Leucoma salicis nucleopolyhedrovirus (LesaNPV) genome sequence shed new light on the origin of the Alphabaculovirus orpseudotsugatae species.

LesaNPV (Leucoma salicis nucleopolyhedrovirus) is an alphabaculovirus group Ib. Potentially, it can be an eco-friendly agent to control the white satin moth Leucoma salicis population. In this study, we have established the relationship between LesaNPV and other closely related alphabaculoviruses. Environmental samples of late instar of white satin moth collected in Poland infected with baculovirus have been homogenized, polyhedra were purified and subjected to scanning and transmission electron microscopy. Viral DNA was sequenced using the Illumina platform and the whole-genome sequence was established by de novo assembly of paired reads. Genome annotation and phylogenetic analyses were performed with the use of bioinformatics tools. The genome of LesaNPV is 132 549 bp long with 154 ORFs and 54.9% GC content. Whole-genome sequencing revealed deletion of dUTPase as well as ribonucleoside reductases small and large subunits region in LesaNPV genome compared to Dasychira pudibunda nucleopolyhedrovirus (DapuNPV) and Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV) where this region is complete. Phylogenetic analysis of Baculoviridae family members showed that LesaNPV is less divergent from a common ancestor than closely related species DapuNPV and OpMNPV. This is interesting because their hosts do not occur in the same area. The baculoviruses described in this manuscript are probably isolates of one species and could be assigned to recently denominated species Alphabaculovirus orpseudotsugatae, historically originating from OpMNPV. This finding could have significant implications for the classification and understanding of the phylogeographical spread of baculoviruses.

Severe Acute Respiratory Syndrome Coronavirus 2 in Farmed Mink (Neovison vison), Poland.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent of coronavirus disease and has been spreading worldwide since December 2019. The virus can infect different animal species under experimental conditions, and mink on fur farms in Europe and other areas are susceptible to SARS-CoV-2 infection. We investigated SARS-CoV-2 infection in 91 mink from a farm in northern Poland. Using reverse transcription PCR, antigen detection, and next-generation sequencing, we confirmed that 15 animals were positive for SARS-CoV-2. We verified this finding by sequencing full viral genomes and confirmed a virus variant that has sporadic mutations through the full genome sequence in the spike protein (G75V and C1247F). We were unable to find other SARS-CoV-2 sequences simultaneously containing these 2 mutations. Country-scale monitoring by veterinary inspection should be implemented to detect SARS-CoV-2 in other mink farms.

Expansion of a SARS-CoV-2 Delta variant with an 872 nt deletion encompassing ORF7a, ORF7b and ORF8, Poland, July to August 2021.

Routine genomic surveillance on samples from COVID-19 patients collected in Poland during summer 2021 revealed the emergence of a SARS-CoV-2 Delta variant with a large 872 nt deletion. This change, confirmed by Sanger and deep sequencing, causes complete loss of ORF7a, ORF7b, and ORF8 genes. The index case carrying the deletion is unknown. The standard pipeline for sequencing may mask this deletion with a long stretch of N's. Effects of this deletion on phenotype or immune evasion needs further study.

Antiviral Activity of Uridine Derivatives of 2-Deoxy Sugars against Tick-Borne Encephalitis Virus.

Tick-borne encephalitis virus (TBEV) is a causative agent of tick-borne encephalitis (TBE), one of the most important human infections involving the central nervous system. Although effective vaccines are available on the market, they are recommended only in endemic areas. Despite many attempts, there are still no specific antiviral therapies for TBEV treatment. Previously, we synthesized a series of uridine derivatives of 2-deoxy sugars and proved that some compounds show antiviral activity against viruses from the Flaviviridae and Orthomyxoviridae families targeting the late steps of the N-glycosylation process, affecting the maturation of viral proteins. In this study, we evaluated a series of uridine derivatives of 2-deoxy sugars for their antiviral properties against two strains of the tick-borne encephalitis virus; the highly virulent TBEV strain Hypr and the less virulent strain Neudoerfl. Four compounds (2, 4, 10, and 11) showed significant anti-TBEV activity with IC50 values ranging from 1.4 to 10.2 µM and low cytotoxicity. The obtained results indicate that glycosylation inhibitors, which may interact with glycosylated membrane TBEV E and prM proteins, might be promising candidates for future antiviral therapies against TBEV.

