Publications by authors named "Alexander Rumyantsev"

18 Publications

  • Page 1 of 1

Analysis of a Queueing Model with Batch Markovian Arrival Process and General Distribution for Group Clearance.

Methodol Comput Appl Probab 2020 Oct 19:1-29. Epub 2020 Oct 19.

Institute of Applied Mathematical Research, Karelian Research Centre of RAS, 11 Pushkinskaya Str., Petrozavodsk, Russia.

In this paper we consider a single server queueing model with under general bulk service rule with infinite upper bound on the batch size which we call . The arrivals occur according to a batch Markovian point process and the services are generally distributed. The customers arriving after the service initiation cannot enter the ongoing service. The service time is independent on the batch size. First, we employ the classical embedded Markov renewal process approach to study the model. Secondly, under the assumption that the services are of phase type, we study the model as a continuous-time Markov chain whose generator has a very special structure. Using matrix-analytic methods we study the model in steady-state and discuss some special cases of the model as well as representative numerical examples covering a wide range of service time distributions such as constant, uniform, Weibull, and phase type.
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http://dx.doi.org/10.1007/s11009-020-09828-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7569101PMC
October 2020

Primary Immunodeficiencies in Russia: Data From the National Registry.

Front Immunol 2020 6;11:1491. Epub 2020 Aug 6.

Tatarstan Pediatric Republican Clinical Hospital, Kazan, Russia.

Primary immunodeficiencies (PID) are a group of rare genetic disorders with a multitude of clinical symptoms. Characterization of epidemiological and clinical data via national registries has proven to be a valuable tool of studying these diseases. The Russian PID registry was set up in 2017, by the National Association of Experts in PID (NAEPID). It is a secure, internet-based database that includes detailed clinical, laboratory, and therapeutic data on PID patients of all ages. The registry contained information on 2,728 patients (60% males, 40% females), from all Federal Districts of the Russian Federation. 1,851/2,728 (68%) were alive, 1,426/1,851 (77%) were children and 425/1,851 (23%) were adults. PID was diagnosed before the age of 18 in 2,192 patients (88%). Antibody defects (699; 26%) and syndromic PID (591; 22%) were the most common groups of PID. The minimum overall PID prevalence in the Russian population was 1.3:100,000 people; the estimated PID birth rate is 5.7 per 100,000 live births. The number of newly diagnosed patients per year increased dramatically, reaching the maximum of 331 patients in 2018. The overall mortality rate was 9.8%. Genetic testing has been performed in 1,740 patients and genetic defects were identified in 1,344 of them (77.2%). The median diagnostic delay was 2 years; this varied from 4 months to 11 years, depending on the PID category. The shortest time to diagnosis was noted in the combined PIDs-in WAS, DGS, and CGD. The longest delay was observed in AT, NBS, and in the most prevalent adult PID: HAE and CVID. Of the patients, 1,622 had symptomatic treatment information: 843 (52%) received IG treatment, mainly IVIG (96%), and 414 (25%) patients were treated with biological drugs. HSCT has been performed in 342/2,728 (16%) patients, of whom 67% are currently alive, 17% deceased, and 16% lost to follow-up. Three patients underwent gene therapy for WAS; all are currently alive. Here, we describe our first analysis of the epidemiological features of PID in Russia, allowing us to highlight the main challenges around PID diagnosis and treatment.
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http://dx.doi.org/10.3389/fimmu.2020.01491DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7424007PMC
May 2021

Magnetic resonance imaging of changes in the brain of children cured of acute lymphoblastic leukemia.

Hematol Rep 2019 Sep 18;11(3):7946. Epub 2019 Sep 18.

Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology, Ministry of Healthcare, Moscow.

