Molecular mimicry of the receptor-binding domain of the SARS-CoV-2 spike protein: from the interaction of spike-specific antibodies with transferrin and lactoferrin to the antiviral effects of human recombinant lactoferrin.

The pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection involves dysregulations of iron metabolism, and although the mechanism of this pathology is not yet fully understood, correction of iron metabolism pathways seems a promising pharmacological target. The previously observed effect of inhibiting SARS-CoV-2 infection by ferristatin II, an inducer of transferrin receptor 1 (TfR1) degradation, prompted the study of competition between Spike protein and TfR1 ligands, especially lactoferrin (Lf) and transferrin (Tf). We hypothesized molecular mimicry of Spike protein as cross-reactivity of Spike-specific antibodies with Tf and Lf. Thus, strong positive correlations (R2 > 0.95) were found between the level of Spike-specific IgG antibodies present in serum samples of COVID-19-recovered and Sputnik V-vaccinated individuals and their Tf-binding activity assayed with peroxidase-labeled anti-Tf. In addition, we observed cross-reactivity of Lf-specific murine monoclonal antibody (mAb) towards the SARS-CoV-2 Spike protein. On the other hand, the interaction of mAbs produced to the receptor-binding domain (RBD) of the Spike protein with recombinant RBD protein was disrupted by Tf, Lf, soluble TfR1, anti-TfR1 aptamer, as well as by peptides RGD and GHAIYPRH. Furthermore, direct interaction of RBD protein with Lf, but not Tf, was observed, with affinity of binding estimated by KD to be 23 nM and 16 nM for apo-Lf and holo-Lf, respectively. Treatment of Vero E6 cells with apo-Lf and holo-Lf (1-4 mg/mL) significantly inhibited SARS-CoV-2 replication of both Wuhan and Delta lineages. Protective effects of Lf on different arms of SARS-CoV-2-induced pathogenesis and possible consequences of cross-reactivity of Spike-specific antibodies are discussed.

Corrigendum to "Assessment of trivalent live influenza vaccines in MDCK cell line" [MethodsX 8 (2021) 101442].

[This corrects the article DOI: 10.1016/j.mex.2021.101442.].

Assessment of trivalent live influenza vaccines in MDCK cell line.

We applied a one-step reverse transcriptase real-time PCR (rRT-PCR) analysis using TaqMan technique to evaluate the infectious titers of vaccine strains containing in trivalent live influenza vaccines (LAIVs). The cold-adapted reassortant influenza viruses A/H1N1 pdm09, A/H3N2, B/Yamagata and B/Victoria, included in the composition of the LAIV in 2015-2016, 2017-2018 and 2018-2019 flu season were studied for reproductive activity in MDCK cells as part of a mono-vaccine and tri-vaccine. For this we have developed a set of specific primers and probes. Method validation was performed using ELISA-test after mouse monoclonal antibodies to hemagglutinin (HA) staining of MDCK monolayer. Influenza B viruses B/Yamagata and B/Victoria were studied in MDCK cells in mono-infection and coinfection with different multiplicity of infection (MOI) using quantitative rRT-PCR.•RT-PCR analysis was adjusted to assess the growth characteristics of cold-adapted reassortant influenza viruses in MDCK cell line. The greatest suppression in the composition of the tri-vaccine was exposed to the H1N1 pdm09 LAIV component.•Influenza B viruses are least suppressed in trivalent LAIV. Influenza viruses B/Yamagata and B/Victoria reproduced as part of a mixed preparation not lower, if not better than as a mono-preparation at an MOI of 0.1. At an MOI of 0.01, the reproduction of both B/Yamagata and B/Victoria in the mixture was reduced compared to mono-vaccine.•The interference of trivalent LAIV vaccine viruses in MDCK cells was minimal at low dilutions. This indicates that it is undesirable to reduce the titers of vaccine viruses, including at the stages of transportation and storage of LAIV.

Evaluation of influenza A and B cold-adapted reassortant virus reproduction in trivalent live influenza vaccines.

