scholarly journals The Influence of an Elevated Production of Extracellular Enveloped Virions of the Vaccinia Virus on Its Properties in Infected Mice

Acta Naturae ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 120-132
Author(s):  
S. N. Shchelkunov ◽  
S. N. Yakubitskiy ◽  
T. V. Bauer ◽  
A. A. Sergeev ◽  
A. S. Kabanov ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Virulence Genes ◽  
Point Mutations ◽  
Intradermal Injection ◽  
Smallpox Vaccination ◽  
Intracerebral Injection ◽  
Research Subject ◽  
Modern Approach ◽  
Injection Test ◽  
Targeted Inactivation

The modern approach to developing attenuated smallpox vaccines usually consists in targeted inactivation of vaccinia virus (VACV) virulence genes. In this work, we studied how an elevated production of extracellular enveloped virions (EEVs) and the route of mouse infection can influence the virulence and immunogenicity of VACV. The research subject was the LIVP strain, which is used in Russia for smallpox vaccination. Two point mutations causing an elevated production of EEVs compared with the parental LIVP strain were inserted into the sequence of the VACV A34R gene. The created mutant LIVP-A34R strain showed lower neurovirulence in an intracerebral injection test and elevated antibody production in the intradermal injection method. This VACV variant can be a promising platform for developing an attenuated, highly immunogenic vaccine against smallpox and other orthopoxvirus infections. It can also be used as a vector for designing live-attenuated recombinant polyvalent vaccines against various infectious diseases.

scholarly journals ROUTE-COUPLED PATHOGENICITY AND IMMUNOGENICITY OF VACCINIA VIRUS VARIANT INOCULATED MICE

2019 ◽  
Author(s):  
S. Shchelkunov ◽  
A. Sergeev ◽  
A. Kabanov ◽  
S. Yakubitskyi ◽  
T. Bauer ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Virus Strain ◽  
Dna Sequences ◽  
Virulence Genes ◽  
Clinical Manifestations ◽  
World Health ◽  
Smallpox Vaccination ◽  
Natural Evolution ◽  
Vaccinia Virus Strain ◽  
Virus Strains

Vaccinia virus had played a key role in the global smallpox eradication. However, in case of mass vaccination with various vaccinia virus strains severe side effects were revealed sometimes ending up with lethal outcomes, especially in immunocompromised humans. Hence, the World Health Organization recommended to cancel smallpox vaccination after declaring in 1980 about smallpox eradication. Over last 40 years, human population virtually lost immunity not only against smallpox, but also against other zoonotic orthopoxvirus infections, such as monkeypox, cowpox, buffalopox, and camel pox. All of them pose an represent increasing threat to human health and heighten a risk of emerging highly contagious viruses due to natural evolution of previous zoonotic orthopoxviruses. In order to prevent development of small outbreaks into spreading epidemics and, thus, to decrease a risk of emergence due to natural evolution of highly pathogenic for humans orthopoxviruses, efforts should be applied to develop safe new generation live vaccines based on vaccinia virus with target virulence genes inactivation. These strains should be examined in laboratory animal models inoculated via different routes. Currently, vaccinia virus often becomes attenuated to create live recombinant vaccines due to inserting target DNA sequences into the virus virulence genes resulting in their inactivation. Vaccinia virus strain LIVP used in the Russian Federation as smallpox vaccine as well as derivative attenuated variant LIVP-GFP created by using genetic engineering methods with inactivating its thymidine kinase gene were examined.Such viruses were intracerebrally inoculated into suckling mice at doses of 101 or 102 PFU/animal for neurovirulence assessment. Adult mice were infected intranasally, subcutaneously or intradermally at doses of 107 or 108 PFU/animal and clinical manifestations were analyzed for 14 days. On 28 day after the onset, blood serum samples were collected from individual mice to measure virus specific antibody level by using ELISA. It was shown that recombinant vaccinia virus strain LIVP-GFP displayed markedly lowered neurovirulence and pathogenicity for mice as compared to parental LIVP. Finally, intradermal route turned out to demonstrate the most safe and effective profile for immunization with both examined vaccinia virus strains.