This study was aimed to systematize magnetic resonance imaging (MRI) presentation of toxic leukoencephalopathy, to find the correlation between method of central nervous system (CNS) leukemia prevention and changes on MRI, to find relationship between existence leukoencephalopathy on imaging and neurocognitive deficits in pediatric patients after anti-leukemic therapy. Brain MRI data of 48 children, who underwent a therapy course according to the ALL-MB intermediate risk protocol, was evaluated. In accordance with two arms of this protocol, they received either radiation therapy, or additional intrathecal administration of chemotherapeutic agents as a prevention of CNS leukemia. Also, neurocognitive tests were performed. According to the results of the performed investigation, 10 (50%) out of 20 children, who received cranial irradiation and 18 (66.6%) out of 27 patients, who received only intrathecal therapy demonstrated abnormal brain changes (leukoencephalopathy) according to MRI data. Leukoencephalopathy was mostly presented by diffuse zones and localized predominantly in the frontal and temporal lobes. There was no correlation between method of CNS prevention and the existence of leukoencephalopathy on MRI. The analysis of our data did not show significant differences in brain damage and severity of cognitive impairment depending on the type of prevention of CNS leukemia. Moreover, in this study no statistical correlation was found between leukoencephalopathy on MRI and neurocognitive impairment according to clinical tests data. Further long-term prospective studies and examinations should be performed to assess late neurotoxic effects.
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http://dx.doi.org/10.4081/hr.2019.7946DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6761460PMC
September 2019

αβ T Cell-Depleted Haploidentical Hematopoietic Stem Cell Transplantation without Antithymocyte Globulin in Children with Chemorefractory Acute Myelogenous Leukemia.

Biol Blood Marrow Transplant 2019 05 21;25(5):e179-e182. Epub 2019 Jan 21.

Department of Hematopoietic Stem Cell Transplantation, Dmitriy Rogachev National Medical Research Center of Pediatric Hematology, Oncology, and Immunology, Moscow, Russia. Electronic address:

We evaluated the outcome of αβ T cell-depleted haploidentical hematopoietic stem cell transplantation (HSCT) in a cohort of children with chemorefractory acute myelogenous leukemia (AML). Twenty-two patients with either primary refractory (n = 10) or relapsed refractory (n = 12) AML in active disease status received a transplant from haploidentical donors. The preparative regimen included cytoreduction with fludarabine and cytarabine and subsequent myeloablative conditioning with treosulfan and thiotepa. Antithymocyte globulin was substituted with tocilizumab in all patients and also with abatacept in 10 patients. Grafts were peripheral blood stem cells engineered by αβ T cell and CD19 depletion. Post-transplantation prophylactic therapy included infusion of donor lymphocytes, composed of a CD45RA-depleted fraction with or without a hypomethylating agent. Complete remission was achieved in 21 patients (95%). The cumulative incidence of grade II-IV acute graft-versus-host disease (GVHD) was 18%, and the cumulative incidence of chronic GVHD was 23%. At 2 years, transplantation-related mortality was 9%, relapse rate was 42%, event-free survival was 49%, and overall survival was 53%. Our data suggest that αβ T cell-depleted haploidentical HSCT provides a reasonable chance of long-term survival in a cohort of children with chemorefractory AML and creates a solid basis for further improvement.
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http://dx.doi.org/10.1016/j.bbmt.2019.01.023DOI Listing
May 2019

PPP3CB contributes to poor prognosis through activating nuclear factor of activated T-cells signaling in neuroblastoma.

Mol Carcinog 2019 03 13;58(3):426-435. Epub 2018 Dec 13.

Chiba Cancer Center Research Institute, Chiba, Japan.

We previously identified a gain-of-function mutation in PPP3CB in a neuroblastoma (NB) with MYCN amplification. Here we investigated the functional and clinical role of PPP3CB in NB. High PPP3CB expression was an independent indicator predicting poor prognosis of NB. Overexpression of wildtype or mutated PPP3CB (PPP3CBmut) promoted cell growth, but PPP3CB knockdown decreased cell growth in NB cells. Forced expressions of PPP3CB and PPP3CBmut activated NFAT2 and NFAT4 transcription factors and inhibited GSK3β activity, resulting in the increase in the expressions of c-Myc, MYCN, and β-catenin, which were downregulated in response to PPP3CB knockdown. Treatment with calcineurin inhibitor cyclosporin A (CsA) or FK506 suppressed cell proliferation and induced apoptotic cell death in both MYCN-amplified and MYCN-non-amplified NB cell lines. Expression of PPP3CB protein was decreased in response to two calcineurin inhibitors. c-Myc, MYCN, and β-catenin were downregulated at the mRNA and protein levels in CsA or FK506-treated NB cells. Our data indicate that elevated expression of PPP3CB and the expression of its constitutively active mutant contribute to the aggressive behavior of NB tumors and therefore suggest that inhibition of calcineurin activity might have therapeutic potential for high-risk NB.
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http://dx.doi.org/10.1002/mc.22939DOI Listing
March 2019

A novel approach to a rabies vaccine based on a recombinant single-cycle flavivirus vector.

