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Background Hepatitis E virus (HEV) is a major causative agent of acute hepatitis worldwide, prompting continuous HEV vaccine efforts. Vaccine development is hampered by the lack of convenient animal models susceptible to infection with different HEV genotypes. We produced recombinant open reading frame 2 protein (pORF2; p551) of HEV genotype (GT) 3 and assessed its immunogenicity and protectivity against HEV challenge in common marmosets (Callithrix jacchus, CM). Methods p551 with consensus sequence corresponding to amino acid residues 110–660 of HEV GT3 pORF2 was expressed in E. coli and purified by affinity chromatography. CMs were immunized intramuscularly with 20 μg of p551 VLPs with alum adjuvant (n = 4) or adjuvant alone (n = 2) at weeks 0, 3, 7 and 19. At week 27, p551-immunized and control animals were challenged with HEV GT1 or GT3 and thereafter longitudinally screened for markers of liver function, anti-HEV IgG and HEV RNA in feces and sera. Results Purified p551 formed VLPs with particle size of 27.71 ± 2.42 nm. Two immunizations with p551 induced anti-HEV IgG mean titer of 1:1810. Immunized CMs challenged with homologous and heterologous HEV genotype did not develop HEV infection during the follow-up. Control CMs infected with both HEV GT1 and GT3 demonstrated signs of HEV infection with virus shedding and elevation of the levels of liver enzymes. High levels of anti-HEV IgG persisted in vaccinated CMs and control CMs that resolved HEV infection, for up to two years post challenge. Conclusions CMs are shown to be a convenient laboratory animal model susceptible to infection with HEV GT1 and GT3. Immunization with HEV GT3 ORF2/p551 triggers potent anti-HEV antibody response protecting CMs from homologous and heterologous HEV challenge. This advances p551 in VLPs as a prototype vaccine against HEV.

Because of their high biocompatibility, stability, ability to negotiate biological barrier passage, and functionalization properties, biological nanoparticles have been actively investigated for many medical applications. Biological nanoparticles, including natural extracellular vesicles (EVs) and synthetic extracellular vesicle-mimetic nanovesicles (EMNVs) represent novel drug delivery vehicles that can accommodate different payloads. In this study, we investigated EVs and EMNVs for their physical, biological and delivery properties and we showed that EMNVs have similar delivery properties compared to EVs. In addition, these nanotherapeutics were analyzed for their cytostatic properties in combination with the FDA-approved drug hydroxychloroquine (HCQ), which increased their cytostatic thanks to its lysosome-destabilizing properties. Altogether, these data demonstrated that, at least in vitro, the use of synthetic biomimetic particles is comparable to the natural counterparts, while their synthesis is significantly faster and more cost effective. In addition, we highlighted the benefits of combining biological nanoparticles with a lysosome destabilizing agent that increased the delivery properties of the particles.

The factors influencing hepatitis E virus (HEV) circulation remain largely unexplored. We investigated HEV seroprevalence in humans and the prevalence of infection in farm pigs and rabbits in different regions of the Russian Federation, as well as the genetic diversity and population dynamics of HEV. Anti-HEV IgG antibody detection rates in the general population increase significantly with age, from 1.5% in children and adolescents under 20 years old to 4.8% in adults aged between 20 and 59 years old, to 16.7% in people aged 60 years and older. HEV seroprevalence varies between regions, with the highest rate observed in Belgorod Region (16.4% compared with the national average of 4.6%), which also has the country’s highest pig population. When compared with the archival data, both increases and declines in HEV seroprevalence have been observed within the last 10 years, depending on the study region. Virus shedding has been detected in 19 out of the 21 pig farms surveyed. On one farm, circulation of the same viral strain for five years was documented. All human and animal strains belonged to the HEV-3 genotype, with its clade 2 sequences being predominant in pigs. Sequences from patients, pigs, and sewage from pig farms clustered together, suggesting a zoonotic infection in humans and possible environmental contamination. The HEV-3 population size predicted using SkyGrid reconstruction demonstrated exponential growth in the 1970s–1990s, with a subsequent decline followed by a short rise around the year 2010, the pattern being similar to the dynamics of the pig population in the country. The HEV-3 reproduction number (Re) predicted using Birth-Death Skyline analysis has fluctuated around 1 over the past 20 years in Russia, but is 10 times higher in Belgorod Region. In conclusion, HEV-3 circulation varies both geographically and temporally, even within a single country. The possible factors contributing to this variability are largely related to the circulation of the virus among farm pigs.

