The vaccinia virus (VACV) has been used for prophylactic immunization against smallpox for many decades. However, the VACV-based vaccine had been highly reactogenic. Therefore, after the eradication of smallpox, the World Health Organization in 1980 recommended that vaccination against this infection be discontinued. As a result, there has been a rise in the occurrence of orthopoxvirus infections in humans in recent years, with the most severe being the 2022 monkeypox epidemic that reached all continents. Thus, it is crucial to address the pressing matter of developing safe and highly immunogenic vaccines for new generations to combat orthopoxvirus infections. In a previous study, we created a LAD strain by modifying the LIVP (L) VACV strain, which is used as a first-generation smallpox vaccine in Russia. This modification involved introducing mutations in the A34R gene to enhance extracellular virion production and deleting the A35R gene to counteract the antibody response to the viral infection. In this study, a strain LADA was created with an additional deletion in the DNA of the LAD strain ati gene. This ati gene directs the production of a major non-virion immunogen. The findings indicate that the LADA VACV variant exhibits lower levels of reactogenicity in BALB/c mice during intranasal infection, as compared to the original L strain. Following intradermal immunization with a 105 PFU dose, both the LAD and LADA strains were found to induce a significantly enhanced cellular immune response in mice when compared to the L strain. At the same time, the highest level of virus-specific IFN-γ producing cells for the LAD variant was detected on the 7th day post-immunization (dpi), whereas for LADA, it was observed on 14 dpi. The LAD and LADA strains induced significantly elevated levels of VACV-specific IgG compared to the original L strain, particularly between 28 and 56 dpi. The vaccinated mice were intranasally infected with the cowpox virus at a dose of 460 LD50 to assess the protective immunity at 62 dpi. The LADA virus conferred complete protection to mice, with the LAD strain providing 70% protection and the parent strain L offering protection to only 60% of the animals.

The purpose of this study is to evaluate the effect of dose and frequency of immunization of BALB/c mice with intradermal (ID) injection of the LIVP strain of vaccinia virus (VACV) on the level of their protection against the ectromelia virus (ECTV), which is highly virulent for mice. Immunization of mice was carried out using a single or double ID injection of VACV LIVP with an interval of 28 days at a dose of 105 plaque-forming units (PFU), as well as a single injection at a dose of 106 PFU. In blood sera taken from mice during their lifetime, the dynamics of biosynthesis of VACV-specific IgG was determined with enzyme-linked immunosorbent assay (ELISA) up to 140 days postvaccination (dpv). At 142 dpv, groups immunized with the LIVP virus and control animals were intranasally infected with a lethal dose of ECTV and the indicators of clinical manifestations of infection, body weight of animals, and their death were monitored. At 28 dpv the level of antibodies in mice vaccinated with VACV LIVP at a dose of 106 PFU significantly exceeded this level in mice vaccinated with the same virus at a dose of 105 PFU. Repeated immunization with VACV LIVP at a dose of 105 PFU on 28 dpv led to a significant increase in the biosynthesis of virus-specific IgG and the level of analyzed antibodies in this group of mice corresponded to a similar indicator for the group of mice vaccinated once with VACV LIVP at a dose of 106 PFU. After infection with a lethal dose of ECTV, all animals in the control group (unvaccinated) died by the eighth day after infection, while those vaccinated with VACV LIVP once at a dose of 105 PFU died by the 14th day. In groups of mice vaccinated twice at a dose of 105 PFU and once at a dose of 106 PFU, 80

Vaccinia virus (VACV) strains caused a more pronounced production of antibodies with intradermal (i.d.) injection compared to scarification (s.s.) inoculation. To test for developed protective immunity at 62 day post vaccination (dpv), mice were intranasally infected with a cowpox virus. The results showed that i.d. injection provided the development of protective immunity in mice to a much greater extent compared to s.s. inoculation with VACV strains.

