[Stochastic Packaging of Cas Proteins into Exosomes].

CRISPR/Cas systems are perspective molecular tools for targeted manipulation with genetic materials, such as gene editing, regulation of gene transcription, modification of epigenome etc. While CRISPR/Cas systems proved to be highly effective for correcting genetic disorders and treating infectious diseases and cancers in experimental settings, clinical translation of these results is hampered by the lack of efficient CRISPR/Cas delivery vehicles. Modern synthetic nanovehicles based on organic and inorganic polymers have many disadvantages, including toxicity issues, the lack of targeted delivery, and complex and expensive production pipelines. In turn, exosomes are secreted biological nanoparticles that exhibit high biocompatibility, physico-chemical stability, and the ability to cross biological barriers. Early clinical trials found no toxicity associated with exosome injections. In the recent years, exosomes have been considered as perspective delivery vehicles for CRISPR/Cas systems in vivo. The aim of this study was to analyze the efficacy of CRISPR/Cas stochastic packaging into exosomes for several human cell lines. Here, we show that Cas9 protein is effectively localized into the compartment of intracellular exosome biogenesis, but stochastic packaging of Cas9 into exosomes turns to be very low (~1%). As such, stochastic packaging of Cas9 protein is very ineffective and cannot be used for gene editing purposes. Developing novel tools and technologies for loading CRISPR/Cas systems into exosomes is needed.

Therapeutic Vaccines Against Human Papilloma Viruses: Achievements and Prospects.

Human papillomaviruses of high carcinogenic risk (HR HPVs) are major etiological agents of malignant diseases of the cervix, vulva, penis, anal canal, larynx, head, and neck. Prophylactic vaccination against HPV, which mainly covers girls and women under 25, does not prevent vertical and horizontal HPV transmission in infants and children and does not have a therapeutic effect. As a result, a significant proportion of the population is not protected from the HPV infection and development of HPV-associated neoplastic transformation and cancer, which indicates the need for development and introduction of therapeutic HPV vaccines. Unlike prophylactic vaccines aimed at the formation of virus-neutralizing antibodies, therapeutic vaccines elicit cellular immune response leading to the elimination of infected and malignant cells expressing viral proteins. The ideal targets for vaccine immunotherapy are highly conserved HR HPV oncoproteins E6 and E7 expressed in precancerous and tumor tissues. Here, we describe expression of these proteins during different stages of HPV infection, their antigenic and immunogenic properties, and T-cell epitopes, the response to which correlates with natural regression of HPV-induced neoplastic changes. The review describes patterns of E6 and E7 oncoproteins presentation to the immune system as components of candidate vaccines along with the results of the most promising preclinical trials and animal models used in these trials. Special attention is paid to vaccine candidates which have shown efficacy in clinical trials in patients with HPV-associated neoplastic changes.

Assessment of the Parameters of Adaptive Cell-Mediated Immunity in Naïve Common Marmosets (Callithrix jacchus).

Common marmosets are small New World primates that have been increasingly used in biomedical research. This report presents efficient protocols for assessment of the parameters of adaptive cell-mediated immunity in common marmosets, including the major subpopulations of lymphocytes and main markers of T- and B-cell maturation and activation using flow cytometry with a multicolor panel of fluorescently labelled antibodies. Blood samples from eight common marmosets were stained with fluorescently labeled monoclonal antibodies against their population markers (CD45, CD3, CD20, CD4, CD8) and lymphocyte maturation and activation markers (CD69, CD62L, CD45RO, CD107a and CD27) and analyzed by flow cytometry. Within the CD45+ population, 22.7±5.5% cells were CD3- CD20+ and 67.6±6.3% were CD3+CD20-. The CD3+ subpopulation included 55.7±5.5% CD3+CD4+CD8- and 34.3±3.7% CD3+CD4-CD8+ cells. Activation and maturation markers were expressed in the following lymphocyte proportions: CD62L on 54.0±10.7% of CD3+CD4+ cells and 74.4±12.1% of CD3+CD8+ cells; CD69 on 2.7±1.2% of CD3+CD4+ cells and 1.2±0.5% of CD3+CD8+ cells; CD45RO on 1.6±0.6% of CD3+CD4+ cells and 1.8±0.7% of CD3+CD8+ cells; CD107a on 0.7±0.5% of CD3+CD4+ cells and 0.5±0.3% of CD3+CD8+ cells; CD27 on 94.6±2.1% of CD3+ cells and 8.9±3.9% CD20+ cells. Female and male subjects differed in the percentage of CD3+CD4+CD45RO+ cells (1.9±0.5 in females vs 1.1±0.2 in males; p < 0.05). The percentage of CD20+CD27+ cells was found to highly correlate with animals' age (r = 0.923, p < 0.005). The basal parameters of adaptive cell-mediated immunity in naïve healthy marmosets without markers of systemic immune activation were obtained. These parameters and the described procedures are crucial in documenting the changes induced in common marmosets by prophylactic and therapeutic immune interventions.