scholarly journals Attenuation of Vaccinia Virus

Acta Naturae ◽  
2015 ◽  
Vol 7 (4) ◽  
pp. 113-121 ◽  
Author(s):  
S. N. Yakubitskiy ◽  
I. V. Kolosova ◽  
R. A. Maksyutov ◽  
S. N. Shchelkunov
Keyword(s):  
Vaccinia Virus ◽  
Vaccine Strain ◽  
Neutralizing Antibodies ◽  
Protective Immunity ◽  
Virulence Genes ◽  
Binding Protein ◽  
Smallpox Vaccination ◽  
Lethal Challenge ◽  
Mammalian Cell Cultures ◽  
Genes Encoding

Since 1980, in the post-smallpox vaccination era the human population has become increasingly susceptible compared to a generation ago to not only the variola (smallpox) virus, but also other zoonotic orthopoxviruses. The need for safer vaccines against orthopoxviruses is even greater now. The Lister vaccine strain (LIVP) of vaccinia virus was used as a parental virus for generating a recombinant 1421ABJCN clone defective in five virulence genes encoding hemagglutinin (A56R), the IFN--binding protein (B8R), thymidine kinase (J2R), the complement-binding protein (C3L), and the Bcl-2-like inhibitor of apoptosis (N1L). We found that disruption of these loci does not affect replication in mammalian cell cultures. The isogenic recombinant strain 1421ABJCN exhibits a reduced inflammatory response and attenuated neurovirulence relative to LIVP. Virus titers of 1421ABJCN were 3 lg lower versus the parent VACV LIVP when administered by the intracerebral route in new-born mice. In a subcutaneous mouse model, 1421ABJCN displayed levels of VACV-neutralizing antibodies comparable to those of LIVP and conferred protective immunity against lethal challenge by the ectromelia virus. The VACV mutant holds promise as a safe live vaccine strain for preventing smallpox and other orthopoxvirus infections.

scholarly journals Mutations in the A34R gene increase the immunogenicity of vaccinia virus

2021 ◽  
Vol 25 (2) ◽  
pp. 139-146
Author(s):  
S. N. Shchelkunov ◽  
T. V. Bauer ◽  
S. N. Yakubitskiy ◽  
A. A. Sergeev ◽  
A. S. Kabanov ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Point Mutations ◽  
World Health ◽  
Smallpox Vaccination ◽  
Virus Infections ◽  
Pathogenic Viruses ◽  
Foreign Genes ◽  
Health Organization ◽  
Human Infections

Vaccination is the most simple and reliable approach of protection to virus infections. The most effective agents are live vaccines, usually low-virulence organisms for humans and closely related to pathogenic viruses or attenuated as a result of mutations/deletions in the genome of pathogenic virus. Smallpox vaccination with live vaccinia virus (VACV) closely related to smallpox virus played a key role in the success of the global smallpox eradication program carried out under the World Health Organization auspices. As a result of the WHO decision as of 1980 to stop smallpox vaccination, humankind has lost immunity not only to smallpox, but also to other zoonotic, orthopoxviruscaused human infections. This new situation allows orthopoxviruses to circulate in the human population and, as a consequence, to alter several established concepts of the ecology and range of sensitive hosts for various orthopoxvirus species. Classic VACV-based live vaccine for vaccination against orthopoxvirus infections is out of the question, because it can cause severe side effects. Therefore, the development of new safe vaccines against orthopoxviral infections of humans and animals is an important problem. VACV attenuation by modern approaches carried out by targeted inactivation of certain virus genes and usually leads to a decrease in the effectiveness of VACV in vivo propagation. As a result, it can cause a diminishing of the immune response after administration of attenuated virus to patients at standard doses. The gene for thymidine kinase is frequently used for insertion/inactivation of foreign genes and it causes virus attenuation. In this research, the effect of the introduction of two point mutations into the A34R gene of attenuated strain LIVP-GFP (ТК–), which increase the yield of extracellular enveloped virions (EEV), on the pathogenicity and immunogenicity of VACV LIVP-GFP-A34R administered intranasally to laboratory mice were studied. It was shown that increase in EEV production by recombinant strain VACV LIVP-GFP-A34R does not change the attenuated phenotype characteristic of the parental strain LIVP-GFP, but causes a significantly larger production of VACV-specific antibodies.

