RESUMO
Human papillomavirus (HPV) infection poses a significant health challenge, particularly in low- and middle-income countries (LMIC), where limited healthcare access and awareness hinder vaccine accessibility. To identify alternative HPV targeting interventions, we previously reported on surfactant protein A (SP-A) as a novel molecule capable of recognising HPV16 pseudovirions (HPV16-PsVs) and reducing infection in a murine cervicovaginal HPV challenge model. Building on these findings, our current study aimed to assess SP-A's suitability as a broad-spectrum HPV-targeting molecule and its impact on innate immune responses. We demonstrate SP-A's ability to agglutinate and opsonise multiple oncogenic HPV-PsVs types, enhancing their uptake and clearance by RAW264.7 murine macrophages and THP-1 human-derived immune cells. The SP-A opsonisation of HPV not only led to increased lysosomal accumulation in macrophages and HaCaT keratinocytes but also resulted in a decreased infection of HaCaT cells, which was further decreased when co-cultured with innate immune cells. An analysis of human innate immune cell cytokine profiles revealed a significant inflammatory response upon SP-A exposure, potentially contributing to the overall inhibition of HPV infection. These results highlight the multi-layered impact of SP-A on HPV, innate immune cells and keratinocytes and lay the basis for the development of alternative prophylactic interventions against diverse HPV types.
Assuntos
Macrófagos , Infecções por Papillomavirus , Proteína A Associada a Surfactante Pulmonar , Humanos , Animais , Camundongos , Infecções por Papillomavirus/prevenção & controle , Infecções por Papillomavirus/imunologia , Infecções por Papillomavirus/virologia , Proteína A Associada a Surfactante Pulmonar/metabolismo , Proteína A Associada a Surfactante Pulmonar/imunologia , Células RAW 264.7 , Macrófagos/imunologia , Macrófagos/metabolismo , Imunidade Inata , Queratinócitos/metabolismo , Queratinócitos/virologia , Queratinócitos/imunologia , Citocinas/metabolismo , Células HaCaT , Células THP-1 , FemininoRESUMO
Infection by oncogenic human papillomavirus (HPV) is the primary cause of cervical cancer and other anogenital cancers. The majority of cervical cancer cases occur in low- and middle- income countries (LMIC). Concurrent infection with Human Immunodeficiency Virus (HIV) further increases the risk of HPV infection and exacerbates disease onset and progression. Highly effective prophylactic vaccines do exist to combat HPV infection with the most common oncogenic types, but the accessibility to these in LMIC is severely limited due to cost, difficulties in accessing the target population, cultural issues, and maintenance of a cold chain. Alternative preventive measures against HPV infection that are more accessible and affordable are therefore also needed to control cervical cancer risk. There are several efforts in identifying such alternative prophylactics which target key molecules involved in early HPV infection events. This review summarizes the current knowledge of the initial steps in HPV infection, from host cell-surface engagement to cellular trafficking of the viral genome before arrival in the nucleus. The key molecules that can be potentially targeted are highlighted, and a discussion on their applicability as alternative preventive means against HPV infection, with a focus on LMIC, is presented.
Assuntos
Alphapapillomavirus/efeitos dos fármacos , Interações Hospedeiro-Patógeno/fisiologia , Infecções por Papillomavirus/etiologia , Infecções por Papillomavirus/prevenção & controle , Alphapapillomavirus/metabolismo , Alphapapillomavirus/patogenicidade , Feminino , Furina/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Terapia de Alvo Molecular/métodos , Polissacarídeos/química , Polissacarídeos/farmacologiaRESUMO
Understanding and modulating the early steps in oncogenic Human Papillomavirus (HPV) infection has great cancer-preventative potential, as this virus is the etiological agent of virtually all cervical cancer cases and is associated with many other anogenital and oropharyngeal cancers. Previous work from our laboratory has identified cell-surface-expressed vimentin as a novel HPV16 pseudovirus (HPV16-PsVs)-binding molecule modulating its infectious potential. To further explore its mode of inhibiting HPV16-PsVs internalisation, we supplemented it with exogenous recombinant human vimentin and show that only the globular form of the molecule (as opposed to the filamentous form) inhibited HPV16-PsVs internalisation in vitro. Further, this inhibitory effect was only transient and not sustained over prolonged incubation times, as demonstrated in vitro and in vivo, possibly due to full-entry molecule engagement by the virions once saturation levels have been reached. The vimentin-mediated delay of HPV16-PsVs internalisation could be narrowed down to affecting multiple steps during the virus' interaction with the host cell and was found to affect both heparan sulphate proteoglycan (HSPG) binding as well as the subsequent entry receptor complex engagement. Interestingly, decreased pseudovirus internalisation (but not infection) in the presence of vimentin was also demonstrated for oncogenic HPV types 18, 31 and 45. Together, these data demonstrate the potential of vimentin as a modulator of HPV infection which can be used as a tool to study early mechanisms in infectious internalisation. However, further refinement is needed with regard to vimentin's stabilisation and formulation before its development as an alternative prophylactic means.
