Circumventing antivector immunity: potential use of nonhuman adenoviral vectors

Adenoviruses are efficient gene delivery vectors based on their ability to transduce a wide variety of cell types and drive high-level transient transgene expression. While there have been advances in modifying human adenoviral (HAdV) vectors to increase their safety profile, there are still pitfalls that need to be further addressed. Preexisting humoral and cellular immunity against common HAdV serotypes limits the efficacy of gene transfer and duration of transgene expression. As an alternative, nonhuman AdV (NHAdV) vectors can circumvent neutralizing antibodies against HAdVs in immunized mice and monkeys and in human sera, suggesting that NHAdV vectors could circumvent preexisting humoral immunity against HAdVs in a clinical setting. Consequently, there has been an increased interest in developing NHAdV vectors for gene delivery in humans. In this review, we outline the recent advances and limitations of HAdV vectors for gene therapy and describe examples of NHAdV vectors focusing on their immunogenicity, tropism, and potential as effective gene therapy vehicles

Evaluation of direct and cell-mediated triple-gene therapy in spinal cord injury in rats

© 2017 Elsevier Inc.Current treatment options for spinal cord injury (SCI) are scarce. One of the most promising innovative approaches include gene-therapy, however no single gene has so far been shown to be of clinical relevance. This study investigates the efficacy of various combinations of vascular endothelial growth factor (VEGF), glial cell-derived neurotrophic factor (GDNF), angiogenin (ANG) and neuronal cell adhesion molecule (NCAM) in rats. Multiple therapeutic genes were administered intrathecally either via adenoviral vectors or by using genetically modified human umbilical cord blood mononuclear cells (hUCBMCs). Following the induction of SCI, serial assessment of cord regeneration was performed, including morphometric analysis of gray and white matters, electrophysiology and behavioral test. The therapeutic gene combinations VEGF + GDNF + NCAM and VEGF + ANG + NCAM had positive outcomes on spinal cord regeneration, with enhanced recovery seen by the cell-based approach when compared to direct gene therapy. The efficacy of the genes and the delivery methods are discussed in this paper, recommending their potential use in SCI

Analysis of recombinant VEGF gene expression by genetically modified umbilical cord blood mononuclear cells in experiment in vivo

To obtain a significant therapeutic effect transplanted genetically modified cells should have an enhanced ability to survive and active expression of the therapeutic gene. In this paper, by using immunofluorescent staining we investigated the functional activity of the gene-cell formulation designed to deliver a therapeutic gene into the area of regeneration. As a model we used transgenic SOD1-G93A mice with amyotrophic lateral sclerosis phenotype which received xenotransplantation of human umbilical cord blood mononuclear cells, genetically modified with adenoviral expression vector encoding vascular endothelial growth factor (VEGF) and the reporter green fluorescent protein (EGFP). Results of the study allowed to establish not only the duration of survival of transplanted cells, but also the efficiency of expression of recombinant genes in genetically modified cells in vivo. Double immunofluorescent staining with antibodies against human nuclear antigen HNA and VEGF detected HNA+/VEGF+ cells in the terminal stage of disease 15 weeks after transplantation. These data suggest that genetically modified umbilical cord blood mononuclear cells, transplanted into SOD1-G93A transgenic mice, are able to penetrate the blood-brain barrier and migrate into the area of degeneration of nerve tissue and survive from the time of transplantation until the death of animals at the terminal stage of disease. At that time adenoviral expression vector encoding therapeutic gene is functionally active in transplanted cells, and secretory products of recombinant gene act on target cells by a paracrine mechanism

Мутации в геноме вирусов гриппа птиц подтипов Н1 и Н5, ответственные за адаптацию к млекопитающим