Vaccine 2017 12 9;35(49 Pt B):6898-6904. Epub 2017 Sep 9.

Sanofi Pasteur, R&D Discovery US, 38 Sidney Street, Cambridge, MA 02139, United States.

The RepliVax® vaccine (RV) platform is based on flavivirus genomes that are rationally attenuated by deletion. These single-cycle RV vaccine candidates targeting flavivirus pathogens have been demonstrated to be safe, highly immunogenic, and efficacious in animal models, including non-human primates. Here we show utility of the technology for delivery of a non-flavivirus immunogen by engineering several West Nile-based RV vectors to express full-length rabies virus G protein. The rabies virus G protein gene was incorporated in place of different West Nile structural protein gene deletions. The resulting RV-RabG constructs were demonstrated to replicate to high titers (8 log infectious particles/ml) in complementing helper cells. Following infection of normal cells, they provided efficient rabies virus G protein expression, but did not spread to surrounding cells. Expression of rabies virus G protein was stable and maintained through multiple rounds of in vitro passaging. A sensitive neurovirulence test in 2-3 day old neonatal mice demonstrated that RV-RabG candidates were completely avirulent indicative of high safety. We evaluated the RV-RabG variants in several animal models (mice, dogs, and pigs) and demonstrated that a single dose elicited high titers of rabies virus-neutralizing antibodies and protected animals from live rabies virus challenge (mice and dogs). Importantly, dogs were protected at both one and two years post-immunization, demonstrating durable protective immunity. The data demonstrates the potential of the RepliVax® technology as a potent vector delivery platform for developing vaccine candidates against non-flavivirus targets.
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http://dx.doi.org/10.1016/j.vaccine.2017.08.055DOI Listing
December 2017

Fermented Milk Consumption and Common Infections in Children Attending Day-Care Centers: A Randomized Trial.

J Pediatr Gastroenterol Nutr 2016 11;63(5):534-543

*Children's Clinical Hospital No. 9 named after G.N. Speransky of Moscow Healthcare Department, Moscow, Russia †Danone Nutricia Research, Centre de Recherche Daniel Carasso, Palaiseau, France ‡University Medicine, Johannes-Gutenberg-University, Mainz, Germany §Federal Budget Institution of Science "Central Research Institute of Epidemiology" of The Federal Service on Customers' Rights Protection and Human Well-being Surveillance ||Federal Government Budget Institution "Federal Scientific Clinical Center of Pediatric Hematology, Oncology and Immunology named after Dmitry Rogachev" of the Ministry of Health of Russian Federation, Moscow, Russia.

Objectives: This multicenter, double-blind, randomized, placebo-controlled clinical trial investigated the effect of a fermented milk product containing the Lactobacillus casei National Collection of Microorganisms and Cell Cultures (CNCM) I-1518 strain on respiratory and gastrointestinal common infectious diseases (CIDs) in children attending day-care centers in Russia.

Methods: Children ages 3 to 6 years received 100 g of a fermented milk product (n = 300) or a control product (n = 299) twice daily for 3 months, followed by a 1-month observation period. The primary outcome was the incidence of CIDs during the product consumption period.

Results: There was no significant difference in the incidence of CIDs between the groups (N = 98 with fermented milk product vs N = 93 with control product). The overall number of CIDs (and no severe cases at all) in both study groups and in all 12 centers, however, was unexpectedly low resulting in underpowering of the study. No differences were found between the groups in the duration or severity of disease, duration of sick leave from day-care centers, parental missed working days, or in quality-of-life dimensions on the PedsQL questionnaire (P > 0.05).There was, however, a significantly lower incidence of the most frequently observed CID, rhinopharyngitis, in children consuming the fermented milk product compared with those consuming the control product (N = 81 vs N = 100, relative risk 0.82, 95% confidence interval 0.69-0.96, P = 0.017) when considering the entire study period.