Vaccines are proven to be an effective tool in prophylaxis of severe COVID-19, but emerging mutated SARS-CoV-2 variants constantly challenge vaccines’ protectivity. We have evaluated the ability of the sera from individuals vaccinated with two variants of inactivated vaccine CoviVac and COVID-19 convalescents (May-December 2020) to neutralize SARS-CoV-2 variants Delta and Omicron. Four groups of serum samples (CoviVac vaccinees; COVID-19 convalescents; mice immunized with CoviVac preparations based on prototype B.1.1 strain and Delta variant) were evaluated in virus neutralization test against SARS-CoV-2 heterologous B.1.1 virus, Delta and Omicron variants. CoviVac preparations based on B.1.1 and Delta induced neutralizing antibodies against SARS-CoV-2 B.1.1 and two variants of concern. We observed a decrease in neutralization capacity in the sera from CoviVac (based on B.1.1 strain) vaccinees: 57.1% samples had detectable neutralizing antibodies against Delta and 61.9% against Omicron variants. Sera samples of all (100%) mice immunized with a candidate vaccine based on the SARS-CoV-2 Delta variant strain had neutralizing antibodies against all tested strains.

Introduction Middle East respiratory syndrome (MERS) is acute inflammatory disease of respiratory system with a high mortality, caused Ьу coronavirus MERS-CoV At present moment, we still lack specific therapeutic preparations and vaccines against MERS Vaccine administration can help to limit the spread of the disease and lower the mortality Duration of vaccine-induced immune response is one of the key characteristics of a vaccine, which is connected with duration of its protective effectiveness Unfortunately, the data on duration of vaccine-induced immune response against MERS is scarce The aim of the study was to determine duration of humoral immune response in mice and primates and duration of protective immune response in mice after immunization with the heterologous virus-vectored vaccine against MERS (BVRS-GamVac-Combi), developed earlier Ьу our research group Material and methods To study duration of humoral immune response, we used mice of C57BL/6 strain and common marmosets Animals were immunized with the vaccine BVRS-GamVac-Combi, based on recomЬinant adenoviral vectors rAd26 and rAd5 Antigen-specific-antibody titers were determined with ELISA, virus-neutralizing antibody titers were measured with virus neutralization assay using MERS-CoV (EMC/2012) To study duration of protective immune response, we used a model of lethal infection on transgenic mice, carrying human DPP4 gene of viral receptor Results In present research, we showed that vaccination of animals with BVRS-GamVac-Combi induced robust humoral immune response, which persisted at least 18 months after immunization In addition, our vaccine protected 100 % of animals from lethal infection for at least 7 months after immunization Concluslon Strength of vaccine-induced immune response is generally connected with a protective effectiveness of a vaccine One of the key problems of vaccine design is to find a way to provide as long and robust immune response as possible Duration of vaccine-induced immune response is one of the key characteristics of a vaccine, which demands quality control during multiple steps of a vaccine development Введение Ближневосточный респираторный синдром (БВРС) - это острое воспалительное заболевание дыхательной системы с высокой летальностью, возбудителем которого является коронавирус БВРС-КоВ В настоящее время в мире не существует специфических профилактических и терапевтических средств против БВРС Вакцино-профилактика позволит ограничить распространение данного заболевания и снизить летальность Одной из ключевых характеристик вакцин является длительность индуцируемого иммунного ответа, от которой зависит продолжительность протективного эффекта вакцины К сожалению, данных по длительности поствакцинального иммунного ответа для вакцин против БВРС сейчас недостаточно Цель исследования - определение длительности гуморального иммунного ответа у грызунов и приматов и протективного иммунного ответа после иммунизации комбинированной векторной вакциной против БВРС (БВРС-ГамВак-Комби), разработанной нами ранее Материал и методы Длительность гуморального иммунитета исследовали на мышах линии C57BL/6 и обыкновенных игрунках Животных иммунизировали вакциной БВРС-ГамВак-Комби на основе рекомбинантных векторов rAd26 и rAd5 Титр антиген-специфических антител определяли методом иммуноферментного анализа (ИФА) Титр вирус-нейтрализующих антител определяли с помощью реакции вирус-нейтрализации против вируса БВРС-КоВ (MERS-CoV EMC/2012) Длительность протективного иммунитета исследовали на модели летальной инфекции у трансгенных мышей, несущих ген человека DPP4, кодирующий рецептор к БВРС-КоВ Результаты Исследование длительности поствакцинального гуморального иммунного ответа у грызунов и приматов показало, что вакцинация животных БВРС-ГамВак-Комби индуцирует формирование напряженного гуморального иммунного ответа к гликопротеину S БВРС-КоВ, который сохраняется на протяжении не менее 18 мес Также было показано, что вакцинация позволяет защитить 100 % животных от летальной инфекции, вызванной БВРС-КоВ (MERS-CoV EMC/2012, 100 ЛД50/мышь), через 7 мес после иммунизации Заключение Напряженность поствакцинального гуморального иммунного ответа, как правило, связана с протективностью вакцины Одной из ключевых задач при дизайне вакцин является обеспечение наиболее длительного напряженного иммунного ответа Длительность поствакцинального иммунного ответа - одна из ключевых характеристик вакцин, которая требует контроля на различных этапах их разработки