Immunization with live nonpathogenic H5N3 duck influenza virus protects chickens against highly pathogenic H5N1 virus.

Development of an effective, broadly-active and safe vaccine for protection of poultry from H5N1 highly pathogenic avian influenza viruses (HPAIVs) remains an important practical goal. In this study we used a low pathogenic wild aquatic bird virus isolate А/duck/Moscow/4182/2010 (H5N3) (dk/4182) as a live candidate vaccine. We compared this virus with four live 1:7 reassortant anti-H5N1 candidate vaccine viruses with modified hemagglutinin from either A/Vietnam/1203/04 (H5N1) or A/Kurgan/3/05 (H5N1) and the rest of the genes from either H2N2 cold-adapted master strain A/Leningrad/134/17/57 (rVN-Len and rKu-Len) or H6N2 virus A/gull/Moscow/3100/2006 (rVN-gull and rKu-gull). The viruses were tested in parallel for pathogenicity, immunogenicity and protective effectiveness in chickens using aerosol, intranasal and oral routes of immunization. All five viruses showed zero pathogenicity indexes in chickens. Viruses rVN-gull and rKu-gull were immunogenic and protective, but they were insufficiently attenuated and caused significant mortality of 1-day-old chickens. The viruses with cold-adapted backbones (rVN-Len and rKu-Len) were completely nonpathogenic, but they were significantly less immunogenic and provided lower protection against lethal challenge with HPAIV A/Chicken/Kurgan/3/05 (H5N1) as compared with three other vaccine candidates. Unlike other four viruses, dk/4182 was both safe and highly immunogenic in chickens of any age regardless of inoculation route. Single administration of 106 TCID50 of dk/4182 virus via drinking water provided complete protection of 30-days-old chickens from 100 LD50 of the challenge virus. Our results suggest that low pathogenic viruses of wild aquatic birds can be used as safe and effective live poultry vaccines against highly pathogenic avian viruses.

[GENUS ANELLOVIRIDAE VIRUSES IN CHRONIC LIVER DISEASE].

AIM: Viruses from genus Anelloviridae (TTV, TTMDV and TTMV) are small DNA viruses that are widespread in human popu- lation. Data on tissue tropism, cell localization and morphometry of anelloviruses are scarce. The purpose of this study was to determine the prevalence of TTV, TTMDV and TTMV in persons with liver disease and in healthy individuals, as well as electron-microscopic verification of Anelloviridae species. METHODS: Detection of anelloviral DNA was performed in serum samples from 203 patients with liver diseases of various etiology and 115 voluntary blood donors using PCR with primers allowing to differentiate TTV, TTMDV TTMV based on the length of amplified fragment. Histopathological and electron microscopic studies were performed for liver biopsy specimens from 203 patients with liver disease. RESULTS: High prevalence (70-90%) of all three anelloviruses in healthy individuals and patients with liver disease was demonstrated, with high frequency of triple TTV, TTMDV and TTMV infection (52.2-55.7%). Electron-microscopic study of liver biopsy specimens from TTMDV monoinfected patients gave a submicroscopic image of TTMDV virions with diameter 35.86 ± 2.04 nm. Electron microscopic studies confirmed the nature of liver damage in TTMDV monoinfection: accumulation of virus in the hepatocytes, significant cyropathy with enlightenment matrix of the cytoplasm and reduction of intracellula organelles involved in protein synthesis, portal and perivascular perisinusoidal fibrosis. TTV, TTMDV and TTMV virions were dentified in hepatocytes, confirming these viruses to be hepatotropic. CONCLUSION: Our results demonstrate that anelloviruses are lymphotropic viruses, individual genotypes of those might be hepatotropic and pathogenic to liver.