Conclusions: Although no other significant differences were shown between the fermented milk and control product groups in this study, lower incidence of rhinopharyngitis may indicate a beneficial effect of this fermented milk product.
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http://dx.doi.org/10.1097/MPG.0000000000001248DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5084641PMC
November 2016

Single-dose vaccine against tick-borne encephalitis.

Proc Natl Acad Sci U S A 2013 Aug 15;110(32):13103-8. Epub 2013 Jul 15.

Sanofi Pasteur, Discovery US, Cambridge, MA 02139, USA.

Tick-borne encephalitis (TBE) virus is the most important human pathogen transmitted by ticks in Eurasia. Inactivated vaccines are available but require multiple doses and frequent boosters to induce and maintain immunity. Thus far, the goal of developing a safe, live attenuated vaccine effective after a single dose has remained elusive. Here we used a replication-defective (single-cycle) flavivirus platform, RepliVax, to generate a safe, single-dose TBE vaccine. Several RepliVax-TBE candidates attenuated by a deletion in the capsid gene were constructed using different flavivirus backbones containing the envelope genes of TBE virus. RepliVax-TBE based on a West Nile virus backbone (RV-WN/TBE) grew more efficiently in helper cells than candidates based on Langat E5, TBE, and yellow fever 17D backbones, and was found to be highly immunogenic and efficacious in mice. Live chimeric yellow fever 17D/TBE, Dengue 2/TBE, and Langat E5/TBE candidates were also constructed but were found to be underattenuated. RV-WN/TBE was demonstrated to be highly immunogenic in Rhesus macaques after a single dose, inducing a significantly more durable humoral immune response compared with three doses of a licensed, adjuvanted human inactivated vaccine. Its immunogenicity was not significantly affected by preexisting immunity against WN. Immunized monkeys were protected from a stringent surrogate challenge. These results support the identification of a single-cycle TBE vaccine with a superior product profile to existing inactivated vaccines, which could lead to improved vaccine coverage and control of the disease.
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http://dx.doi.org/10.1073/pnas.1306245110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3740852PMC
August 2013

Therapy of advanced-stage mature B-cell lymphoma and leukemia in children and adolescents with rituximab and reduced intensity induction chemotherapy (B-NHL 2004M protocol): the results of a multicenter study.

J Pediatr Hematol Oncol 2014 Jul;36(5):395-401

*Federal Research Centre of Pediatric Hematology, Oncology and Immunology named after Dmitry Rogachev †Russian Pediatric Clinical Hospital, Moscow ‡Municipal Clinical Hospital No. 31, Saint-Petersburg §Regional Pediatric Clinical Hospital, Yekaterinburg ∥Regional Pediatric Clinical Hospital, Nizhniy Novgorod ¶Territorial Pediatric Clinical Hospital, Perm, Russia.

Pediatric mature B-cell non-Hodgkin lymphomas (B-NHLs) are highly aggressive malignant tumors that are curable with chemotherapy (ChT). High-dose methotrexate (MTX) is considered indispensable for successful treatment, but this therapy frequently induces severe mucositis and infectious complications, especially in induction, which can cause treatment failure. A prospective multicenter trial of combined immunochemotherapy for advanced-stage B-NHL with rituximab and the modified NHL-BFM-90 protocol was conducted. The major differences from the original protocol were a decrease in the dose of MTX from 5000 to 1000 mg/m/24 h in the first 2 ChT blocks and the addition of rituximab at 375 mg/m to each of the first 4 blocks of ChT. Eighty-three newly diagnosed patients with a median age of 8.84 years with Burkitt lymphoma/leukemia and diffuse large B-cell lymphomas stage III to IV were included. Four patients died during induction ChT due to tumor lysis syndrome and infection. Two additional patients died subsequently due to tumor resistance. Complete remission was achieved in 77 (92.8%) patients; 2 patients relapsed at 1 and 3 months, and 2 developed secondary malignancies at 1 and 6.5 years, respectively, after the completion of therapy. The overall survival probability was 82%±8% with a median follow-up of 65.2 months. Combined therapy with rituximab and intensive ChT with a reduced MTX dose of 1 g/m in the 2 induction courses was feasible and produced high cure rates in patients with pediatric advanced-stage mature B-NHL.
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http://dx.doi.org/10.1097/MPH.0b013e31829d4900DOI Listing
July 2014

Characterization of the RepliVax platform for replication-defective flavivirus vaccines.