Avian influenza viruses of H1 and H5 subtypes were involved in the formation of highly pathogenic viruses that caused pandemics and panzootics in the 20th–21st centuries. In order to assess the zoonotic potential of viruses of these subtypes, two viruses of H1N1 and H5N3 have been isolated from wild ducks in Moscow and adapted to growth in mouse lungs. Their phenotypic properties were studied, and the genetic changes that occurred during adaptation were identified. The original A/duck/Moscow/4970/2013 (H1N1) and A/duck/Moscow/4182-C/2010 (H5N3) viruses were apathogenic for mice but became pathogenic after 7–10 passages in mouse lungs. Complete genome sequencing revealed 2 amino acid substitutions in the proteins of the H1N1 mouse-adapted variant (Glu627Lys in PB2 and Asp35Asn in hemagglutinin (HA) – numbering according to H3) and 6 mutations in the proteins of H5N3 virus (Glu627lys in PB2, Val113Ala in PB1, Ser82Pro in PB1-F2, Lys52Arg in HA2, Arg65Lys in NP, and Ser59Ile in NA). The increase in virulence is most likely due to a common substitution in the protein PB2 Glu627Lys as revealed in both viruses. The replacement of Asp35Asn in HA of the mouse-adapted H1N1 virus is associated with an increase in the pH value of the HA transition from 5.0 for 5.5 in comparison to the HA of parent virus. The found mutations in HA, NA, and PB1-F2 proteins of the adapted H5N3 variant are unique. The mutations Glu627Lys in PB2, Arg65Lys in NP, and Val113Ala in PB1 are most likely host adaptive.Вирусы гриппа птиц подтипов Н1 и Н5 участвовали в формировании высокопатогенных вариантов вирусов, вызвавших пандемии и  панзоотии в  XX–XXI  веках. С  целью оценки зоонозного потенциала вирусов этих подтипов, выделенных от диких уток в черте Москвы, была проведена адаптация вирусов к размножению в легких мышей, изучены их фенотипические свойства и идентифицированы генетические изменения, возникшие при адаптации. Изначально апатогенные для мышей вирусы A/duck/Moscow/4970/2013 (H1N1) и A/duck/Moscow/4182‑C/2010 (H5N3) после 7–10 пассажей через легкие мышей изменили фенотип на патогенный. Полногеномное секвенирование выявило в адаптированных к мышам вирусах 2 аминокислотные замены в вирусе гриппа H1N1 (Glu627Lys в белке PB2 и Asp35Asn в гемагглютинине (HA) — нумерация по H3) и 6 мутаций в белках вируса H5N3 (Glu627Lys в PB2, Val113Ala в PB1, Ser82Pro в PB1‑F2, Lys52Arg в HA2, Arg65Lys в NP и Ser59Ile в NA). Возрастание вирулентности для мышей, скорее всего, обусловлено общей для обоих вирусов заменой – Glu627Lys в  белке PB2. Замена Asp35Asn в  HA адаптированного к  мышам вируса гриппа H1N1  ассоциирована с возрастанием значения рН конформационного перехода HA с 5.0 до 5.5 относительно HA дикого вируса. Обнаруженные в адаптированном варианте H5N3 мутации в белках НА, NA и PB1‑F2 — уникальные. Мутации Glu627Lys в PB2, Arg65Lys в NP и Val113Ala в PB1, скорее всего, носят адаптационный характер

Protecting Mice from H7 Avian Influenza Virus by Immunisation with a Recombinant Adenovirus Encoding Influenza A Virus Conserved Antigens

Influenza is a highly contagious disease that causes annual epidemics and occasional pandemics. Birds are believed to be the source of newly emerging pandemic strains, including highly pathogenic avian influenza viruses of the subtype H7. The aim of the study: to evaluate the ability of the recombinant human adenovirus, serotype 5, which expresses genes of influenza A highly conserved antigens (ion channel M2 and nucleoprotein NP), to provide protection to laboratory mice against infection with a lethal dose of avian influenza virus, subtype H7. To achieve this goal, it was necessary to adapt influenza A virus, subtype H7 for reproduction in the lungs of mice, to characterise it, and to use it for evaluation of the protective properties of the recombinant adenovirus. Materials and methods: avian influenza virus A/Chicken/NJ/294508-12/2004 (H7N2) was adapted for reproduction in the lungs of mice by repeated passages. The adapted strain was sequenced and assessed using hemagglutination test, EID50 and LD50 for laboratory mice. BALB/c mice were immunised once with Ad5-tet-M2NP adenovirus intranasally, and 21 days after the immunisation they were infected with a lethal dose (5 LD50) of influenza virus A/Chicken/NJ/294508-12/2004 (H7N2) in order to assess the protective properties of the recombinant adenovirus. The level of viral shedding from the lungs of the infected mice was evaluated by titration of the lung homogenates in MDCK cell culture on days 3 and 6 after infection. The level of specific antibodies to H7 avian influenza virus was determined by indirect enzyme immunoassay. Results: the use of Ad5-tet-M2NP adenovirus for immunisation of the mice ensured 100% survival of the animals that had disease symptoms (weight loss) after their infection with the lethal dose (5 LD50) of H7 avian influenza virus. The study demonstrated a high post-vaccination level of humoral immune response to H7 avian influenza virus. The virus titer decreased significantly by day 6 in the lungs of mice that had been immunised with Ad5-tet-M2NP compared to the control group. Conclusion: the Ad5-tetM2NP recombinant adenovirus can be used to create a candidate pandemic influenza vaccine that would protect against avian influenza viruses, subtype H7, in particular