Among the nonvirion proteins of the vaccinia virus (VACV), a 94-kDa long protein is most abundantly present; the protein is a truncated form of the 150-kDa A-type inclusion (ATI) protein of the cowpox virus encoded by the ati gene. This VACV protein does not form intracellular ATIs, being as it is a major immunogen upon infection/immunization of humans or animals with the VACV. Antibodies specific to this protein are not virus-neutralizing. The present study focused on the effect of the production of this nonstructural major immunogenic VACV protein on the manifestation of pathogenicity and immunogenicity of the virus in the BALB/c mouse model of infection. In order to introduce a targeted deletion into the VACV LIVP genome, the recombinant integration/deletion plasmid p∆ati was constructed and further used to generate the recombinant virus LIVP∆ati. The pathogenicity of the VACV LIVP and LIVP∆ati strains was studied in 3-week-old mice. The mice were intranasally infected with the viruses at a dose of 107 pfu; 50% of the animals infected with the parent LIVP strain died, while infection with the LIVP∆ati strain led to the death of only 20% of the mice. Intradermal vaccination of mice aged 67 weeks with the LIVP∆ati virus statistically significantly increased the production of VACV-specific IgG, compared to that after intradermal vaccination with VACV LIVP. Meanwhile, no differences were noted in the cell-mediated immune response to the vaccination of mice with VACV LIVP or LIVP∆ati, which was assessed by ELISpot according to the number of splenocytes producing IFN- in response to stimulation with virus-specific peptides. Intranasal infection of mice with lethal doses of the cowpox virus or the ectromelia virus on day 60 post-immunization with the studied VACV variants demonstrated that the mutant LIVP∆ati elicits a stronger protective response compared to the parent LIVP.

The protein encoded by the vaccinia virus D4R gene has base excision repair uracil–DNA N-glycosylase (vvUNG) activity and also acts as a processivity factor in the viral replication complex. The use of a protein unlike PolN/PCNA sliding clamps is a unique feature of orthopoxviral replication, providing an attractive target for drug design. However, the intrinsic processivity of vvUNG has never been estimated, leaving open the question whether it is sufficient to impart processivity to the viral polymerase. Here, we use the correlated cleavage assay to characterize the translocation of vvUNG along DNA between two uracil residues. The salt dependence of the correlated cleavage, together with the similar affinity of vvUNG for damaged and undamaged DNA, support the one-dimensional diffusion mechanism of lesion search. Unlike short gaps, covalent adducts partly block vvUNG translocation. Kinetic experiments show that once a lesion is found it is excised with a probability ~0.76. Varying the distance between two uracils, we use a random walk model to estimate the mean number of steps per association with DNA at ~4200, which is consistent with vvUNG playing a role as a processivity factor. Finally, we show that inhibitors carrying a tetrahydro-2,4,6-trioxopyrimidinylidene moiety can suppress the processivity of vvUNG.

The monkeypox epidemic, which became unusually widespread among humans in 2022, has brought awareness about the necessity of smallpox vaccination of patients in the risk groups. The modern smallpox vaccine variants are introduced either intramuscularly or by skin scarification. Intramuscular vaccination cannot elicit an active immune response, since tissues at the vaccination site are immunologically poor. Skin has evolved into an immunologically important organ in mammals; therefore, intradermal delivery of a vaccine can ensure reliable protective immunity. Historically, vaccine inoculation into scarified skin (the s.s. route) was the first immunization method. However, it does not allow accurate vaccine dosing, and high-dose vaccines need to be used to successfully complete this procedure. Intradermal (i.d.) vaccine injection, especially low-dose one, can be an alternative to the s.s. route. This study aimed to compare the s.s. and i.d. smallpox immunization routes in a mouse model when using prototypic second- and fourth-generation low-dose vaccines (104 pfu). Experiments were conducted using BALB/c mice; the LIVP or LIVP-GFP strains of the vaccinia virus (VACV) were administered into the tail skin via the s.s. or i.d. routes. After vaccination (7, 14, 21, 28, 42, and 56 days post inoculation (dpi)), blood samples were collected from the retro-orbital venous sinus; titers of VACV-specific IgM and IgG in the resulting sera were determined by ELISA. Both VACV strains caused more profound antibody production when injected via the i.d. route compared to s.s. inoculation. In order to assess the level of the elicited protective immunity, mice were intranasally infected with a highly lethal dose of the cowpox virus on 62 dpi. The results demonstrated that i.d. injection ensures a stronger protective immunity in mice compared to s.s. inoculation for both VACV variants.

