Inhibitory Effects Of Extracellular Vesicles From Symbiotic Vaginal Lactobacilli Against Human Papillomavirus Infection

Human papillomavirus（HPVs）are non-enveloped DNA viruses that infect epithelial cells.High-risk HPV infection can lead to the development of cervical cancer and represent a significant health burden worldwide.Preventive vaccines against certain HPV types are currently available on the market,but have limited role in already infected populations,which means that new antiviral infection drugs need to be developed to treat established HPV infections and associated diseases.In recent years,it has been increasingly recognized that the composition of the vaginal microbiome may influence the persistence of HPV infection and the development of cervical diseases.The bacterial community in the vagina is an important defense against pathogens including sexually transmitted infections.Probiotics can improve host health by shaping the vaginal microecological balance and immune homeostasis.In most women,the vaginal and cervical microbiome is dominated by the Lactobacillus species（spp.）,that benefit the host through a symbiotic relationship.Extracellular vesicles（EVs）have been found to be particles released by all cells and are enriched with various active molecules from the parent cell.Bacteria can use their extracellular vesicles to interact with neighboring bacteria,thereby regulating the microbial environment and the bacteria themselves,playing an important role in the physiology and pathogenesis of cells.In this study,we used Lactobacillus gasseri,a highly immunoreactive bacterium in the female vagina,as a target strain to explore whether this strain can produce EVs and whether these EVs can inhibit the infection of host cells by human papillomavirus,with the aim of developing a new local anti-infective immune adjuvant for diseases associated with HPV infection.The specific findings of the study are as follows:1.Preparation and characterization of extracellular vesicles of Lactobacillus gasseri.A standard procedure for the isolation and purification of EVs from Lactobacillus gasseri culture supernatants was established based on ultracentrifugation.Then the purified L.gasseri-derived EVs were analyzed by transmission electron microscopy,nanoparticle tracking analysis（NTA）and Western Blot.The results showed that L.gasseri-derived EVs could secrete nanosized extracellular vesicles,and the particle size distribution of most EVs was between 90-130 nm.NTA analysis showed that the particle concentration of EVs was 1.4×10¹⁰particles/m L,and the average particle size was 102.1±4.1 nm.Western Blot results showed that the maeker protein TSG101 was detected in EVs.The ability of L.gasseri-derived EVs to deliver and release metronidazole was further verified by inhibition experiments.Firstly,metronidazole was successfully encapsulated by extracellular vesicles through ultrasonic degradation method to obtain EVs-metronidazole,and the encapsulation rate was 13.49%and the drug loading rate was 25.49%measured using spectrophotometer.The size and Zeta potential of EVs and EVs-metronidazole nanoparticles were examined by nanoparticle sizer,and the average particle sizes of empty-loaded EVs and EVs-metronidazole were found to be 101.5±8.7 nm and 125.6±3.6 nm,with Zeta potentials of-10.1±1.81 m V and-17.3±2.28 m V,respectively.In the drug release experiment,the results showed that EVs had a certain sustained release effect on drugs.Preliminary studies on the bacterial inhibitory activity of EVs and EVs-metronidazole were conducted by the double-layer agar method,and found that they had a positive effect on E.coli,S.aureus and L.monocytogenes,with the strongest inhibitory effect on L.monocytogenes.The hemolysis assay demonstrated that both EVs and EVs-metronidazole did not exhibit haemolysis and have a good biosafety profile.2.Establishment of a human papillomavirus pseudovirus assay system.In this experiment,the HPV structural protein expression plasmid p16L1L2 and fluorescent reporter plasmid EGFP-N2 were simultaneously transfected into cells using Lipofectamine 2000 to successfully produce a high titer HPV16 pseudovirus with good infectivity.Electron microscopy confirmed that the pseudovirus was a well-assembled icosahedral non-enveloped particle with a uniform diameter of about 60 nm,containing structural proteins L1 and L2.This is consistent with the reported properties of HPV16 pseudoviruses,indicating that the obtained pseudoviruses can be used in the study of HPV biology instead of natural viruses.3.Inhibitory effects of Lactobacillus gasseri against high risk papillomavirus types in vitro.Vaginal microorganisms,mainly Lactobacillus gasseri,play a key role in the prevention of HPV transmission and here we investigated whether the anti-HPV infection effect of Lactobacillus gasseri is mediated by extracellular vesicles released by these bacteria.In this experiment,human cervical cancer cells（He La cells）and human embryonic kidney epithelial cells（293FT cells）were infected with HPV16pseudovirus,and treated with EVs released by L.gasseri.Firstly,the cytotoxicity of L.gasseri-derived EVs was measured by MTT assay,which showed no significant cell killing effect at a certain concentration range,suggesting that the EVs have good potential for drug delivery with very low toxicity.Confocal microscopy results showed that EVs can be effectively internalized into cells to exter their antiviral infection effects.Subsequently,the effect of EVs on HPV16 infection was evaluated at different concentrations and time points,the results showed that EVs treatment significantly reduced intracellular green fluorescence protein expression in a dose-dependent manner.Pretreatment of HPV16 pseudovirus with EVs prior to infection or addition of EVs during adsorption both significantly reduced HPV16 pseudovirus infection of cells.Therefore,we hypothesize that EVs can interact directly with HPV viral particles and block some early steps of the life cycle of HPV16 after binding to cells.In summary,the probiotics Lactobacillus gasseri were selected in this study,which can effectively prevent viral infection,and confirmed to produce and secrete extracellular vesicles containing the active components of the parent bacteria.Using metronidazole as a drug model,it was demonstrated that EVs released by L.gasseri could effectively encapsulate,deliver and release drugs with good antibacterial effect on E.coli,S.aureus and L.monocytogenes.EVs inhibited human papillomavirus infection in vitro by constructing a cell model constructed by HPV16 pseudovirus.These results provide new insights into the prevention and treatment of HPV virus infection.