Vaccine 2011 Jul 26;29(32):5184-94. Epub 2011 May 26.

Sanofi Pasteur, Discovery US, 38 Sidney Street, Cambridge, MA 02139, USA.

RepliVax, a novel replication-defective vaccine platform has recently been described as a suitable means of generating potent vaccines targeting flaviviruses. In this study, we directly compared attenuation, immunogenicity and efficacy of several prototype RepliVax constructs to available, well characterized live attenuated (LAV) and inactivated (INV) flavivirus vaccine controls in mice and hamsters. Other important aspects of general mechanisms and properties of RepliVax vaccines were also studied. The prototypes were found to be nonpathogenic in sensitive suckling mouse neurovirulence tests, and highly immunogenic and efficacious in mice and hamsters, with evidence that immunogenicity can be comparable to LAV controls in terms of both magnitude and durability of response. Our data also suggest that choice of inoculation route can be beneficial for maximizing RepliVax immunogenicity. Additionally, different vaccine constructs can be administered as cocktail formulations without compromising immunogenicity of individual components. RepliVax constructs were determined to induce a Th1 biased immune response, similar to LAVs, and different from INV inducing a Th2 type response. The results presented validate the utility of the RepliVax platform for development of novel flavivirus vaccines.
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http://dx.doi.org/10.1016/j.vaccine.2011.05.032DOI Listing
July 2011

The neurovirulence and neuroinvasiveness of chimeric tick-borne encephalitis/dengue virus can be attenuated by introducing defined mutations into the envelope and NS5 protein genes and the 3' non-coding region of the genome.

Virology 2010 Sep 1;405(1):243-52. Epub 2010 Jul 1.

Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

Tick-borne encephalitis (TBE) is a severe disease affecting thousands of people throughout Eurasia. Despite the use of formalin-inactivated vaccines in endemic areas, an increasing incidence of TBE emphasizes the need for an alternative vaccine that will induce a more durable immunity against TBE virus (TBEV). The chimeric attenuated virus vaccine candidate containing the structural protein genes of TBEV on a dengue virus genetic background (TBEV/DEN4) retains a high level of neurovirulence in both mice and monkeys. Therefore, attenuating mutations were introduced into the envelope (E(315)) and NS5 (NS5(654,655)) proteins, and into the 3' non-coding region (Delta30) of TBEV/DEN4. The variant that contained all three mutations (vDelta30/E(315)/NS5(654,655)) was significantly attenuated for neuroinvasiveness and neurovirulence and displayed a reduced level of replication and virus-induced histopathology in the brains of mice. The high level of safety in the central nervous system indicates that vDelta30/E(315)/NS5(654,655) should be further evaluated as a TBEV vaccine.
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http://dx.doi.org/10.1016/j.virol.2010.06.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2914112PMC
September 2010

Direct random insertion of an influenza virus immunologic determinant into the NS1 glycoprotein of a vaccine flavivirus.

Virology 2010 Jan 12;396(2):329-38. Epub 2009 Nov 12.

Sanofi pasteur, Discovery US, 38 Sidney Street, Cambridge, MA 02139, USA.