Relevance. The epidemiological situation in the world is characterized by an increase in the incidence of orthopoxvirus infections in humans and animals. In this regard, it is necessary to develop new safe vaccines against these infections.Aim. Conducting preclinical studies on the specific activity of the live vaccine against smallpox and other orthopoxvirus infections VACΔ6 based on the vaccinia virus (VACV) with six deleted virulence genes are presented.Matherials and methods. The studies were performed in accordance with the requirements of the Guidelines for conducting preclinical studies of drugs (immunobiological preparations), the State Pharmacopoeia XIII and the European Pharmacopoeia 7.0.Results and discussion. The vaccine strain VACΔ6 VACV showed significantly reduced neurovirulence in the model of intracerebral administration to suckling mice, and reduced inflammatorynecrotic activity in the model of intradermal administration to rabbits compared to the classical first­generation smallpox live vaccine approved for use in Russia. Preclinical studies of three series of the finished dosage form of the VAC∆6 vaccine showed its authenticity, thermal stability, non­pyrogenicity and safety. Double intradermal vaccination of rabbits at a dose of 106 PFU/animal, a 100% protective effect was provided against the intranasal infection of rabbits with VACV strain HB­-92 at a dose of 1995 LD50 and a double intradermal vaccination of mice at a dose of 106 PFU/animal, full protection was provided against the intranasal infection of mice with ectromelia virus strain K1 at a dose of 56 LD50.Conclusion. Based on the conducted complex of studies, it can be concluded that the created vaccine of the fourth generation VACΔ6 is safer compared to the live smallpox vaccine of the first generation and is not inferior to it in immunogenic and protective properties.

Considering that vaccination against smallpox with live vaccinia virus led to serious adverse effects in some cases, the WHO, after declaration of the global eradication of smallpox in 1980, strongly recommended to discontinue the vaccination in all countries. This led to the loss of immunity against not only smallpox but also other zoonotic orthopoxvirus infections in humans over the past years. An increasing number of human infections with zoonotic orthopoxviruses and, first of all, monkeypox, force us to reconsider a possible re-emergence of smallpox or a similar disease as a result of natural evolution of these viruses. The review contains a brief analysis of the results of studies on genomic organization and evolution of human pathogenic orthopoxviruses, development of modern methods for diagnosis, vaccination, and chemotherapy of smallpox, monkeypox, and other zoonotic human orthopoxvirus infections.

Among nonvirion proteins of the vaccinia virus (VACV), a 94-kDa protein (a truncated form of ATI protein) is synthesized in the largest quantity. This protein is a major immunogen upon infection of humans and animals with VACV. Meanwhile, antibodies specific to this protein are not virus-neutralizing. The present study aimed to investigate the effect of production of this protein on manifestation of pathogenicity and immunogenicity of VACV in a mouse model.

Due to cessation of mass smallpox vaccination in 1980, the collective immunity of humans against orthopoxvirus infections has virtually been lost. Therefore, the risk of spreading zoonotic human orthopoxvirus infections caused by monkeypox and cowpox viruses has increased in the world. First-generation smallpox vaccines based on Vaccinia virus (VAC) are reactogenic and therefore not suitable for mass vaccination under current conditions. This necessitates the development of modern safe live vaccines based on VAC using genetic engineering. We created the VACΔ6 strain by transient dominant selection. In the VACΔ6 genome, five virulence genes were intentionally deleted, and one gene was inactivated by inserting a synthetic DNA fragment. The virus was passaged 71 times in CV-1 cells to obtain the VACΔ6 strain from the VAC LIVP clonal variant. Such a long passage history might have led to additional off-target mutations in VACΔ6 compared to the original LIVP variant. To prevent this, we performed a genome-wide sequencing of VAC LIVP, VACΔ6, and five intermediate viral strains to assess possible off-target mutations. A comparative analysis of complete viral genomes showed that, in addition to target mutations, only two nucleotide substitutions occurred spontaneously when obtaining VACΔ4 from the VACΔ3 strain; the mutations persisting in the VACΔ5 and VACΔ6 genomes. Both nucleotide substitutions are located in intergenic regions (positions 1431 and 189738 relative to LIVP), which indicates an extremely rare occurrence of off-target mutations when using transient dominant selection to obtain recombinant VAC variants with multiple insertions/deletions. To assess the genome stability of the resulting attenuated vaccine strain, 15 consecutive cycles of cultivation of the industrial VACΔ6 strain were performed in 4647 cells certified for vaccine production in accordance with the “Guidelines for Clinical Trials of Medicinal Products”. PCR and sequencing analysis of six DNA fragments corresponding to the regions of disrupted genes in VACΔ6 showed that all viral DNA sequences remained unchanged after 15 passages in 4647 cells.