BACKGROUND: The objective of the present study was to compare reproduction of trivalent LAIV vaccine strains in MDCK cells and to perform quantitative RT-PCR analysis of trivalent LAIV replication after inoculation in mice. METHODS: We applied a reverse transcriptase real-time PCR (rRT-PCR) analysis using TaqMan technique to evaluate the infectious titers of vaccine strains containing in trivalent live influenza vaccines (LAIVs). We confirmed the PCR data in ELISA using staining of MDCK monolayer with mouse monoclonal antibodies to hemagglutinin. RESULTS: The viral load during the reproduction of mono-vaccines and trivalent LAIV in MDCK cells was similar at low dilutions. The content of vaccine viruses was evaluated using quantitative RT-PCR analysis in the nasal turbinate and lungs of CBA mice on day 3 after intranasal immunization. It was shown that despite the almost complete absence of reproduction of the A/H3N2 virus in mice, the immune response of A/H3N2-specific antibodies was formed at the same level as to other viruses. In MDCK cells, a decreased infectious titers of vaccine viruses in trivalent LAIV compared to mono-vaccines was demonstrated except for B/Yamagata virus. CONCLUSION: RT-PCR analysis is applicable to assess the growth characteristics of cold-adapted reassortant influenza viruses in vitro and in mice. The interference of trivalent LAIV vaccine viruses in MDCK cells was minimal at low dilutions. In mice, decrease in infectious titers did not lead to a decline of the immunogenicity.

Genetic Basis of Attenuation of Cold-Adapted Influenza Strain B/Leningrad/14/17/55 - Backup Master Donor Virus for Influenza Type B Live Attenuated Vaccines.

The reassortant vaccine strain of live attenuated influenza vaccine inherits temperature sensitivity and areactogenicity from cold-adapted attenuated master donor virus. In Russia, B/ USSR/60/69 master donor virus (B60) is currently in use for the preparation of live attenuated type B influenza vaccine candidates. Trivalent live attenuated influenza vaccine based on A/ Leningrad/134/17/57 and B60 are licensed for the use in Russia for single dose vaccination of adults and children over 3 years. B/Leningrad/14/17/55 (B14) cold-adapted virus is a backup master donor virus for live attenuated type B influenza vaccine. According to our preliminary estimates, it is more attenuated than B60, which can allow expanding applicability of this vaccine for children under 3 years of age. In this paper, the role of B14 genes in its attenuation was assessed. Representative collection of reassortants of B14 with epidemic influenza B viruses was obtained, a phenotypic analysis of reassortants was performed, and their pathogenicity for animals was assessed. The leading role of PB2 and PA genes in attenuation of B14 master donor virus was proven.

Combined immunization with attenuated live influenza vaccine and chimeric pneumococcal recombinant protein improves the outcome of virus-bacterial infection in mice.

Influenza and its bacterial complications are a leading cause of morbidity and mortality worldwide. The effect of combined immunization with live influenza vaccine and recombinant chimeric pneumococcal protein in dual infection caused by influenza H1N1 and S. pneumoniae (serotype 3) has been studied. The combined vaccine consisted of the strain A/California/2009/38 (H1N1) pdm and chimeric recombinant protein PSPF composed of immunodominant fragments of the surface virulence factors of S. pneumoniae-PsaA, PspA, and Shr1875-associated with modified salmonella flagellin. Vaccinated mice were infected with the influenza virus 24 hours before or 24 hours after the onset of pneumococcal infection. The protective effect of combined vaccination was shown on both models of viral-bacterial infection.

Experimental Study of Genetic Constellation of Cold-Adapted Master Donor Viruses for Live Attenuated Influenza Vaccine Type B.

We studied the constellation of genes encoding polymerase complex proteins of master donor viruses for Russian live attenuated influenza vaccine type B. Reassortants of the reserve attenuation donor B/Leningrad/14/17/55 with B/USSR/60/69 master donor virus currently used for manufacturing seasonal influenza vaccine were prepared and examined. Most reassortants obtained by the classical reassortment method inherited all genes from the B/Leningrad/14/17/55 virus except the gene encoding PB1 subunit of the polymerase complex. One reassortant was selected for further evaluation of the role of PB1 gene. Greater attenuation of the strain for experimental animals (mice) in comparison with the original strains was demonstrated. This indicates high degree of constellation of genes of cold-adapted master donor viruses and the important compensating role of amino acid substitutions in the PB1 protein of B/Leningrad/14/17/55 donor virus in preventing viral hyperattenuation.