Assuntos
Papillomavirus Humano 16/fisiologia , Vimentina/farmacologia , Internalização do Vírus , Alphapapillomavirus/fisiologia , Animais , Membrana Celular/virologia , Feminino , Células HEK293 , Proteoglicanas de Heparan Sulfato/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Papillomavirus/virologia , Conformação Proteica , Receptores Virais/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Vimentina/química , Pseudotipagem Viral , Vírion/fisiologiaRESUMO
Abstract: Infection by oncogenic human papillomavirus (HPV) is the principle cause of cervical cancer and other anogenital cancers. The majority of cervical cancer cases occur in low- and middle-income countries (LMIC). Prophylactic vaccines exist to combat HPV infection but accessibility to these in LMIC is limited. Alternative preventative measures against HPV infection are therefore also needed to control cervical cancer risk. HPV employs multiple mechanisms to evade the host immune response. Therefore, an approach to promote HPV recognition by the immune system can reduce infection. Surfactant proteins A and D (SP-A and SP-D) are highly effective innate opsonins of pathogens. Their function is primarily understood in the lung, but they are also expressed at other sites of the body, including the female reproductive tract (FRT). We hypothesized that raised levels of SP-A and/or SP-D may enhance immune recognition of HPV and reduce infection. Co-immunoprecipitation and flow cytometry experiments showed that purified human SP-A protein directly bound HPV16 pseudovirions (HPV16-PsVs), and the resulting HPV16-PsVs/SP-A complex enhanced uptake of HPV16-PsVs by RAW264.7 murine macrophages. In contrast, a recombinant fragment of human SP-D bound HPV16-PsVs weakly and had no effect on viral uptake. To assess if SP-A modulates HPV16-PsVs infection in vivo, a murine cervicovaginal challenge model was applied. Surprisingly, neither naïve nor C57BL/6 mice challenged with HPV16-PsVs expressed SP-A in the FRT. However, pre-incubation of HPV16-PsVs with purified human SP-A at a 1:10 (w/w) ratio significantly reduced the level of HPV16-PsV infection. When isolated cells from FRTs of naïve C57BL/6 mice were incubated with HPV16-PsVs and stained for selected innate immune cell populations by flow cytometry, significant increases in HPV16-PsVs uptake by eosinophils, neutrophils, monocytes, and macrophages were observed over time using SP-A-pre-adsorbed virions compared to control particles. This study is the first to describe a biochemical and functional association of HPV16 virions with the innate immune molecule SP-A. We show that SP-A impairs HPV16-PsVs infection and propose that SP-A is a potential candidate for use in topical microbicides which provide protection against new HPV infections.
RESUMO
BACKGROUND: To determine if variations exist in the KSHV host receptor EPHA2's coding region that affect KSHV infectivity and/or KS prevalence among South African HIV-infected patients. METHODS: A retrospective candidate gene association study was performed on 150 patients which were randomly selected from a total of 756 HIV-infected patients and grouped according to their KS status and KSHV serodiagnosis; namely group 1: KS+/KSHV+; group 2: KS-/KSHV+; group 3: KS-/KSHV-. Peripheral blood DNA was used to extract DNA and PCR amplify and sequence the entire EPHA2 coding region, which was compared to the NCBI reference through multiple alignment. RESULTS: 100% (95% CI 92.9-100%) of the KS positive patients, and 31.6% (95% CI 28.3-35.1%) of the KS negative patients were found to be KSHV seropositive. Aggregate variation across the entire EPHA2 coding region identified an association with KS (OR = 6.6 (95% CI 2.8, 15.9), p = 2.2 × 10-5). This was primarily driven by variation in the functionally important protein tyrosine kinase domain (Pkinase-Tyr; OR = 4.9 (95% CI 1.9, 12.4), p = 0.001) and the sterile-α-motif (SAM; OR = 13.8 (95% CI 1.7, 111.6), p = 0.014). Mutation analysis revealed two novel, non-synonymous heterozygous variants (c.2254 T > C: OR undefined, adj. p = 0.02; and c.2990 G > T: OR undefined, adj. p = 0.04) in Pkinase-Tyr and SAM, respectively, to be statistically associated with KS; and a novel heterozygous transition (c.2727C > T: OR = 6.4 (95% CI 1.4, 28.4), adj. p = 0.03) in Pkinase-Tyr to be statistically associated with KSHV. CONCLUSIONS: Variations in the KSHV entry receptor gene EPHA2 affected susceptibility to KSHV infection and KS development in a South African HIV-infected patient cohort.