The conventional live smallpox vaccine based on the vaccinia virus (VACV) cannot be widely used today because it is highly reactogenic. Therefore, there is a demand for designing VACV variants possessing enhanced immunogenicity, making it possible to reduce the vaccine dose and, therefore, significantly eliminate the pathogenic effect of the VACV on the body. In this study, we analyzed the development of the humoral and T cell-mediated immune responses elicited by immunizing mice with low-dose VACV variants carrying the mutant A34R gene (which increases production of extracellular virions) or the deleted A35R gene (whose protein product inhibits antigen presentation by the major histocompatibility complex class II). The VACV LIVP strain, which is used as a smallpox vaccine in Russia, and its recombinant variants LIVP-A34R*, LIVP-dA35R, and LIVP-A34R*-dA35R, were compared upon intradermal immunization of BALB/c mice at a dose of 104 pfu/animal. The strongest T cell-mediated immunity was detected in mice infected with the LIVP-A34R*-dA35R virus. The parental LIVP strain induced a significantly lower antibody level compared to the strains carrying the modified A34R and A35R genes. Simultaneous modification of the A34R gene and deletion of the A35R gene in VACV LIVP synergistically enhanced the immunogenic properties of the LIVP-A34R*-dA35R virus.

The spread of the monkeypox virus infection among humans in many countries outside of Africa, which started in 2022, is now drawing the attention of the medical and scientific communities to the fact that immunization against this infection is sorely needed. According to current guidelines, immunization of people with the first-generation smallpox vaccine based on the vaccinia virus (VACV) LIVP strain, which is licensed in Russia, should be performed via transepidermal inoculation (skin scarification, s.s.). However, the long past experience of using this vaccination technique suggests that it does not ensure virus inoculation into patients' skin with enough reliability. The procedure of intradermal (i.d.) injection of a vaccine can be an alternative to s.s. inoculation. The effectiveness of i.d. vaccination can depend on the virus injection site on the body. Therefore, the aim of this study was to compare the development of the humoral and cellular immune responses in BALB/c mice immunized with the LIVP VACV strain, which was administered either by s.s. inoculation or i.d. injection into the same tail region of the animal. A virus dose of 105 pfu was used in both cases. ELISA of serum samples revealed no significant difference in the dynamics and level of production of VACV-specific IgM and IgG after i.d. or s.s. vaccination. A ELISpot analysis of splenocytes from the vaccinated mice showed that i.d. administration of VACV LIVP to mice induces a significantly greater T-cell immune response compared to s.s. inoculation. In order to assess the protective potency, on day 45 post immunization, mice were intranasally infected with lethal doses of either the cowpox virus (CPXV) or the ectromelia virus (ECTV), which is evolutionarily distant from the VACV and CPXV. Both vaccination techniques ensured complete protection of mice against infection with the CPXV. However, when mice were infected with a highly virulent strain of ECTV, 50% survived in the i.d. immunized group, whereas only 17% survived in the s.s. immunized group. It appears, therefore, that i.d. injection of the VACV can elicit a more potent protective immunity against orthopoxviruses compared to the conventional s.s. technique.