Morphological, genetic and biological characterisation of a novel alphabaculovirus isolated from Cryptophlebia peltastica (Lepidoptera: Tortricidae).

Cryptophlebia peltastica is an agricultural pest of litchis and macadamias in South Africa with phytosanitary status for certain markets. Current control methods rely on chemical, cultural and classical biological control. However, a microbial control option has not been developed. An Alphabaculovirus from C. peltastica was recovered from a laboratory reared colony and morphologically characterised by transmission electron microscopy (TEM). Analysis of occlusion bodies indicated a single NPV (SNPV) varying in size from 421 to 1263 nm. PCR amplification and sequencing of the polh gene region using universal primers followed by BLAST analysis revealed a 93% similarity to a partial polh gene sequence from Epinotia granitalis NPV. Further genetic characterisation involving single restriction endonuclease (REN) digestion of genomic DNA was carried out to generate profiles for comparison against other baculovirus species and potential new isolates of the same virus. The complete genome of the virus was sequenced, assembled and analysed for a more comprehensive genetic analysis. The genome was 115728 base pairs (bp) in length with a GC content of 37.2%. A total of 126 open reading frames (ORFs) were identified with minimal overlap and no preference in orientation. Bioassays were used to determine the virulence of the NPV against C. peltastica. The NPV was virulent against C. peltastica with an LC50 value of 6.46 × 103 OBs/ml and an LC90 value of 2.46 × 105 OBs/ml, and time mortality ranging between 76.32 h and 93.49 h. This is the first study to describe the isolation and genetic characterisation of a novel SNPV from C. peltastica, which has potential for development into a biopesticide for the control of this pest in South Africa.

Anti-Hepatitis C Virus Activity of Uridine Derivatives of 2-Deoxy Sugars.

Hepatitis C virus (HCV), the etiological agent of the most common and dangerous diseases of the liver, is a major health problem worldwide. Despite many attempts, there is still no vaccine available. Although many drugs have been approved for use mostly in combination regimen, their high costs make them out of reach in less developed regions. Previously, we have synthesized a series of compounds belonging to uridine derivatives of 2-deoxy sugars and have proved that some of them possess antiviral activity against influenza A virus associated with N-glycosylation inhibition. Here, we analyze the antiviral properties of these compounds against HCV. Using cell culture-derived HCV (HCVcc), HCV pseudoparticles (HCVpp), and replicon cell lines, we have shown high anti-HCV activity of two compounds. Our results indicated that compounds 2 and 4 significantly reduced HCVcc propagation with IC50 values in low µM range. Further experiments using the HCVpp system confirmed that both compounds significantly impaired the infectivity of produced HCVpp due to the inhibition of the correct maturation of viral glycoproteins. Overall, our results suggest that inhibiting the glycosylation process might be a good target for new therapeutics not only against HCV, but other important viral pathogens which contain envelopes with highly glycosylated proteins.

Novel Uridine Glycoconjugates, Derivatives of 4-Aminophenyl 1-Thioglycosides, as Potential Antiviral Compounds.

A novel series of uridine glycoconjugates, derivatives of 4-aminophenyl 1-thioglycosides, was designed and synthesized. All compounds were evaluated in vitro for their antiviral activity against hepatitis C virus (HCV) and classical swine fever virus (CSFV), two important human and animal viral pathogens for which new or improved therapeutic options are needed. The antiviral activity of all synthesized compounds was confirmed using pseudo-plaque reduction assays in which a significant arrest of CSFV and HCV growth was observed in the presence of these compounds. Two of the synthesized compounds, 9 and 12, displayed a significant inhibitory effect on HCV and CSFV propagation with IC50 values of 4.9 and 13.5 µM for HCV and 4.2 and 4 µM for CSFV, respectively, with low cytotoxicity. Using various infection and replication models, we have shown that both compounds were able to significantly reduce viral genome replication by up to 90% with IC50 values in the low micromolar range. A structure activity analysis of the synthesized compounds showed that the high antiviral activity was attributed to the hydrophobicity of glycoconjugates and the introduction of elements capable to coordinate metal ions into the spacer connecting the sugar and uridine moiety, which can be useful in the development of new antiviral compounds in the future.

Biological Evaluation of Uridine Derivatives of 2-Deoxy Sugars as Potential Antiviral Compounds against Influenza A Virus.