Способ получения рекомбинантных антител, продуцируемых клеточной линией, трансдуцированной рекомбинантными аденовирусами

Objectives. To develop a technology for obtaining recombinant antibodies in a suspension culture of human HEK293 cells using transduction with recombinant adenovirus serotype 5 (rAd5) carrying genes expressing heavy and light chains of antibodies on the example of two broadspectrum anti-influenza antibodies 27F3 and CR9114.Methods. Ad5-27F3-H, Ad5-CR9114-H, and Ad5-27F3-L recombinant adenoviruses carrying the 27F3 antibody heavy chain gene, CR9114 antibody heavy chain gene, and 27F3 light chain gene, respectively, were generated using the AdEasy™ Adenoviral vector system. To accumulate preparative amounts of recombinant r27F3 and rCR9114 antibodies, the HEK293 suspension cell line was transduced with recombinant adenoviruses carrying genes for heavy and light chains of antibodies. The cells were cultured in a wave-type bioreactor. Chromatography was used to purify recombinant antibodies from the culture medium. After analyzing the molecular weights of purified antibodies using protein electrophoresis, their ability to interact with influenza A and B viruses was analyzed using the Western blot technique, while their ability to neutralize influenza A and B viruses was evaluated using the virus neutralization assay.Results. A method for the accumulation and purification of recombinant r27F3 and CR9114 antibodies from the culture medium of a suspension culture of human cells following transduction with its recombinant adenoviruses carrying the genes for heavy and light chains of these antibodies was developed. The ability of the r27F3 antibody to interact with and neutralize influenza A viruses of group 1 (except influenza A virus subtype H2) and group 2 was shown. The ability of the rCR9114 antibody to interact with influenza A viruses of group 1 and influenza B viruses, as well as to neutralize influenza A viruses of group 1, was demonstrated.Conclusions. A technology for obtaining recombinant antibodies in a suspension culture of HEK293 cells using transduction with recombinant adenoviruses carrying genes expressing heavy and light chains of antibodies was developed along with a confirmation of their specificity.Цели. Разработать технологию получения рекомбинантных антител в суспензионной культуре клеток человека HEK293 с помощью трансдукции рекомбинантными аденовирусами человека пятого серотипа, несущими гены, экспрессирующие тяжелые и легкие цепи антител, на примере двух противогриппозных антител широкого спектра действия 27F3 и CR9114.Методы. Рекомбинантные аденовирусы Ad5-27F3-H, Ad5-CR9114-H и Ad5-27F3-L, несущие ген тяжелой цепи антитела 27F3, ген тяжелой цепи антитела CR9114 и ген легкой цепи 27F3, были получены с помощью набора AdEasy™ Adenoviral vector system. Для накопления препаративных количеств рекомбинантных антител r27F3 и rCR9114 суспензионную клеточную линию HEK293 трансдуцировали рекомбинантными аденовирусами, несущими гены тяжелых и легких цепей антител, и культивировали клетки в биореакторе волнового типа. Рекомбинантные антитела очищали из культуральной жидкости хроматографическим методом. Молекулярную массу полученных антител анализировали с помощью белкового электрофореза, их способность взаимодействовать с вирусами гриппа А и В методом вестерн-блот анализа, а способность нейтрализовать вирусы гриппа А и В с помощью реакции вирус-нейтрализации.Результаты. Отработана методика накопления и очистки рекомбинантных антител r27F3 и CR9114 из культуральной жидкости суспензионной культуры клеток человека после трансдукции ее рекомбинантными аденовирусами, несущими гены тяжелых и легких цепей этих антител. Показана способность антитела r27F3 взаимодействовать с вирусами гриппа А подгруппы 1 (кроме вируса грипп А субтипа H2) и подгруппы 2 и нейтрализовать их. Показана способность антитела rCR9114 взаимодействовать с вирусами гриппа А подгруппы 1 и вирусами гриппа В, а также нейтрализовать вирусы гриппа А подгруппы 1.Выводы. Отработана технология получения рекомбинантных антител в суспензионной культуре клеток HEK293 с помощью трансдукции рекомбинантными аденовирусами, несущими гены, экспрессирующие тяжелые и легкие цепи антител, и показана их специфичность