Chronic HCV infection and associated liver cancer impose a heavy burden on the healthcare system. Direct acting antivirals eliminate HCV, unless it is drug resistant, and partially reverse liver disease, but they cannot cure HCV-related cancer. Possible remedy could be a multi-component immunotherapeutic vaccine targeting both HCV-infected and malignant cells, also those not infected with HCV. To meet this need we developed a two-component DNA vaccine based on the highly conserved core protein of HCV to target HCV-infected cells, and a renowned tumor associated antigen telomerase reverse transcriptase (TERT) based on the rat TERT, to target malignant cells. Their synthetic genes were expression-optimized, and HCV core was truncated after aa 152 (Core152opt) to delete the domain interfering with immunogenicity. Core152opt and TERT DNA were highly immunogenic in BALB/c mice, inducing IFN-γ/IL-2/TNF-α response of CD4+ and CD8+ T cells. Also, DNA-immunization with TERT enhanced cellular immune response against luciferase encoded by a co-delivered plasmid (Luc DNA). However, DNA-immunization with Core152opt and TERT mix resulted in abrogation of immune response against both components. A loss of bioluminescent signal after co-delivery of TERT and Luc DNA into mice indicated that TERT affects the in vivo expression of luciferase directed by the immediate early cytomegalovirus and interferon-β promoters. Panel of mutant TERT variants was created and tested for their expression effects. TERT with deleted N-terminal nucleoli localization signal and mutations abrogating telomerase activity still suppressed the IFN-β driven Luc expression, while the inactivated reverse transcriptase domain of TERT and its analogue, enzymatically active HIV-1 reverse transcriptase, exerted only weak suppressive effects, implying that suppression relied on the presence of the full-length/nearly full-length TERT, but not its enzymatic activity. The effect(s) could be due to interference of the ectopically expressed xenogeneic rat TERT with biogenesis of mRNA, ribosomes and protein translation in murine cells, affecting the expression of immunogens. HCV core can aggravate this effect, leading to early apoptosis of co-expressing cells, preventing the induction of immune response.

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 naive 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.