A live chimeric vaccine virus against Japanese encephalitis (JE), ChimeriVax-JE, was used to define methods for optimal, random insertion of foreign immunologic determinants into flavivirus glycoproteins. The conserved M2e peptide of influenza A virus was randomly inserted into the yellow fever-specific NS1 glycoprotein of ChimeriVax-JE. A technique combining plaque purification with immunostaining yielded a recombinant virus that stably expressed M2e at NS1-236 site. The site was found permissive for other inserts. The insertion inhibited NS1 dimerization in vitro, which had no significant effect on virus replication in vitro and immunogenicity in vivo. Two different NS1-specific monoclonal antibodies and a polyclonal antibody efficiently recognized only the NS1 protein dimer, but not monomer. Adaptation of the virus to Vero cells resulted in two amino acid changes upstream from the insert which restored NS1 dimerization. Immunized mice developed high-titer M2e-specific antibodies predominantly of the IgG2A isotype indicative of a Th1-biased response.
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http://dx.doi.org/10.1016/j.virol.2009.10.033DOI Listing
January 2010

Safety evaluation of chimeric Langat/Dengue 4 flavivirus, a live vaccine candidate against tick-borne encephalitis.

J Med Virol 2009 Oct;81(10):1777-85

Chumakov Institute of Poliomyelitis and Viral Encephalitides RAMSc, Moscow, Russia.

The chimeric flavivirus LGT/DEN4 containing prM and E genes of naturally attenuated Langat virus with remaining sequence derived from low neuroinvasive Dengue 4 virus was previously produced and assessed as a candidate for live vaccine against tick-borne encephalitis (TBE) [Pletnev and Men (1998): Proc Natl Acad Sci USA 95:1746-1751; Pletnev et al. (2000): Virology 274:23-31; Pletnev et al. (2001): J Virol 75:8259-8267; Wright et al. (2008): Vaccine 26:882-890]. In this article we compared two animal species: mice and monkeys, in order to select most sensitive models for safety evaluation of new vaccine candidates against TBE. Direct neurovirulence in suckling mice, neuroinvasiveness upon peripheral inoculation, rate of virus multiplication and expansion in CNS and its ability to persist in the central nervous system (CNS) were studied in adult mice; virological and pathomorphological examination of the CNS and visceral organs after intrathalamic virus inoculation was selected as a safety neurovirulence test in monkeys. The chimera was substantially less virulent in both animal models compared to the Absettarov strain of TBE virus. LGT/DEN4 was highly attenuated in suckling and adult mice with no evidence of viral persistence in CNS. In contrast to the mouse model, the chimera was able to reproduce in the CNS of monkeys to moderate titers, caused pathomorphological lesions in two and even illness in one of four animals, and was registered in simian brain on the 30th day post-infection. The presented data show that tests in mice solely might not be a sufficient model for safety testing of chimeric viruses.
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http://dx.doi.org/10.1002/jmv.21587DOI Listing
October 2009

Construction and biological characterization of artificial recombinants between a wild type flavivirus (Kunjin) and a live chimeric flavivirus vaccine (ChimeriVax-JE).

Vaccine 2007 Sep 27;25(37-38):6661-71. Epub 2007 Jul 27.

Acambis, Inc., 38 Sidney St., Cambridge, MA 02139, USA.

Although the theoretical concern of genetic recombination has been raised related to the use of live attenuated flavivirus vaccines [Seligman, Gould, Lancet 2004;363:2073-5], it has little foundation [e.g., Monath TP, Kanesa-Thasan N, Guirakhoo F, Pugachev K, Almond J, Lang J, et al. Vaccine 2005;23:2956-8]. To investigate biological effects of recombination between a chimeric yellow fever (YF) 17D/Japanese encephalitis (JE) vaccine virus (ChimeriVax-JE) and a wild-type flavivirus Kunjin (KUN-cDNA), the prM-E envelope protein genes were swapped between the two viruses, resulting in new YF 17D/KUN(prM-E) and KUN/JE(prM-E) chimeras. The prM-E genes are easily exchangeable between flavivirues, and thus the exchange was expected to yield the most replication-competent chimeras, while other rationally designed recombinants would be more likely to be crippled or non-viable. The new chimeras proved highly attenuated in comparison with the KUN-cDNA parent, as judged by plaque size and growth kinetics in cell culture, low viremia in hamsters, and reduced neurovirulence/neuroinvasiveness in mice. These data provide strong experimental evidence that the potential of recombinants, should they ever emerge, to cause disease or spread (compete in nature with wild-type flaviviruses) would be indeed extremely low.
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http://dx.doi.org/10.1016/j.vaccine.2007.07.016DOI Listing
September 2007

Microevolution of tick-borne encephalitis virus in course of host alternation.