The adaptive potential of North American subtype H7N2 avian influenza viruses to mammals

Introduction. H7 subtype avian influenza viruses causing severe epizootics among birds are phylogenetically different in the Eastern and Western hemispheres. Numerous human infections caused by these viruses in the Eastern hemisphere indicate that H7 viruses can overcome the interspecies barrier and pose a potential threat of a new pandemic.The H7N2 viruses with deletion of amino acids 221–228 (H3 numbering) in hemagglutinin (HA) had been circulating among poultry in the Western Hemisphere during 1996–2006, and had once again been detected in 2016 in an animal shelter, where they caused cat diseases. The objective of this study is to elucidate the mechanism of adaptation to mammals of North American H7N2 influenza viruses with deletion in HA. Materials and methods. The A/chicken/New Jersey/294598-12/2004 (H7N2) virus was adapted to mice by the lung passages. Complete genomes of original and mouse-adapted viruses were analyzed. The receptor specificity and thermostability of viruses, HA activation pH and virulence for mice were determined. Results. The non-pathogenic H7N2 avian influenza virus became pathogenic after 10 passages in mice. Amino acid substitutions occurred in five viral proteins: one in PB2 (E627K), NA (K127N), NEP (E14Q), four in HA and six in NS1. Mutations in HA slightly changed receptor specificity but increased the pH of HA activation by 0.4 units. The NS1 protein undergone the greatest changes in the positions (N73T, S114G, K118R, G171A, F214L and G224R), where amino acid polymorphisms were observed in the original virus, but only minor amino acid variants have been preserved in the mouse adapted variant. Conclusion. The results show that H7N2 viruses have the potential to adapt to mammals. The increase in virulence is most likely due to the adaptive E627K mutation in PB2 and possibly in HA

ВОЗМОЖНО ЛИ ВЛИЯНИЕ МИКОПЛАЗМЕННОЙ ИНФЕКЦИИ НА ПАТОГЕНЕЗ РАКА ПРЕДСТАТЕЛЬНОЙ ЖЕЛЕЗЫ?