Telomerase reverse transcriptase (TERT) is a classic tumor-associated antigen overexpressed in majority of tumors. Several TERT-based cancer vaccines are currently in clinical trials, but immune correlates of their antitumor activity remain largely unknown. Here, we characterized fine specificity and lytic potential of immune response against rat TERT in mice. BALB/c mice were primed with plasmids encoding expression-optimized hemagglutinin-tagged or nontagged TERT or empty vector and boosted with same DNA mixed with plasmid encoding firefly luciferase (Luc DNA). Injections were followed by electroporation. Photon emission from booster sites was assessed by in vivo bioluminescent imaging. Two weeks post boost, mice were sacrificed and assessed for IFN-&gamma, interleukin-2 (IL-2), and tumor necrosis factor alpha (TNF-&alpha, ) production by T-cells upon their stimulation with TERT peptides and for anti-TERT antibodies. All TERT DNA-immunized mice developed cellular and antibody response against epitopes at the N-terminus and reverse transcriptase domain (rtTERT) of TERT. Photon emission from mice boosted with TERT/TERT-HA+Luc DNA was 100 times lower than from vector+Luc DNA-boosted controls. Bioluminescence loss correlated with percent of IFN-&gamma, /IL-2/TNF-&alpha, producing CD8+ and CD4+ T-cells specific to rtTERT, indicating immune clearance of TERT/Luc-coexpressing cells. We made murine adenocarcinoma 4T1luc2 cells to express rtTERT by lentiviral transduction. Expression of rtTERT significantly reduced the capacity of 4T1luc2 to form tumors and metastasize in mice, while not affecting in vitro growth. Mice which rejected the tumors developed T-cell response against rtTERT and low/no response to the autoepitope of TERT. This advances rtTERT as key component of TERT-based therapeutic vaccines against cancer.

Highlights • There were multiple importations and several patterns of spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Russia. • There are no signs of direct virus importations from China. • SARS-CoV-2 variants circulating in Russia mostly contain seven common mutations., Objectives The outbreak of coronavirus disease 2019 (COVID-19) started in December 2019 in China and then spread worldwide over the following months, involving 188 countries. The objective of this study was to determine the molecular epidemiology of the COVID-19 outbreak in Russia. Methods In this study, two severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains were isolated and genetically characterized. A phylogenetic analysis of all available Russian sequences was then performed and these were compared to the epidemiological data on COVID-19 incidence to evaluate the molecular epidemiology and pattern of virus spread in the territory of Russia. Results and conclusions Whole genome analysis of the isolates obtained in this study and 216 others isolated in Russia revealed a set of seven common mutations when compared to the original Wuhan virus, including amino acid substitutions in spike protein S and nucleoprotein N, possibly affecting their properties. Phylogenetic analysis of all Russian sequences and 8717 sequences from other countries showed multiple importations of the virus into Russia, local circulation, and several patterns of virus spread.

Background: Covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is the major cause of viral persistence in patients with chronic HBV infection. Understanding the mechanisms underlying stability and persistence of HBV cccDNA in hepatocytes is critical for developing novel therapeutics and managing chronic hepatitis B. In this study, we observed an unexpected increase in HBV cccDNA levels upon suppression of transcription by de novo DNA methyltransferase DNMT3A and uncovered additional mechanisms potentially involved in HBV cccDNA maintenance. Methods: HBV-expressing cell lines were transfected with a DNMT3A-expressing plasmid. Real-time PCR and HBsAg assays were used to assess the HBV replication rate. Cell cycling was analyzed by fluorescent cell sorting. CRISPR/Cas9 was utilized to abrogate expression of APOBEC3A and APOBEC3B. Alterations in the expression of target genes were measured by real-time PCR. Results: Similar to previous studies, HBV replication induced DNMT3A expression, which in turn, led to reduced HBV transcription but elevated HBV cccDNA levels (4- to 6-fold increase). Increased levels of HBV cccDNA were not related to cell cycling, as DNMT3A accelerated proliferation of infected cells and could not contribute to HBV cccDNA expansion by arresting cells in a quiescent state. At the same time, DNMT3A suppressed transcription of innate immunity factors including cytidine deaminases APOBEC3A and APOBEC3B. CRISPR/Cas9-mediated silencing of APOBEC3A and APOBEC3B transcription had minor effects on HBV transcription, but significantly increased HBV cccDNA levels, similar to DNMT3A. In an attempt to further analyze the detrimental effects of HBV and DNMT3A on infected cells, we visualized &gamma, H2AX foci and demonstrated that HBV inflicts and DNMT3A aggravates DNA damage, possibly by downregulating DNA damage response factors. Additionally, suppression of HBV replication by DNMT3A may be related to reduced ATM/ATR expression. Conclusion: Formation and maintenance of HBV cccDNA pools may be partially suppressed by the baseline expression of host inhibitory factors including APOBEC3A and APOBEC3B. HBV inflicts DNA damage both directly and by inducing DNMT3A expression.