Virology 2007 May 26;362(1):75-84. Epub 2007 Jan 26.

M.P. Chumakov Institute of Poliomyelitis and Viral Encephalitides, Russian Academy of Medical Sciences, Moscow Region 142782, Russia.

Two tick-borne encephalitis (TBE) virus variants were studied: mouse brain-adapted strain EK-328 and its derivate adapted to Hyalomma marginatum ticks. The tick-adapted virus exhibited small-plaque phenotype and slower replication in PEK cells, higher yield in ticks, decreased neuroinvasiveness in mice, increased binding to heparin-sepharose. A total of 15 nucleotide substitutions distinguished genomes of these variants, six substitutions resulted in protein sequence alterations, and two were in 5'NTR. Two amino acid substitutions in E protein were responsible for the observed phenotypic differences. Data obtained during reverse passaging of the tick-adapted virus in vivo and in vitro suggest that TBE virus exists as a heterogeneous population that contains virus variants most adapted to reproduction in either ticks or mammals. Host switch results in a change in the ratio of these variants in the population. Plaque purification of the tick-adapted virus resulted in the prompt emergence of new mutants with different virulence for mammals.
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http://dx.doi.org/10.1016/j.virol.2006.12.013DOI Listing
May 2007

Chimeric West Nile/dengue virus vaccine candidate: preclinical evaluation in mice, geese and monkeys for safety and immunogenicity.

Vaccine 2006 Sep 21;24(40-41):6392-404. Epub 2006 Jun 21.

Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-8133, USA.

A live attenuated virus vaccine is being developed to protect against West Nile virus (WN) disease in humans. Previously, it was found that chimeric West Nile/dengue viruses (WN/DEN4 and WN/DEN4Delta30) bearing the membrane precursor and envelope protein genes of WN on a backbone of dengue type 4 virus (DEN4) with or without a deletion of 30 nucleotides (Delta30) in the 3' noncoding region of the DEN4 part of the chimeric genome were attenuated and efficacious in mice and monkeys against WN challenge. Here, we report the generation of a clinical lot of WN/DEN4Delta30 virus and its further preclinical evaluation for safety and immunogenicity in mice, geese and monkeys. The vaccine candidate had lost neuroinvasiveness in highly sensitive immunodeficient mice inoculated intraperitoneally and had greatly reduced neurovirulence in suckling mice inoculated intracerebrally (IC). Compared to the wild-type WN parent, the chimeric virus was highly restricted in replication in both murine and human neuroblastoma cells as well as in brains of suckling mice. The WN/DEN4Delta30 virus failed to infect geese, indicating that chimerization of WN with DEN4 completely attenuated WN for this avian host. This observation suggests that the WN/DEN4 chimeric viruses would be restricted in their ability to be transmitted from vaccinees to domestic or wild birds. In monkeys, the WN/DEN4Delta30 vaccine candidate was highly immunogenic despite its low level of replication with undetectable viremia. Furthermore, the WN/DEN4Delta30 vaccine virus was safe and readily induced neutralizing antibodies against WN in monkeys immune to each of the four serotypes of dengue virus. These studies confirm the attenuation of WN/DEN4Delta30 for non-human primates, including dengue-immune monkeys, and demonstrate both a highly restricted replication (>10(8)-fold decrease) in the brain of mice inoculated IC and an absence of infectivity for birds, findings that indicate this vaccine should be safe for both the recipient and the environment.
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http://dx.doi.org/10.1016/j.vaccine.2006.06.008DOI Listing
September 2006

A tick-borne Langat virus mutant that is temperature sensitive and host range restricted in neuroblastoma cells and lacks neuroinvasiveness for immunodeficient mice.

J Virol 2006 Feb;80(3):1427-39

Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases/NIH, 12735 Twinbrook Parkway, Twinbrook 3, Room 3W13, MSC 8133, Bethesda, MD 20892-8133, USA.