scholarly journals Enhancing the Protective Immune Response to Administration of a LIVP-GFP Live Attenuated Vaccinia Virus to Mice

Pathogens ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 377
Author(s):  
Sergei N. Shchelkunov ◽  
Stanislav N. Yakubitskiy ◽  
Kseniya A. Titova ◽  
Stepan A. Pyankov ◽  
Alexander A. Sergeev
Keyword(s):  
Genetic Engineering ◽  
Vaccinia Virus ◽  
Fluorescent Protein ◽  
Point Mutations ◽  
Protein Product ◽  
Smallpox Vaccination ◽  
Green Fluorescent Protein Gene ◽  
Kinase Gene ◽  
Foreign Genes ◽  
Green Fluorescent

Following the WHO announcement of smallpox eradication, discontinuation of smallpox vaccination with vaccinia virus (VACV) was recommended. However, interest in VACV was soon renewed due to the opportunity of genetic engineering of the viral genome by directed insertion of foreign genes or introduction of mutations or deletions into selected viral genes. This genomic technology enabled production of stable attenuated VACV strains producing antigens of various infectious agents. Due to an increasing threat of human orthopoxvirus re-emergence, the development of safe highly immunogenic live orthopoxvirus vaccines using genetic engineering methods has been the challenge in recent years. In this study, we investigated an attenuated VACV LIVP-GFP (TK-) strain having an insertion of the green fluorescent protein gene into the viral thymidine kinase gene, which was generated on the basis of the LIVP (Lister-Institute for Viral Preparations) strain used in Russia as the first generation smallpox vaccine. We studied the effect of A34R gene modification and A35R gene deletion on the immunogenic and protective properties of the LIVP-GFP strain. The obtained data demonstrate that intradermal inoculation of the studied viruses induces higher production of VACV-specific antibodies compared to their levels after intranasal administration. Introduction of two point mutations into the A34R gene, which increase the yield of extracellular enveloped virions, and deletion of the A35R gene, the protein product of which inhibits presentation of antigens by MHC II, enhances protective potency of the created LIVP-TK--A34R*-dA35R virus against secondary lethal orthopoxvirus infection of BALB/c mice even at an intradermal dose as low as 103 plaque forming units (PFU)/mouse. This virus may be considered not only as a candidate attenuated live vaccine against smallpox and other human orthopoxvirus infections but also as a vector platform for development of safe multivalent live vaccines against other infectious diseases using genetic engineering methods.

scholarly journals Persisting Humoral Antiviral Immunity within the Japanese Population after the Discontinuation in 1976 of Routine Smallpox Vaccinations

2005 ◽  
Vol 12 (4) ◽  
pp. 520-524 ◽  
Author(s):  
Shuji Hatakeyama ◽  
Kyoji Moriya ◽  
Masayuki Saijo ◽  
Yuji Morisawa ◽  
Ichiro Kurane ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Japanese Population ◽  
Neutralizing Antibodies ◽  
Antiviral Immunity ◽  
Smallpox Vaccination ◽  
Considerable Proportion ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specific Immunoglobulin ◽  
Specific Igg ◽  
Induced Immunity

ABSTRACT Concerns have arisen recently about the possible use of smallpox for a bioterrorism attack. Routine smallpox vaccination was discontinued in Japan in 1976; however, it is uncertain exactly how long vaccination-induced immunity lasts. We sought to evaluate the seroprevalence and intensity of anti-smallpox immunity among representatives of the present Japanese population. The subjects included 876 individuals who were born between 1937 and 1982. Vaccinia virus-specific immunoglobulin G (IgG) levels were measured by enzyme-linked immunosorbent assay (ELISA), and 152 of 876 samples were also tested for the presence of neutralizing antibodies. Of the subjects who were born before 1962, between 1962 and 1968, and between 1969 and 1975, 98.6, 98.6, and 66.0%, respectively, still retained the vaccinia virus-specific IgG with ELISA values for optical density at 405 nm (OD405) of ≥0.10. The corresponding figures for retained IgGs with OD405 values of ≥0.30 were 91.0, 90.3, and 58.2%, respectively. Neutralizing antibodies were also maintained. The sera with OD405 values of ≥0.30 showed 89% sensitivity and a 93% positive predictive value for detection of neutralizing antibodies (≥4). Thus, approximately 80% of persons born before 1969 and 50% of those born between 1969 and 1975 were also found to have maintained neutralizing antibodies against smallpox. A considerable proportion of the previous vaccinated individuals still retain significant levels of antiviral immunity. This long-lasting immunity may provide some protective benefits in the case of reemergence of smallpox, and the disease may not spread as widely and fatally as generally expected.