Influenza virus infection is a major cause of morbidity and mortality worldwide. Due to the limited ability of currently available treatments, there is an urgent need for new anti-influenza drugs with broad spectrum protection. We have previously shown that two 2-deoxy sugar derivatives of uridine (designated IW3 and IW7) targeting the glycan processing steps during maturation of viral glycoproteins show good anti-influenza virus activity and may be a promising alternative approach for the development of new anti-influenza therapy. In this study, a number of IW3 and IW7 analogues with different structural modifications in 2-deoxy sugar or uridine parts were synthesized and evaluated for their ability to inhibit influenza A virus infection in vitro. Using the cytopathic effect (CPE) inhibition assay and viral plaque reduction assay in vitro, we showed that compounds 2, 3, and 4 exerted the most inhibitory effect on influenza virus A/ostrich/Denmark/725/96 (H5N2) infection in Madin-Darby canine kidney (MDCK) cells, with 50% inhibitory concentrations (IC50) for virus growth ranging from 82 to 100 (µM) without significant toxicity for the cells. The most active compound (2) showed activity of 82 µM with a selectivity index value of 5.27 against type A (H5N2) virus. Additionally, compound 2 reduced the formation of HA glycoprotein in a dose-dependent manner. Moreover, an analysis of physicochemical properties of studied compounds demonstrated a significant linear correlation between lipophilicity and antiviral activity. Therefore, inhibition of influenza A virus infection by conjugates of uridine and 2-deoxy sugars is a new promising approach for the development of new derivatives with anti-influenza activities.

New Method for Differentiation of Granuloviruses (Betabaculoviruses) Based on Multitemperature Single Stranded Conformational Polymorphism.

Baculoviruses have been used as biopesticides for decades. Recently, due to the excessive use of chemical pesticides there is a need for finding new agents that may be useful in biological protection. Sometimes few isolates or species are discovered in one host. In the past few years, many new baculovirus species have been isolated from environmental samples, thoroughly characterized and thanks to next generation sequencing methods their genomes are being deposited in the GenBank database. Next generation sequencing (NGS) methodology is the most certain way of detection, but it has many disadvantages. During our studies, we have developed a method based on Polymerase chain reaction (PCR) followed by Multitemperature Single Stranded Conformational Polymorphism (MSSCP) which allows for distinguishing new granulovirus isolates in only a few hours and at low-cost. On the basis of phylogenetic analysis of betabaculoviruses, representative species have been chosen. The alignment of highly conserved genes-granulin and late expression factor-9, was performed and the degenerate primers were designed to amplify the most variable, short DNA fragments flanked with the most conserved sequences. Afterwards, products of PCR reaction were analysed by MSSCP technique. In our opinion, the proposed method may be used for screening of new isolates derived from environmental samples.

Genome Analysis and Genetic Stability of the Cryptophlebia leucotreta Granulovirus (CrleGV-SA) after 15 Years of Commercial Use as a Biopesticide.

Thaumatotibia leucotreta Meyrick (Lepidoptera: Tortricidae) is an indigenous pest in southern Africa which attacks citrus fruits and other crops. To control T. leucotreta in South Africa, an integrated pest management (IPM) programme incorporating the baculovirus Cryptophlebialeucotreta granulovirus (CrleGV-SA) as a biopesticide has been implemented. This study investigated the genetic stability of a commercially produced CrleGV-SA product that has been applied in the field since 2000. Seven representative full-genome sequences of the CrleGV-SA isolate spanning a 15-year period were generated and compared with one another. Several open reading frames (ORFs) were identified to have acquired single nucleotide polymorphisms (SNPs) during the 15-year period, with three patterns observed and referred to as "stable", "reversion", and "unstable switching". Three insertion events were also identified, two of which occurred within ORFs. Pairwise multiple alignments of these sequences showed an identity ranging from 99.98% to 99.99%. Concentration-response bioassays comparing samples of CrleGV-SA from 2000 and 2015 showed an increase in virulence toward neonate T. leucotreta larvae. The CrleGV-SA genome sequence generated from the 2015 sample was compared to the Cape Verde reference genome, CrleGV-CV3. Several fusion events were identified between ORFs within these genomes. These sequences shared 96.7% pairwise identity, confirming that CrleGV-SA is a genetically distinct isolate. The results of this study indicate that the genome of CrleGV-SA has remained stable over many years, with implications for its continued use as a biopesticide in the field. Furthermore, the study describes the first complete baculovirus genome to be sequenced with the MinION (Oxford Nanopore, Oxford, UK) platform and the first complete genome sequence of the South African CrleGV isolate.

