of the first novel laboratory-mouse PV, designated MusPV. This agent, recently identified in the tissues from florid and asymmetrical papillomas on the face of nude mice (NMRI-Foxn1(nu)/Foxn1(nu)), was demonstrated to be transmissible to immunocompetent mice (Ingle et al., 2010). The MusPV genome is 7510 bp in length, is organized similarly to those of other PVs and has at least seven ORFs (E1, E2, E4, E6, E7, L1 and L2). Phylogenetic analysis indicates that MusPV belongs to the π genus together with four other rodent PVs (McPV2, MaPV1, MmiPV and RnPV1). Of the rodent PVs, MusPV appears most closely related to Mastomys coucha PV (McPV2), with 65â% genomic homogeneity and 80â% L1 amino acid similarity. Rodent PVs, except for MnPV1, do not contain any identifiable retinoblastoma protein (RB) binding sites. MusPV has one putative RB-binding site on the E6 protein but not on the E7 protein. Non-coding regions (NCRs) of PVs maintain multiple binding sites for transcription factors (TFs). The NCR of MusPV has numerous sites for TF binding, of which at least 13 TFs are common to all PVs in the π genus. MusPV provides a potentially valuable, novel mouse model to study mechanisms of infection, oncology and novel preventive and therapeutic approaches in mice that can be translated to diseases caused by human PVs.
Asunto(s)
ADN Viral/química , ADN Viral/genética , Genoma Viral , Papillomaviridae/genética , Papillomaviridae/aislamiento & purificación , Infecciones por Papillomavirus/veterinaria , Enfermedades de los Roedores/virología , Animales , Clonación Molecular , Análisis por Conglomerados , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Infecciones por Papillomavirus/virología , Filogenia , Análisis de Secuencia de ADN , Homología de SecuenciaRESUMEN
Serological assays intended for diagnosis, sero-epidemiologic assessment, and measurement of protective antibody titers upon infection or vaccination are essential for managing the SARS-CoV-2 pandemic. Serological assays measuring the antibody responses against SARS-CoV-2 antigens are readily available. However, some lack appropriate characteristics to accurately measure SARS-CoV-2 antibodies titers and neutralization. We developed an Enzyme-linked Immunosorbent Assay (ELISA) methods for measuring IgG, IgA, and IgM responses to SARS-CoV-2, Spike (S), receptor binding domain (RBD), and nucleocapsid (N) proteins. Performance characteristics of sensitivity and specificity have been defined. ELISA results show positive correlation with microneutralization and Plaque Reduction Neutralization assays with infectious SARS-CoV-2. Our ELISA was used to screen healthcare workers in Louisville, KY during the first wave of the local pandemic in the months of May and July 2020. We found a seropositive rate of approximately 1.4% and 2.3%, respectively. Our analyses demonstrate a broad immune response among individuals and suggest some non-RBD specific S IgG and IgA antibodies neutralize SARS-CoV-2.
Asunto(s)
Anticuerpos Antivirales/sangre , COVID-19/epidemiología , SARS-CoV-2/inmunología , Área Bajo la Curva , COVID-19/sangre , COVID-19/virología , Proteínas de la Nucleocápside de Coronavirus/inmunología , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunoglobulina A/sangre , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Kentucky/epidemiología , Pandemias , Fosfoproteínas/inmunología , Curva ROC , SARS-CoV-2/aislamiento & purificación , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/inmunologíaRESUMEN
Certain types of human papillomavirus (HPV) induce cancers, especially cervical cancers in women. A meta-analysis of the literature suggests that HPV is also associated with 20%-25% of non small cell lung carcinoma (NSCLC). Merkel cell Polyomavirus (MCPyV) causes most Merkel cell carcinomas in immunocompromised hosts, and is associated with some squamous carcinomas of skin in immunocompetent individuals. Since both oncogenic viruses appear to involve the tonsils and, therefore, have clear access to the lungs, we examined that the possible association of HPV and MCPyV infections with lung cancers, especially, NSCLC. DNAs were extracted from 51 frozen tissues from 30 lung cancer patients, and examined for the presence of HPV and MCPyV by PCR and DNA sequencing analysis. Clinical data was correlated with the viral status. HPVs were only detected in 5 adenocarcinomas (16.7% of all lung cancers examined). Three were positive for HPV-16, 1 for HPV-11 and 1 had an unknown HPV type DNA. None was identified in benign tissue. MCPyV DNA was detected in 5 NSCLCs (16.7%). Three of the 5 were identified in squamous carcinomas, 1 in adenocarcinoma, and 1 in an unspecified NSCLC. Two additional samples were positive for MCPyV DNA within benign adjacent lung tissue only. In one adenocarcinoma, HPV-11 was identified in an adenocarcinoma, and MCPyV DNA was detected in the adjacent "benign" tissue. HPV and MCPyV were directly associated with 33.3% of NSCLC. Further studies are necessary to determine if polyomavirus and papillomavirus are necessary risk factors for some cases of NSCLC.