Amino Acid Substitutions N123D and N149D in Hemagglutinin Molecule Enhance Immunigenicity of Live Attenuated Influenza H7N9 Vaccine Strain in Experiment.

We compared three cold-adapted live attenuated influenza vaccine strains prepared by reverse genetics methods on the basis of master donor virus A/Leningrad/134/17/57 and influenza H7N9 strains A/Anhui/1/2013 and A/Shanghai/1/2013. Two strains based on A/Anhui/1/2013 differed by amino acid positions 123 and 149 in HA1 (123N/149N; 123D/149D). All strains efficiently replicated in developing chicken embryos; A/Shanghai/1/2013-based strain and A/Anhui/1/2013-123N/149N variant were characterized by reduced replication in MDCK cells. Strains based on A/Anhui/1/2013 virus agglutinated erythrocytes with α2,3- and α2,6-linked sialic acid residues, whereas strain A/Shanghai/1/2013 only α2,3. In experiments with BALB/c mice, Anhui-123D/149D strain was most immunogenic and induced high crossreactive humoral immune response, therefore it can be recommended as the model virus for the construction of recombinant vector vaccines based on live attenuated influenza vaccine.

Development of Pyrosequencing Assay for Evaluation of Genetic Stability of Vaccine Strains of Live Attenuated Influenza Type B Vaccine.

We developed a pyrosequencing protocol for monitoring of stability of attenuating mutations in the genome of vaccine reassortants based on master donor virus of Russian live attenuated influenza vaccine B/USSR/60/69. The developed protocol allows rapid and accurate assessment of mutations and can be used for analysis of genetic stability of reassortants during vaccine strain development and manufacturing, as well as genetic stability of vaccine isolates of influenza B virus during pre-clinical and clinical trials.

Composition of the Stabilizer and Conditions of Lyophilization for Preserving Infectious Activity of Influenza Virus.

For stabilization of vaccine preparations, they are lyophilized. The composition of the protective medium is an important parameter affecting the quality of the vaccine after drying. In view of the risk of spreading prion diseases, the use of media containing animal proteins is not recommended. In this study, protective media containing no animal proteins and lyophilization regimen were determined. The optimum lyophilization regimen consisted of three stages: freezing at -70°C, main stage at -35°C, and drying at 24°C. Protective medium containing 4% trehalose or protective medium with 10% sucrose and 5% soy peptone ensured highest stability of the lyophilized vaccine preparation in temperature range of 4-24°C. This can help to overcome possible break in the cold chain, which is important during transporting or storage of vaccine preparations.

Modeling of 3D Structure of Chimeric Constructs Based on Hemagglutinin of Influenza Virus and Immunogenic Epitopes of Streptococcus Agalactiae.

A project of an experimental recombinant vector vaccine for prevention of diseases caused by pathogenic streptococci based on ScaAB lipoprotein of Streptococcus agalactiae and a coldadapted strain of live influenza vaccine as a vector was developed. The sequence of ScaAB lipoprotein was analyzed and fragments forming immunodominant epitopes were determined. Chimeric molecules of influenza virus hemagglutinin H7 carrying insertions of bacterial origin were constructed. Based on the results of simulation, the most promising variants were selected; they represented fragments of lipoprotein ScaAB lacking N-terminal domain bound to hemagglutinin via a flexible linker. These insertions should minimally modulate the properties of the influenza strain, while retaining potential immunogenicity to a wide group of pathogenic streptococci.

Live Attenuated Influenza Vaccines engineered to express the nucleoprotein of a recent isolate stimulate human influenza CD8+ T cells more relevant to current infections.