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.

Chronic hepatitis B virus infection (CHB) caused by the hepatitis B virus (HBV) is one of the most common viral infections in the world. Reactivation of HBV infection is a life-threatening condition observed in patients with CHB receiving chemotherapy or other medications. Although HBV reactivation is commonly attributed to immune suppression, other factors have long been suspected to play a role, including intracellular signaling activated in response to DNA damage. We investigated the effects of DNA-damaging factors (doxorubicin and hydrogen peroxide) on HBV reactivation/replication and the consequent DNA-damage response. Dose-dependent activation of HBV replication was observed in response to doxorubicin and hydrogen peroxide which was associated with a marked elevation in the mRNA levels of ataxia-telangiectasia mutated (ATM) and ATM- and RAD3-related (ATR) kinases. Downregulation of ATM or ATR expression by shRNAs substantially reduced the levels of HBV RNAs and DNA. In contrast, transcriptional activation of ATM or ATR using CRISPRa significantly increased HBV replication. We conclude that ATM and ATR are essential for HBV replication. Furthermore, DNA damage leading to the activation of ATM and ATR transcription, results in the reactivation of HBV replication.

HIV-induced immune suppression results in the high prevalence of HIV/AIDS-associated malignancies including Kaposi sarcoma, non-Hodgkin lymphoma, and cervical cancer. HIV-infected people are also at an increased risk of “non-AIDS-defining” malignancies not directly linked to immune suppression but associated with viral infections. Their incidence is increasing despite successful antiretroviral therapy. The mechanism behind this phenomenon remains unclear. Here, we obtained daughter clones of murine mammary gland adenocarcinoma 4T1luc2 cells expressing consensus reverse transcriptase of HIV-1 subtype A FSU_A strain (RT_A) with and without primary mutations of drug resistance. Inin vitrotests, mutations of resistance to nucleoside inhibitors K65R/M184V reduced the polymerase, and to nonnucleoside inhibitors K103N/G190S, the RNase H activities of RT_A. Expression of these RT_A variants in 4T1luc2 cells led to increased production of the reactive oxygen species (ROS), lipid peroxidation, enhanced cell motility in the wound healing assay, and upregulation of expression ofVimentinandTwist. These properties, particularly, the expression ofTwist, correlated with the levels of expression RT_A and/or the production of ROS. When implanted into syngeneic BALB/C mice, 4T1luc2 cells expressing nonmutated RT_A demonstrated enhanced rate of tumor growth and increased metastatic activity, dependent on the level of expression of RT_A andTwist. No enhancement was observed for the clones expressing mutated RT_A variants. Plausible mechanisms are discussed involving differential interactions of mutated and nonmutated RTs with its cellular partners involved in the regulation of ROS. This study establishes links between the expression of HIV-1 RT, production of ROS, induction of EMT, and enhanced propagation of RT-expressing tumor cells. Such scenario can be proposed as one of the mechanisms of HIV-induced/enhanced carcinogenesis not associated with immune suppression.

The hepatitis C virus (HCV) has remarkable genetic diversity and exists as eight genotypes (1 to 8) with distinct geographic distributions. No complete genome sequence of HCV subtype 2b (HCV-2b) is available from Latin American countries, and the factors underlying its emergence and spread within the continent remain unknown. The present study was conducted to determine the first full-length genomic sequences of HCV-2b isolates from Latin America and reconstruct the spatial and temporal diversification of this subtype in Brazil. Nearly complete HCV-2b genomes isolated from two Brazilian patients were obtained by direct sequencing of long PCR fragments and analyzed together with reference sequences using the Bayesian coalescent and phylogeographic framework approaches. The two HCV-2b genomes were 9318 nucleotides (nt) in length (nt 37&ndash, 9354). Interestingly, the long RT-PCR technique was able to detect co-circulation of viral variants that contained an in-frame deletion of 2022 nt encompassing E1, E2, and p7 proteins. Spatiotemporal reconstruction analyses suggest that HCV-2b had a single introduction in Brazil during the early 1980s, displaying an epidemic history characterized by a low and virtually constant population size until the present time. These results coincide with epidemiological data in Brazil and may explain the low national prevalence of this subtype.