At the present time, vast majority of human population lacks immunity against orthopoxvirus infections caused by variola (smallpox), monkeypox, cowpox, or buffalopox viruses. More and more mass outbreaks of orthopoxvirus infections are yearly registered among humans on different continents. To prevent transition of these outbreaks to widespread epidemics, we should develop appropriate immunoprophylaxis strategies. Currently, massive usage of the classic live vaccine based on vaccinia virus is not acceptable, due to its high reactogenicity. Therefore, it is necessary to develop the variants of vaccinia virus with reduced virulence and increased immunogenicity/protectivity. The aim of this work was to study protective effects against a lethal orthopoxvirus infection occuring after low-dose immunization of mice with vaccinia virus variants, i.e., carrying mutant A34R gene causing increased production of extracellular virions, or a A35R gene deletion encoding protein product inhibiting antigen presentation by the major histocompatibility complex class II. The LIVP viral strain used in Russia as a smallpox vaccine, and its recombinant variants (LIVP-A34R*, LIVP-dA35R and LIVP-A34R*-dA35R) were compared with intranasal or intradermal immunization of BALB/c mice at the doses of 105 or 103 PFU. 28 days following administration of viral preparations (experimental groups) or saline (control groups), the mice underwent intravital blood sampling from retroorbital venous sinus. The levels of virion-specific antibodies were determined in individual serum samples by enzyme immunoassay. On the day 30 of experiment, the mice were infected with cowpox virus at a dose of 32 LD50, which caused total death of control mice on days 6-10. In the groups immunized with the studied viruses at a dose of 105 PFU, all the animals survived, regardless of strain, or immunization method. Upon intradermal immunization (103 PFU) of mice immunized with the original LIVP virus, 83% of the animals survived, whereas all mutant strains of the vaccinia virus provided 100% protection of the mice from subsequent cowpox virus infection. Intranasal immunization of mice at a dose of 103 PFU with LIVP strain protected only 33% of animals from lethal infection with cowpox virus, while the mutant strains LIVP-A34R* and LIVP-A34R*-dA35R provided 67% protection, and the LIVP-dA35R strain has resqued 75% of the mice. The studied mutant vaccinia viruses can be considered not only new candidate vaccines against smallpox and other human orthopoxvirus infections, but also as vector platforms for creating live multivalent vaccines against other infectious diseases.

Uracil–DNA glycosylases are enzymes that excise uracil bases appearing in DNA as a result of cytosine deamination or accidental dUMP incorporation from the dUTP pool. The activity of Family 1 uracil–DNA glycosylase (UNG) activity limits the efficiency of antimetabolite drugs and is essential for virulence in some bacterial and viral infections. Thus, UNG is regarded as a promising target for antitumor, antiviral, antibacterial, and antiprotozoal drugs. Most UNG inhibitors presently developed are based on the uracil base linked to various substituents, yet new pharmacophores are wanted to target a wide range of UNGs. We have conducted virtual screening of a 1,027,767-ligand library and biochemically screened the best hits for the inhibitory activity against human and vaccinia virus UNG enzymes. Although even the best inhibitors had IC50 ≥ 100 μM, they were highly enriched in a common fragment, tetrahydro-2,4,6-trioxopyrimidinylidene (PyO3). In silico, PyO3 preferably docked into the enzyme’s active site, and in kinetic experiments, the inhibition was better consistent with the competitive mechanism. The toxicity of two best inhibitors for human cells was independent of the presence of methotrexate, which is consistent with the hypothesis that dUMP in genomic DNA is less toxic for the cell than strand breaks arising from the massive removal of uracil. We conclude that PyO3 may be a novel pharmacophore with the potential for development into UNG-targeting agents.

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, in 1980 the World Health Organization recommended to cancel smallpox vaccination after declaring about smallpox eradication. Over the 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 camelpox. All of them pose a 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 the 28th 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.

Самым простым и надежным способом защиты от вирусных инфекций является вакцинопрофилактика. При этом наибольшей протективной эффективностью обладают живые вакцины, в основе которых используют слабовирулентные для человека вирусы, близкородственные патогенным, или аттенуированные (ослабленные за счет мутаций/делеций в вирусном геноме) варианты патогенного для человека вируса. Вакцинация против оспы с использованием живого вируса осповакцины (vaccinia virus, VACV), близкородственного вирусу натуральной оспы, сыграла важнейшую роль в успехе программы глобальной ликвидации оспы, которая осуществлялась под эгидой Всемирной организации здравоохранения. Прекращение после 1980 г. противооспенной вакцинации привело к тому, что огромная часть населения Земли в настоящее время не имеет иммунитета не только к оспе, но и любым другим зоонозным ортопоксвирусным инфекциям. Это создает возможность циркуляции зоонозных ортопоксвирусов в человеческой популяции и, как следствие, приводит к изменению экологии и круга чувствительных хозяев для разных видов ортопоксвирусов. При этом использование классической живой вакцины на основе VACV для защиты от этих инфекций в настоящее время не приемлемо, так как она может обусловливать тяжелые побочные реакции. В связи с этим все более актуальной становится разработка новых безопасных вакцин против ортопоксвирусных инфекций человека и животных. Аттенуация (ослабление вирулентности) VACV достигается в результате направленной инактивации определенных генов вируса и обычно приводит к уменьшению эффективности размножения VACV in vivo. Следствием этого может быть снижение иммунного ответа при введении аттенуированного вируса пациентам в стандартных дозах. Часто используемым для встройки/инактивации в геноме VACV является ген тимидинкиназы, нарушение которого приводит к аттенуации вируса. В данной работе изучено, как введение двух точечных мутаций в ген A34R аттенуированного штамма LIVP-GFP (ТК-), увеличивающих выход внеклеточных оболочечных вирионов (EEV), влияет на свойства пато- и иммуногенности варианта VACV LIVP-GFP-A34R при интраназальном заражении лабораторных мышей. Показано, что увеличение продукции EEV рекомбинантным штаммом VACV LIVP-GFP-A34R не меняет аттенуированный фенотип, характерный для родительского штамма LIVP-GFP, но приводит к существенно большей продукции VACV-специфичных антител. Ключевые слова: вирус осповакцины; направленные мутации; аттенуация; иммуногенность.