Langat virus (LGT), the naturally attenuated member of the tick-borne encephalitis virus (TBEV) complex, was tested extensively in clinical trials as a live TBEV vaccine and was found to induce a protective, durable immune response; however, it retained a low residual neuroinvasiveness in mice and humans. In order to ablate or reduce this property, LGT mutants that produced a small plaque size or temperature-sensitive (ts) phenotype in Vero cells were generated using 5-fluorouracil. One of these ts mutants, clone E5-104, exhibited a more than 10(3)-fold reduction in replication at the permissive temperature in both mouse and human neuroblastoma cells and lacked detectable neuroinvasiveness for highly sensitive immunodeficient mice. The E5-104 mutant possessed five amino acid substitutions in the structural protein E and one change in each of the nonstructural proteins NS3 and NS5. Using reverse genetics, we demonstrated that a Lys(46)-->Glu substitution in NS3 as well as a single Lys(315)-->Glu change in E significantly impaired the growth of LGT in neuroblastoma cells and reduced its peripheral neurovirulence for SCID mice. This study and our previous experience with chimeric flaviviruses indicated that a decrease in viral replication in neuroblastoma cells might serve as a predictor of in vivo attenuation of the neurotropic flaviviruses. The combination of seven mutations identified in the nonneuroinvasive E5-104 mutant provided a useful foundation for further development of a live attenuated TBEV vaccine. An evaluation of the complete sequence of virus recovered from brain of SCID mice inoculated with LGT mutants identified sites in the LGT genome that promoted neurovirulence/neuroinvasiveness.
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http://dx.doi.org/10.1128/JVI.80.3.1427-1439.2006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1346960PMC
February 2006

Comparison of live and inactivated tick-borne encephalitis virus vaccines for safety, immunogenicity and efficacy in rhesus monkeys.

Vaccine 2006 Jan 9;24(2):133-43. Epub 2005 Aug 9.

Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, MSC 8133, Bethesda, MD 20892-8133, USA.

Three antigenic chimeric live attenuated tick-borne encephalitis virus (TBEV) vaccine candidates were compared for level of replication in murine and human neuroblastoma cells, for neurovirulence and neuroinvasiveness in mice, and for safety, immunogenicity and efficacy in rhesus monkeys. Two chimeric viruses were generated by replacing the membrane precursor and envelope protein genes of dengue type 4 virus (DEN4) with the corresponding genes of a Far Eastern TBEV, Sofjin strain, in the presence (TBEV/DEN4Delta30) or absence (TBEV/DEN4) of a 30 nucleotide deletion (Delta30) in the 3' noncoding region of the DEN4 part of the chimeric genome. A third chimeric TBEV vaccine candidate was based on the antigenically distant, but naturally attenuated Langat virus (LGT). Chimerization of LGT with DEN4 resulted in decreased neurovirulence and neuroinvasiveness in mice and highly restricted viremia in rhesus monkeys. Also, the LGT/DEN4 chimera was highly restricted in replication in both murine and human neuroblastoma cells. In contrast, TBEV/DEN4 and TBEV/DEN4Delta30 were neither attenuated for neurovirulence in the mice nor restricted in replication in the neuroblastoma cells. However, both were highly attenuated for neuroinvasiveness in mice. TBEV/DEN4 replicated to moderately high titer in rhesus monkeys (mean peak viremia=10(3.1)PFU/ml) indicating that the TBEV/DEN4 chimerization had only a modest, if any, attenuating effect in monkeys. However, the addition of the Delta30 mutation to TBEV/DEN4 greatly attenuated the chimeric virus for rhesus monkeys (mean peak viremia=10(0.7)PFU/ml) and induced a higher level of antibody against the TBEV than did LGT/DEN4. A single dose of either highly attenuated TBEV/DEN4Delta30 or LGT/DEN4 vaccine candidate or three doses of an inactivated TBEV vaccine were efficacious in monkeys against wild-type LGT challenge. These results indicate that both TBEV/DEN4Delta30 and LGT/DEN4 are safe and efficacious in rhesus monkeys and should be further evaluated as vaccine candidates for use in humans.
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http://dx.doi.org/10.1016/j.vaccine.2005.07.067DOI Listing
January 2006