Optimization of DNA vaccine delivery improves the potency of the immune response and is crucial to clinical success. Here, we inquired how such optimization impacts the magnitude of the response, its specificity and type. BALB/c mice were DNA-immunized with two model immunogens, HIV-1 protease and reverse transcriptase by intramuscular or intradermal injections with electroporation. DNA immunogens were co-delivered with DNA encoding luciferase. Delivery and expression were monitored by in vivo bioluminescence imaging (BLI). The endpoint immune responses were assessed by IFN-γ/IL-2 FluoroSpot, multiparametric flow cytometry and antibody ELISA. Expression and immunogenicity were compared in relation to the delivery route. Regardless of the route, protease generated mainly IFN-γ, and reverse transcriptase, IL-2 and antibody response. BLI of mice immunized with protease- or reverse transcriptase/reporter plasmid mixtures, demonstrated significant loss of luminescence over time. The rate of decline of luminescence strongly correlated with the magnitude of immunogen-specific response, and depended on the immunogenicity profile and the immunization route. In vitro and in vivo BLI-based assays demonstrated that intradermal delivery strongly improved the immunogenicity of protease, and to a lesser extent, of reverse transcriptase. Immune response polarization and epitope hierarchy were not affected. Thus, by changing delivery/immunogen expression sites, it is possible to modulate the magnitude, but not the type or fine specificity of the induced immune response.

Wild ducks are known to be able to carry avian influenza viruses over long distances and infect domestic ducks, which in their turn infect domestic chickens. Therefore, prevention of virus transmission between ducks and chickens is important to control the spread of avian influenza. Here we used a low pathogenic wild aquatic bird virus A/duck/Moscow/4182/2010 (H5N3) for prevention of highly pathogenic avian influenza virus (HPAIV) transmission between ducks and chickens. We first confirmed that the ducks orally infected with H5N1 HPAIV A/chicken/Kurgan/3/2005 excreted the virus in feces. All chickens that were in contact with the infected ducks became sick, excreted the virus, and died. However, the ducks orally inoculated with 104 50% tissue culture infective doses of A/duck/Moscow/4182/2010 and challenged 14 to 90 days later with H5N1 HPAIV did not excrete the challenge virus. All contact chickens survived and did not excrete the virus. Our results suggest that low pathogenic virus of wild aquatic birds can be used for prevention of transmission of H5N1 viruses between ducks and chickens.

Vaccination of newborns against hepatitis B (HB) was introduced in Russia in 1998. Since then the incidence of acute HB has rapidly declined. However, prevalence of chronic HB remains stable. The aim of this study was to estimate the effect of vaccination on HBV-associated morbidity, and to assess the prevalence of HBV immune escape variants after 10 years of vaccination. Methods 6,217 sera samples collected from volunteers in six epidemiologically different regions of Russia were tested for serological and molecular markers of HBV infection. A mathematical model developed by the U.S. Centers for Disease Control and Prevention was used to estimate the effect of vaccination and birth dose coverage on the incidence of HB and adverse outcomes of infection. Results Prevalence of HBsAg in the study population varied from 1.2% to 8.2%; anti-HBc detection rates were 13.0–46.2%. HBsAg detection rates in epidemiologically significant cohorts were 0.6–10.5% in women of childbearing age; 0–2.4% in children ≤5 years old; 1.9–8.1% in adults ≥30 years old. Mathematical modeling demonstrated that the current 96.1–99.6% level of birth dose coverage increased the effectiveness of vaccination 10–21 times compared to 50% and 0% birth dose coverage scenarios. HBV DNA was detected in 63 sera samples. The frequency of amino acid substitutions in HBsAg was 38% (24/63). Only in 3% (2/63) the mutations were within the a-determinant of HBsAg (M133T and G145S, one case each). None of the identified mutations eluded HBsAg detection, since all these samples tested positive for HBsAg by commercial ELISA. Conclusion Despite a significant decline in acute HB incidence after the introduction of universal vaccination, many undiagnosed potential sources of infection remain. Low prevalence of HBV immune escape variants is a favorable predictor of vaccine effectiveness in the future.