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.

Smallpox eradication and lack of adequate animal model for smallpox infection underlies a necessity to assess immunogenic and protective properties of genetic engineering-created live attenuated smallpox vaccines in several animal models of orthopoxviral infections. Here we compared immunogenic and protective properties of the recombinant vaccinia virus (VACV) LIVP-GFP intradermally (i.d.) inoculated to mice, guinea pigs and rabbits. LIVP-GFP immunization in all animal species was applied at dose of 2 × 104 or 2 × 106 PFU. Control animals were injected with saline. Blood sampling was performed on day 28 after virus LIVP-GFP or saline inoculation. Blood samples were taken intravitally from the retro-orbital venous sinus in mice, heart in guinea pigs or marginal ear vein in rabbits. Serum samples were isolated by precipitating blood cells via centrifugation. The serum anti-VACV IgG titers were determined by ELISA. On day 30 post-immunization animals were intranasally challenged with lethal dose of host specific orthopoxvirus species. Mice were infected by cowpox virus (CPXV) strain GRI-90 at dose 68 LD50, guinea pigs – by VACV GPA at dose 56 LD50, rabbits — by VACV HB-92 at dose 100 LD50. All animals in control group died afterwards, whereas all animals immunized by attenuated recombinant virus LIVP-GFP at dose 2 × 106 PFU survived. In case of the LIVP-GFP immunization at dose 2 × 104 PFU, 88% of mice, 67% of rabbits and 50% of guinea pigs survived after being challenged with species-specific CPXV, VACV HB-92, and VACV GPA. ELISA data for the blood serum samples revealed a correlation between level of VACV-specific antibodies and level of protection in animal species. Based on the data obtained, it could be concluded that all three “animal–orthopoxvirus” models allow to provide with a proper evaluation of immunogenicity and protectiveness for generated modern attenuated vaccines against smallpox and other orthopoxviral human infections. Upon that, it was shown that BALB/c mouse strain was the most convenient investigational host species.

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.

Mass vaccination has played a critical role in the global eradication of smallpox. Various vaccinia virus (VACV) strains, whose origin has not been clearly documented in most cases, have been used as live vaccines in different countries. These VACV strains differed in pathogenicity towards various laboratory animals and in reactogenicity exhibited upon vaccination of humans. In this work, we studied the development of humoral and cellular immune responses in BALB/c mice inoculated intranasally (i.n.) or intradermally (i.d.) with the VACV LIVP strain at a dose of 10(5) PFU/mouse, which was used in Russia as the first generation smallpox vaccine. Active synthesis of VACV-specific IgM in the mice occurred on day 7 after inoculation, reached a maximum on day 14, and decreased by day 29. Synthesis of virus-specific IgG was detected only from day 14, and the level increased significantly by day 29 after infection of the mice. Immunization (i.n.) resulted in significantly higher production of VACV-specific antibodies compared to that upon i.d. inoculation of LIVP. There were no significant differences in the levels of the T cell response in mice after i.n. or i.d. VACV administration at any time point. The maximum level of VACV-specific T-cells was detected on day 14. By day 29 of the experiment, the level of VACV-specific T-lymphocytes in the spleen of mice significantly decreased for both immunization procedures. On day 30 after immunization with LIVP, mice were infected with the cowpox virus at a dose of 46 LD50. The i.n. immunized mice were resistant to this infection, while 33% of i.d. immunized mice died. Our findings indicate that the level of the humoral immune response to vaccination may play a decisive role in protection of animals from orthopoxvirus reinfection.