Human Gb3/CD77 synthase reveals specificity toward two or four different acceptors depending on amino acid at position 211, creating P(k), P1 and NOR blood group antigens.

Human Gb3/CD77 synthase (α1,4-galactosyltransferase, P(k) synthase), encoded by A4GALT gene, is known for synthesis of Gal(α1-4)Gal moiety in globotriaosylceramide (Gb3Cer, CD77, P(k) blood group antigen), a glycosphingolipid of the globo series. Recently, it was shown that c.631C > G mutation in A4GALT, which causes p.Q211E substitution in the open reading frame of the enzyme, broadens the enzyme specificity, making it able also to synthesize Gal(α1-4)GalNAc moiety, which constitutes the defining terminal disaccharide of the NOR antigen (carried by two glycosphingolipids: NOR1 and NOR2). Terminal Gal(α1-4)Gal disaccharide is also present in another glycosphingolipid blood group antigen, called P1, which together with P(k) and NOR comprises the P1PK blood group system. Despite several attempts, it was never clearly shown that P1 antigen is synthesized by Gb3/CD77 synthase, leaving open an alternative hypothesis that there are two homologous α1,4-galactosyltransferases in humans. In this study, using recombinant Gb3/CD77 synthase produced in insect cells, we show that the consensus enzyme synthesizes both the P(k) and P1 antigens, while its p.Q211E variant additionally synthesizes the NOR antigen. This is the first direct biochemical evidence that Gb3/CD77 synthase is able to synthesize two different glycosphingolipid antigens: P(k) and P1, and when p.Q211E substitution is present, the NOR antigen is also synthesized.

Expression of recombinant human bifunctional peptidylglycine α-amidating monooxygenase in CHO cells and its use for insulin analogue modification.

The availability of catalytically active peptidylglycine α-amidating monooxygenase (PAM) should provide the means to examine its potential use for the chemienzymatic synthesis of bioactive peptides for the purpose of pharmacological studies. Hypoglycemic activity is one of the most important features of insulin derivatives. Insulin glargine amide was found to show a time/effect profile which is distinctly more flat and thus more advantageous than insulin glargine itself. The aim of the study was to obtain recombinant PAM and use it for insulin analogue amidation. We stably expressed a recombinant PAM in CHO dhfr-cells in culture. Recombinant PAM was partially purified by fractional ammonium sulphate precipitation and ion-exchange chromatography. The enzyme was used to modify glycine-extended A22(G)-B31(K)-B32(R) human insulin analogue (GKR). Alpha-amidated insulin was analyzed by HPLC and mass spectrometry. Hypoglycemic activity of amidated and non-amidated insulin was compared. The pharmacodynamic effect was based on glucose concentration measurement in Wistar rats with hyperglycemia induced by streptozotocin. The overall glycemic profile up to 36 h was evaluated after subcutaneous single dosing at a range of 2.5-7.5 U/kg b.w. The experiment on rats confirmed with a statistical significance (P < 0.05) hypoglycemic activity of GKR-NH2 in comparison to a control group receiving 0.9% NaCl. Characteristics for GKR-NH2 profile was a rather fast beginning of action (0.5-2.0 h) and quite prolonged return to initial values. GKR-NH2 is a candidate for a hypoglycemic drug product in diabetes care. In addition, this work also provides a valuable alternative method for preparing any other recombinant bioactive peptides with C-terminal amidation.

An alphabaculovirus isolated from dead Lymantria dispar larvae shows high genetic similarity to baculovirus previously isolated from Lymantria monacha - An example of adaptation to a new host.

A new isolate of baculovirus, Lymantria dispar multiple nucleopolyhedrovirus-BNP (LdMNPV-BNP), was found in dead gypsy moth (L. dispar) caterpillars collected in the Biebrzanski National Park in Poland. Here, we examined its biological activity, structure, genetic content and phylogeny. Multiple nucleocapsids of LdMNPV-BNP are enveloped together in 2-26 virions embedded in occluded bodies (OBs) very similar to the OBs previously described in viruses infecting Lymantriinae. This isolate kills pest larvae in a relatively short time (LT50 of approximately 9days for a dose of 2×10(7)OBs/ml), highlighting the possibility for its use as a biopesticide. Next-generation sequencing of LdMNPV-BNP revealed gene content (e.g. DNA photolyase) that is not present in any LdMNPV isolate sequenced to date. The genome is 157,270 base pairs long and has a notably lower G+C content in comparison to other LdMNPVs (50.3% G+C content compared to an average of 57.4% among other LdMNPVs). According to our phylogenetic analysis based on 37 core genes, LdMNPV-BNP is a member of group II alphabaculoviruses, which are closely related to LdMNPV and LyxyMNPV (Lymantria xylina multiple nucleopolyhedrovirus). Molecular evolution inference based on the partial sequence of lef-8, lef-9 and polh genes shows that LdMNPV-BNP and isolates of Lymantria monacha nucleopolyhedrovirus (LymoNPV) may share a very recent common ancestor or be isolates of the same virus species. LdMNPV-BNP, like other baculoviruses, could be beneficial as an active component of biopesticides that can be used during forest integrated pest management.