scholarly journals Emergence of a Viral RNA Polymerase Variant during Gene Copy Number Amplification Promotes Rapid Evolution of Vaccinia Virus

2016 ◽  
Vol 91 (4) ◽  
Author(s):  
Kelsey R. Cone ◽  
Zev N. Kronenberg ◽  
Mark Yandell ◽  
Nels C. Elde
Keyword(s):  
Rna Polymerase ◽  
Vaccinia Virus ◽  
Copy Number ◽  
Point Mutations ◽  
Gene Copy Number ◽  
Viral Rna ◽  
Gene Copy ◽  
Trade Offs ◽  
Number Variation ◽  
Copy Number Amplification

ABSTRACT Viruses are under relentless selective pressure from host immune defenses. To study how poxviruses adapt to innate immune detection pathways, we performed serial vaccinia virus infections in primary human cells. Independent courses of experimental evolution with a recombinant strain lacking E3L revealed several high-frequency point mutations in conserved poxvirus genes, suggesting important roles for essential poxvirus proteins in innate immune subversion. Two distinct mutations were identified in the viral RNA polymerase gene A24R, which seem to act through different mechanisms to increase virus replication. Specifically, a Leu18Phe substitution encoded within A24R conferred fitness trade-offs, including increased activation of the antiviral factor protein kinase R (PKR). Intriguingly, this A24R variant underwent a drastic selective sweep during passaging, despite enhanced PKR activity. We showed that the sweep of this variant could be accelerated by the presence of copy number variation (CNV) at the K3L locus, which in multiple copies strongly reduced PKR activation. Therefore, adaptive cases of CNV can facilitate the accumulation of point mutations separate from the expanded locus. This study reveals how rapid bouts of gene copy number amplification during accrual of distant point mutations can potently facilitate poxvirus adaptation to host defenses. IMPORTANCE Viruses can evolve quickly to defeat host immune functions. For poxviruses, little is known about how multiple adaptive mutations emerge in populations at the same time. In this study, we uncovered a means of vaccinia virus adaptation involving the accumulation of distinct genetic variants within a single population. We identified adaptive point mutations in the viral RNA polymerase gene A24R and, surprisingly, found that one of these mutations activates the nucleic acid sensing factor PKR. We also found that gene copy number variation (CNV) can provide dual benefits to evolving virus populations, including evidence that CNV facilitates the accumulation of a point mutation distant from the expanded locus. Our data suggest that transient CNV can accelerate the fixation of mutations conferring modest benefits, or even fitness trade-offs, and highlight how structural variation might aid poxvirus adaptation through both direct and indirect actions.

scholarly journals Effect of the Route of Administration of the Vaccinia Virus Strain LIVP to Mice on Its Virulence and Immunogenicity

Viruses ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 795
Author(s):  
Sergei N. Shchelkunov ◽  
Stanislav N. Yakubitskiy ◽  
Alexander A. Sergeev ◽  
Alexei S. Kabanov ◽  
Tatiana V. Bauer ◽  
...  
Keyword(s):  
Comparative Analysis ◽  
Vaccinia Virus ◽  
Vaccination Campaign ◽  
Lethal Dose ◽  
Clinical Manifestations ◽  
Immune Defense ◽  
Smallpox Vaccination ◽  
Laboratory Animals ◽  
Optimal Method ◽  
Clonal Variant