Objective: to define a possible correlation between Mycoplasma infection persistence and prostate cancer (PC). Subjects and methods. Two hundred and fifty males aged 45 to 83 years (mean age 65.50.71 years) with suspected PC were examined. In all the patients, polyfocal prostate biopsy from 12 points was carried out, by additionally taking 2 tissue columns from the peripheral area of both lobes. The basic material was referred for morphological study; the two additional columns were tested for Mycoplasma DNA by a polymerase chain reaction (PCR) and real-time PCR. The study was blind. According to the morphological findings, the patients were divided into 2 groups: 1) those with chronic prostatitis, prostate adenoma, low-grade prostatic interstitial neoplasia (PIN); 2) those with high-grade PIN (HG-PIN), PC. There were no age differences between the groups (p = 0.05). Results. The standard procedure for PCR was applied to 127 subjects. Twenty-six (20.5%) of the 127 subjects with suspected PC were found to have Mycoplasma infection, Mycoplasma being detected in 21 (26.2%) of the 81 patients with verified HG-PIN and PC. Mycoplasma hominis was encountered in 19 (15%) patients of the 127 subjects with suspected PC and this infection was present in 16 (20%) of the 81 patients with verified HG-PIN and PC. Comparison of the frequency of HG-PIN and PC in the patients of general group (60%) and in those with Mycoplasma infection (80.8%) revealed significant differences (p = 0.031). HG-PIN and PC were also significantly more frequently seen in the patients with Mycoplasma hominis (84.2%) that in the general patient group (60%) (p = 0.033). There were no significant differences in the frequency of HG-PIN and PC between the patients from the general group (60%) and those with Mycoplasma genitalium (71.4%) (p = 0.05). The patients with verified PC and HG-PIN were more frequently found to have Mycoplasma hominis (20%) than Mycoplasma genitalium (6.2%), which further drew our closer attention to just this pathogen. The real-time PCR was used in 123 subjects to detect Mycoplasma. HG-PIN and prostate adenocarcinoma were revealed in 63 of the 123 patients with suspected PC, Mycoplasma hominis was seen in 46 (37%). The frequency (n=46) was 73.9%. The frequency of HG-PIN and PC was significantly higher in the patients with isolated Mycoplasma hominis DNA that in those without this pathogen (p < 0.001). Conclusion. Thus, the investigation showed a significantly higher correlation in the frequency of HG-PIN and PC in the patients with Mycoplasma infection that in the general study patients with suspected PC. This was supported by the use of both the standard procedure for Mycoplasma DNA determination and real-time PCR diagnosis.Objective: to define a possible correlation between Mycoplasma infection persistence and prostate cancer (PC). Subjects and methods. Two hundred and fifty males aged 45 to 83 years (mean age 65.50.71 years) with suspected PC were examined. In all the patients, polyfocal prostate biopsy from 12 points was carried out, by additionally taking 2 tissue columns from the peripheral area of both lobes. The basic material was referred for morphological study; the two additional columns were tested for Mycoplasma DNA by a polymerase chain reaction (PCR) and real-time PCR. The study was blind. According to the morphological findings, the patients were divided into 2 groups: 1) those with chronic prostatitis, prostate adenoma, low-grade prostatic interstitial neoplasia (PIN); 2) those with high-grade PIN (HG-PIN), PC. There were no age differences between the groups (p = 0.05). Results. The standard procedure for PCR was applied to 127 subjects. Twenty-six (20.5%) of the 127 subjects with suspected PC were found to have Mycoplasma infection, Mycoplasma being detected in 21 (26.2%) of the 81 patients with verified HG-PIN and PC. Mycoplasma hominis was encountered in 19 (15%) patients of the 127 subjects with suspected PC and this infection was present in 16 (20%) of the 81 patients with verified HG-PIN and PC. Comparison of the frequency of HG-PIN and PC in the patients of general group (60%) and in those with Mycoplasma infection (80.8%) revealed significant differences (p = 0.031). HG-PIN and PC were also significantly more frequently seen in the patients with Mycoplasma hominis (84.2%) that in the general patient group (60%) (p = 0.033). There were no significant differences in the frequency of HG-PIN and PC between the patients from the general group (60%) and those with Mycoplasma genitalium (71.4%) (p = 0.05). The patients with verified PC and HG-PIN were more frequently found to have Mycoplasma hominis (20%) than Mycoplasma genitalium (6.2%), which further drew our closer attention to just this pathogen. The real-time PCR was used in 123 subjects to detect Mycoplasma. HG-PIN and prostate adenocarcinoma were revealed in 63 of the 123 patients with suspected PC, Mycoplasma hominis was seen in 46 (37%). The frequency (n=46) was 73.9%. The frequency of HG-PIN and PC was significantly higher in the patients with isolated Mycoplasma hominis DNA that in those without this pathogen (p < 0.001). Conclusion. Thus, the investigation showed a significantly higher correlation in the frequency of HG-PIN and PC in the patients with Mycoplasma infection that in the general study patients with suspected PC. This was supported by the use of both the standard procedure for Mycoplasma DNA determination and real-time PCR diagnosis

Защита мышей от заражения вирусом гриппа птиц субтипа H7 с помощью иммунизации рекомбинантным аденовирусом, кодирующим консервативные антигены вируса гриппа А