The genome of Dasychira pudibunda nucleopolyhedrovirus (DapuNPV) reveals novel genetic connection between baculoviruses infecting moths of the Lymantriidae family.

BACKGROUND: DapuNPV (Dasychira pudibunda nucleopolyhedrovirus), presented in this report, belongs to Alphabaculovirus group Ib. Its full, newly sequenced genome shows close relationship to baculovirus OpMNPV isolated from douglas-fir tussock moth Orgyia pseudotsugata. Baculovirus DapuNPV is a natural limiter of pale tussock moth Dasychira pudibunda L. (syn. Calliteara pudibunda L.)(Lepidoptera, Lymantriidae), which can occur in a form of an outbreak on many species of deciduous trees and may cause significant economic losses in the forests. METHODS: Late instars dead larvae of pale tussock moth were mechanically homogenized and polyhedra were purified during series of ultracentrifugation. Viral DNA was extarcted and sequenced using Miseq Illumina platform. 294,902 paired reads were used for de novo assembling. Genome annotation, multiple allingment to others baculoviruses and phylogegentic analises were perform with the use of multiple bioinformatic tools like: Glimmer3, HMMER web server, Geneious 7 and MEGA6. RESULTS: The genome of DapuNPV is 136,761 bp long with AT pairs content 45.6 %. The predicted number of encoded putative open reading frames (ORFs) is 161 and six of them demonstrate low or no homology to ORFs previously found in baculoviruses. DapuNPV genome shows very high similarity to OpMNPV in a nucleotide sequence (91.1 % of identity) and gene content (150 homologous ORFs), though some major differences (e.g. lack of he65 in OpMNPV) have also been noted. CONCLUSIONS: Similarly to other members of the Baculoviridae family, DapuNPV baculovirus possesses highly conserved core genes. Among them, there is a second copy of occluded derived virus envelope 27 protein (odv-e27), which was previously found only in a member of Alphabaculovirus group II - LyxyMNPV (Lymantria xylina MNPV). Surprisingly enough, DapuNPV and LyxyMNPV genomes share also another feature. Phylogenetic analysis of chitin binding family protein (cbpl) indicates significant similarity of those two baculoviruses from distinct evolutionary groups which infect the same hosts from Lymantriidae. The ubiquitin like family gene (ubil), which has not been described until now, is another characteristic component of DapuNPV genome.

Synthesis and antiviral activity of a novel glycosyl sulfoxide against classical swine fever virus.

A novel compound-2″,3″,4″,6″-tetra-O-acetyl-ß-d-galactopyranosyl-(1→4)-2',3',6'-tri-O-acetyl-1-thio-ß-d-glucopyranosyl-(5-nitro-2-pyridyl) sulfoxide-designated GP6 was synthesized and assayed for cytotoxicity and in vitro antiviral properties against classical swine fever virus (CSFV) in this study. We showed that the examined compound effectively arrested CSFV growth in swine kidney cells (SK6) at a 50% inhibitory concentration (IC50) of 5 ± 0.12 µg/ml without significant toxicity for mammalian cells. Moreover, GP6 reduced the viral E2 and E(rns) glycoproteins expression in a dose-dependent manner. We have excluded the possibility that the inhibitor acts at the replication step of virus life cycle as assessed by monitoring of RNA level in cells and culture medium of SK6 cells after single round of infection as a function of GP6 treatment. Using recombinant E(rns) and E2 proteins of classical swine fever virus produced in baculovirus expression system we have demonstrated that GP6 did not influence glycoprotein production and maturation in insect cells. In contrast to mammalian glycosylation pathway, insect cells support only the ER-dependent early steps of this process. Therefore, we concluded that the late steps of glycosylation process are probably the main targets of GP6. Due to the observed antiviral effect accompanied by low cytotoxicity, this inhibitor represents potential candidate for the development of antiviral agents for anti-flavivirus therapy. Further experiments are needed for investigating whether this compound can be used as a safe antiviral agent against other viruses from unrelated groups.