The mass smallpox vaccination campaign has played a crucial role in smallpox eradication. Various strains of the vaccinia virus (VACV) were used as a live smallpox vaccine in different countries, their origin being unknown in most cases. The VACV strains differ in terms of pathogenicity exhibited upon inoculation of laboratory animals and reactogenicity exhibited upon vaccination of humans. Therefore, each generated strain or clonal variant of VACV needs to be thoroughly studied in in vivo systems. The clonal variant 14 of LIVP strain (LIVP-14) was the study object in this work. A comparative analysis of the virulence and immunogenicity of LIVP-14 inoculated intranasally (i.n.), intradermally (i.d.), or subcutaneously (s.c.) to BALB/c mice at doses of 108, 107, and 106 pfu was carried out. Adult mice exhibited the highest sensitivity to the i.n. administered LIVP-14 strain, although the infection was not lethal. The i.n. inoculated LIVP-14 replicated efficiently in the lungs. Furthermore, this virus was accumulated in the brain at relatively high concentrations. Significantly lower levels of LIVP-14 were detected in the liver, kidneys, and spleen of experimental animals. No clinical manifestations of the disease were observed after i.d. or s.c. injection of LIVP-14 to mice. After s.c. inoculation, the virus was detected only at the injection site, while it could disseminate to the liver and lungs when delivered via i.d. administration. A comparative analysis of the production of virus-specific antibodies by ELISA and PRNT revealed that the highest level of antibodies was induced in i.n. inoculated mice; a lower level of antibodies was observed after i.d. administration of the virus and the lowest level after s.c. injection. Even at the lowest studied dose (106 pfu), i.n. or i.d. administered LIVP-14 completely protected mice against infection with the cowpox virus at the lethal dose. Our findings imply that, according to the ratio between such characteristics as pathogenicity/immunogenicity/protectivity, i.d. injection is the optimal method of inoculation with the VACV LIVP-14 strain to ensure the safe formation of immune defense after vaccination against orthopoxviral infections.

Absence of oropharyngeal vaccinia virus after vaccinia (smallpox) vaccination

2005 ◽  
Vol 94 (6) ◽  
pp. 682-685 ◽  
Author(s):  
Mary M. Klote ◽  
George V. Ludwig ◽  
Melanie P. Ulrich ◽  
Lisa A. Black ◽  
Dallas C. Hack ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Smallpox Vaccination

scholarly journals Failure of the Smallpox Vaccine To Develop a Skin Lesion in Vaccinia Virus-Naïve Individuals Is Related to Differences in Antibody Profiles before Vaccination, Not After

2012 ◽  
Vol 19 (3) ◽  
pp. 418-428 ◽  
Author(s):  
Xiaolin Tan ◽  
Sookhee Chun ◽  
Jozelyn Pablo ◽  
Philip Felgner ◽  
Xiaowu Liang ◽  
...  
Keyword(s):  
Skin Lesion ◽  
Antibody Response ◽  
Vaccinia Virus ◽  
Smallpox Vaccination ◽  
Unknown Etiology ◽  
Before And After ◽  
Antibody Titers ◽  
Antibody Profiling ◽  
Different Doses ◽  
Protein Enzyme

ABSTRACTSuccessful vaccination against smallpox with conventional vaccinia virus is usually determined by the development of a vesicular skin lesion at the site of vaccinia inoculation, called a “take.” Although previous vaccination is known to be associated with attenuation of the take, the immunology that underlies a no-take in vaccinia-naïve individuals is not well understood. We hypothesized that antibody profiling of individuals before and after receiving vaccinia virus would reveal differences between takes and no-takes that may help better explain the phenomenon. Using vaccinia virus proteome microarrays and recombinant protein enzyme-linked immunosorbent assays (ELISAs), we first examined the antibody response in vaccinia-naïve individuals that failed to take after receiving different doses of the replication-competent DryVax and Aventis Pasteur (APSV) smallpox vaccines. Most that received diluted vaccine failed to respond, although four no-takes receiving diluted vaccine and four receiving undiluted vaccine mounted an antibody response. Interestingly, their antibody profiles were not significantly different from those of controls that did show a take. However, we did find elevated antibody titers in no-takes prior to receiving DryVax that were significantly different from those of takes. Although the sample size studied was small, we conclude the failure to take in responders correlates with preexisting immunity of unknown etiology that may attenuate the skin reaction in a way similar to previous smallpox vaccination.

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