Influenza is a highly contagious disease that causes annual epidemics and occasional pandemics. Birds are believed to be the source of newly emerging pandemic strains, including highly pathogenic avian influenza viruses of the subtype H7. The aim of the study: to evaluate the ability of the recombinant human adenovirus, serotype 5, which expresses genes of influenza A highly conserved antigens (ion channel M2 and nucleoprotein NP), to provide protection to laboratory mice against infection with a lethal dose of avian influenza virus, subtype H7. To achieve this goal, it was necessary to adapt influenza A virus, subtype H7 for reproduction in the lungs of mice, to characterise it, and to use it for evaluation of the protective properties of the recombinant adenovirus. Materials and methods: avian influenza virus A/Chicken/NJ/294508-12/2004 (H7N2) was adapted for reproduction in the lungs of mice by repeated passages. The adapted strain was sequenced and assessed using hemagglutination test, EID50 and LD50 for laboratory mice. BALB/c mice were immunised once with Ad5-tet-M2NP adenovirus intranasally, and 21 days after the immunisation they were infected with a lethal dose (5 LD50) of influenza virus A/Chicken/NJ/294508-12/2004 (H7N2) in order to assess the protective properties of the recombinant adenovirus. The level of viral shedding from the lungs of the infected mice was evaluated by titration of the lung homogenates in MDCK cell culture on days 3 and 6 after infection. The level of specific antibodies to H7 avian influenza virus was determined by indirect enzyme immunoassay. Results: the use of Ad5-tet-M2NP adenovirus for immunisation of the mice ensured 100% survival of the animals that had disease symptoms (weight loss) after their infection with the lethal dose (5 LD50) of H7 avian influenza virus. The study demonstrated a high post-vaccination level of humoral immune response to H7 avian influenza virus. The virus titer decreased significantly by day 6 in the lungs of mice that had been immunised with Ad5-tet-M2NP compared to the control group. Conclusion: the Ad5-tetM2NP recombinant adenovirus can be used to create a candidate pandemic influenza vaccine that would protect against avian influenza viruses, subtype H7, in particular.Грипп – высококонтагиозное заболевание, вызывающее ежегодные эпидемии и через неравные интервалы времени – пандемии. Источником вновь возникающих пандемичных штаммов, как правило, являются птицы, а наибольшее беспокойство в настоящее время вызывают высокопатогенные вирусы гриппа птиц субтипа H7. Цель работы: оценить способность рекомбинантного аденовируса человека пятого серотипа, экспрессирующего гены высококонсервативных антигенов вируса гриппа А (ионного канала М2 и нуклеопротеина NP), обеспечивать защиту от заражения лабораторных мышей летальной дозой вируса гриппа птиц субтипа H7. Для достижения цели необходимо было адаптировать для размножения в легких мышей вирус гриппа А субтипа Н7, охарактеризовать и с его помощью оценить защитные свойства рекомбинантного аденовируса. Материалы и методы: вирус гриппа птиц A/Chicken/NJ/294508-12/2004 (H7N2) был адаптирован для размножения в легких мышей путем многократного пассирования. Этот штамм был секвенирован и охарактеризован в реакции гемагглютинации, установлены его ЭИД50 и ЛД50 для лабораторных мышей. Для изучения защитных свойств рекомбинантного аденовируса мыши линии BALB/c были иммунизированы аденовирусом Ad5-tet-M2NP однократно интраназально и через 21 сутки после иммунизации заражены летальной дозой (5 ЛД50) вируса гриппа птиц A/Chicken/NJ/294508-12/2004 (H7N2). Уровень вирусовыделения из легких мышей был оценен на 3 и 6 сутки после заражения с помощью титрования гомогенатов легких на культуре клеток MDCK. Уровень специфических антител к вирусу гриппа птиц субтипа Н7 определяли методом непрямого иммуноферментного анализа. Результаты: иммунизация мышей аденовирусом Ad5-tet-M2NP при наличии симптомов заболевания (снижение массы тела) обеспечила 100% выживаемость животных после заражения летальной дозой (5 ЛД50) вируса гриппа птиц субтипа H7. Продемонстрирован высокий поствакцинальный уровень гуморального иммунного ответа к вирусу гриппа птиц субтипа H7. Показано, что в легких мышей из группы, иммунизированной Ad5-tet-M2NP, уже к 6 суткам наблюдалось существенное снижение титра вируса гриппа птиц субтипа H7 по сравнению с контрольной группой. Заключение: рекомбинантный аденовирус Ad5-tet-M2NP может быть использован для создания потенциальной пандемичной противогриппозной вакцины, в том числе и от вирусов гриппа птиц субтипа H7