Serological characterisation of Lagovirus virus-like particles originating from native and mutated VP60 of rabbit haemorrhagic disease virus 2 and European brown hare syndrome virus.

Introduction: Since lagoviruses cannot be cultivated in vitro, using expression systems is an alternative and promising way of producing diagnostic viral antigens. It opens up their use as active immunogens for vaccine production. Material and Methods: Virus-like particles (VLPs) were produced in a baculovirus expression system in Spodoptera frugiperda 9 (Sf9) insect cells based on wild-type and mutated variants of the virus capsid VP60 protein from a Polish strain of European brown hare syndrome virus (EBHSV) and wild-type and mutated versions of this protein from a Polish strain of rabbit haemorrhagic disease virus 2 (RHDV2). The mutations were the substitution of an arginylglycylaspartic acid (Arg-Gly-Asp/RGD) motif in the P2 subdomain and, in the S or P2 domain, the substitution of three lysines. The VLPs were purified with sucrose gradient ultracentrifugation. Results: Protein production was confirmed by Western blot analysis using rabbit or hare sera and ELISA tests with different types of monoclonal antibody. The haemagglutination properties of some VLPs were also evaluated. Electron microscopy of wild-type EBHSV, wild-type RHDV2 and the four VP60 variants produced in this experiment revealed the formation of characteristic VLP structures. Conclusion: For the first time, mutated VLPs of RHDV2 with an RGD motif in the VP60 sequence were obtained, which could potentially be used to deliver cargo to eukaryotic cells. Virus-like particles based on the VP60 proteins of EBHSV and RHDV with a three-lysine substitution in the S or P2 domains were also obtained. Potential exists for VLPs of EBHSV and RHDV2 as vaccine candidates.

Influence of Dosing Regimen and Adjuvant Type on the Immunogenicity of Novel Recombinant Zika Virus-Like Particles.

Zika virus (ZIKV) is a reemerging mosquito-borne flavivirus that causes febrile illness and is also linked to Guillain-Barré syndrome as well as to microcephaly in newborns. Due to the risk of fetuses developing microcephaly, ZIKV is a serious problem for pregnant women. Although different types of vaccine antigens have been investigated, there is still no approved vaccine that prevents ZIKV. The aim of this study was to produce a potential anti-Zika virus vaccine candidate based on virus-like particles (VLPs) in mammalian cells and to analyze the role of dosing regimen and adjuvant type on the immunogenicity of the obtained antigen. Novel recombinant VLPs (F2A) were designed by introducing the optimized signal sequence of prM protein and by adding a self-cleavage peptide 2A between proteins prM and E. These modifications improved the formation of the glycoprotein E dimer. It has been shown that the increasing dosing regimen generates a significantly higher titer of antibodies; however, the adjuvant type does not affect this process. Sera from mice immunized using an increasing dosing schedule also showed higher neutralization activity against both Zika strains (H/PAN/2016/BEI-259634, a pandemic strain belonging to Asian lineage, and MR766, a reference strain from African lineage). In summary, this is the first report showing the influence of vaccination schedules and adjuvants on the immunogenicity of ZIKV virus-like particles. IMPORTANCE Considering the transmission of ZIKV and the risk of another epidemic as well as the neurological complications that follow ZIKV infection, the virus remains a serious problem for the human population, especially pregnant women. Therefore, there is a great need to develop new effective vaccine candidates. Although different types of vaccine antigens have been used in preclinical studies worldwide, there is still no approved vaccine to prevent ZIKV. VLPs are among the most potent antigens, but to use VLPs, adjuvants must be added to the formulation and appropriate administration must be performed. In this study, we show for the first time the influence of vaccination schedules and adjuvants on the immunogenicity of recombinant ZIKV VLPs. The obtained results can be used in new vaccine designs not only against ZIKV but also against other important viral pathogens.

Functional characterization and immunogenicity of a novel vaccine candidate against tick-borne encephalitis virus based on Leishmania-derived virus-like particles.

Tick-borne encephalitis virus (TBEV) is a major cause of neurological infections in many regions of central, eastern and northern Europe and northern Asia. In approximately 15% of cases, TBEV infections lead to the development of severe encephalitis or meningitis. The main route of TBEV transmission is tick bites; however, ingestion of dairy products from infected animals (goats, cattle and sheep) is also a frequent cause of the disease. Therefore, vaccination of livestock in virus endemic regions could also contribute to the decrease in TBEV infection among humans. Although few vaccines against TBEV based on inactivated viruses are available for humans, due to high costs, vaccination is not mandatory in most of the affected countries. Moreover, there is still no vaccine for veterinary use. Here, we present a characterization and immunogenicity study of a new potential TBEV vaccine based on virus-like particles (VLPs) produced in Leishmania tarentolae cells. VLPs, which mimic native viral particles but do not contain genetic material, show good immunogenic potential. For the first time, we showed that the protozoan L. tarentolae expression system can be successfully used for the production of TBEV virus-like particles with highly efficient production. We confirmed that TBEV recombinant structural proteins (prM/M and E) from VLPs are highly recognized by neutralizing antibodies in in vitro analyses. Therefore, VLPs in combination with AddaVax adjuvant were used in immunization studies in a mouse model. VLPs proved to be highly immunogenic and induced the production of high levels of neutralizing antibodies. In a challenge experiment, immunization with VLPs provided full protection from lethal TBE in mice. Thus, we suggest that Leishmania-derived VLPs may be a good candidate for a safe alternative human vaccine with high efficiency of production. Moreover, this potential vaccine candidate may constitute a low-cost candidate for veterinary use.

The Influence of Adjuvant Type on the Immunogenicity of RBD/N Cocktail Antigens as a Vaccine Candidate against SARS-CoV-2 Virus.

The emerging virus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2 virus), agent of COVID-19, appeared in December 2019 in Wuhan, China, and became a serious threat to global health and public safety. Many COVID-19 vaccines have been approved and licensed around the world. Most of the developed vaccines include S protein and induce an antibody-based immune response. Additionally, T-cell response to the SARS-CoV-2 antigens could be beneficial for combating the infection. The type of immune response is greatly dependent not only on the antigen, but also on adjuvants used in vaccine formulation. Here, we compared the effect of four different adjuvants (AddaS03, Alhydrogel/MPLA, Alhydrogel/ODN2395, Quil A) on the immunogenicity of a mixture of recombinant RBD and N SARS-CoV-2 proteins. We have analyzed the antibody and T-cell response specific to RBD and N proteins and assessed the impact of adjuvants on virus neutralization. Our results clearly indicated that Alhydrogel/MPLA and Alhydrogel/ODN2395 adjuvants elicited the higher titers of specific and cross-reactive antibodies to S protein variants from various SARS-CoV-2 and SARS-CoV-1 strains. Moreover, Alhydrogel/ODN2395 stimulated high cellular response to both antigens, as assessed by IFN-γ production. Importantly, sera collected from mice immunized with RBD/N cocktail in combination with these adjuvants exhibited neutralizing activity against the authentic SARS-CoV-2 virus as well as particles pseudotyped with S protein from various virus variants. The results from our study demonstrate the immunogenic potential of RBD and N antigens and point out the importance of adjuvants selection in vaccine formulation in order to enhance the immunological response. IMPORTANCE Although several COVID-19 vaccines have been approved worldwide, continuous emergence of new SARS-CoV-2 variants calls for new efficient vaccines against them, providing long-lasting immunity. As the immune response after vaccination is dependent not only on antigen used, but also on other vaccine components, e.g., adjuvants, the purpose of this work was to study the effect of different adjuvants on the immunogenicity of RBD/N SARS-CoV-2 cocktail proteins. In this work, it has been shown that immunization with both antigens plus the different adjuvants studied elicited higher Th1 and Th2 responses against RBD and N, which contributed to higher neutralization of the virus. The obtained results can be used for design of new vaccines, not only against SARS-CoV-2, but also against other important viral pathogens.

Oseltamivir analog with boron cluster modulator.

Synthesis of novel neuraminidase inhibitor -- carborane ester of oseltamivir carboxylic acid is described, and its physicochemical and spectral characteristics is provided. Surprisingly, carborane analog of oseltamivir is of an order of magnitude less active than its precursor, the corresponding ethyl ester, which is the active principle of pharmaceutical preparations used in influenza prophylactics and therapy.