Live attenuated influenza vaccines (LAIV) induce CD8+ T lymphocyte responses that play an important role in killing virus-infected cells. Despite the relative conservation of internal influenza A proteins, the epitopes recognized by T cells can undergo drift under immune pressure. The internal proteins of Russian LAIVs are derived from the master donor virus A/Leningrad/134/17/57 (Len/17) isolated 60 years ago and as such, some CD8+ T cell epitopes may vary between the vaccine and circulating wild-type strains. To partially overcome this issue, the nucleoprotein (NP) gene of wild-type virus can be incorporated into LAIV reassortant virus, along with the HA and NA genes. The present study compares the human CD8+ T cell memory responses to H3N2 LAIVs with the Len/17 or the wild-type NP using an in vitro model.

Analysis of Immune Epitopes of Respiratory Syncytial Virus for Designing of Vectored Vaccines Based on Influenza Virus Platform.

The immunoepitope database was used for analysis of experimentally detected epitopes of the respiratory syncytial virus (RSV) proteins and for selection of the epitope combinations for subsequent designing of recombinant vectored anti-RSV vaccines based on attenuated influenza viruses. Three cassettes containing the most promising B- and T-cell RSV epitopes were selected: peptide F (243-294) supporting the formation of humoral immunity in animals; fragment M2-1 (70-101+114-146) containing two MHC I epitopes (82-90 and 127-135); and MHC II-epitope (51-66). The selected constructions contained no neoepitopes causing undesirable effects of vaccination, such as immunotolerance or autoimmunity.

[DEVELOPMENT OF THE QUADRIVALENT LIVE ATTENUATED INFLUENZA VACCINE INCLUDING TWO INFLUENZA B LINEAGES--VICTORIA AND YAMAGATA].

This work is devoted to the research of the live attenuated influenza vaccine (LAIV) comprising two reassortant B/USSR/60/69-based vaccine influenza viruses Victoria and Yamagata. The intranasal immunization of the CBA mice with both Victoria and Yamagata strains induced 100% lung protection against the subsequent infection with the wild-type influenza B viruses of any antigen lineage. The quadrivalent LAIV (qLAIV) comprising both reassortant influenza B viruses Victoria and Yamagata were safe and areactogenic in adult volunteers. Following qLAIV administration the immune response was achieved to both Victoria and Yamagata lineages.

[A preclinical trial of the reassortant influenza virus vaccine strain A/17/Quail/Hong Kong/97/84 (H9N2)].

In this work, we examined the reassortant influenza virus strain A/17/Quail/Hong Kong/97/84 (H9N2) prepared at the Virology Department, Institute of Experimental Medicine, Russian Academy of Medical Sciences. The A/ Leningrad/134/17 (H2N2)-based vaccine candidate contained hemagglutinin and the neuraminidase from the nonpathogenic avian influenza A virus A(H9N2) of the G1 antigenic lineage. The vaccine candidate showed the ts-properties and cold adaptation. When administered intranasally to mice, the vaccine strain A(H9N2) was attenuated. It did not multiply in the lungs but was reproduced well in the nasal cavity, causing the production of the post-vaccination antibody. The A/17/Quail/Hong Kong/97/84(H9N2) virus was immunogenic when administered to mice as a LAIV intranasally or as a IIV intramuscularly. Intranasal A(H9N2) LAIV stimulated local production of the antibodies, which resulted in reduction in lung titers of the challenge virus G9.

[Testing of apathogenic influenza virus H5N3 as a poultry live vaccine].

Four H5N2 experimental vaccine strains and the apathogenic wild duck H5N3 influenza virus A/duck/ Moscow/4182/2010 (dk/4182) were tested as a live poultry vaccine. Experimental strains had the hemagglutinin of the A/Vietnam/1203/04 strain lacking the polybasic HA cleavage site or the hemagglutinin from attenuated virus (Ku/ at) that was derived from the highly pathogenic influenza virus A/chicken/Kurgan/3/2005 (H5N1). The hemagglutinin of the Ku-at has the amino acid substitutions Asp54/Asn and Lys222/Thr in HA1 and Val48/Ile and Lys131/Thr in HA2, while maintaining the polybasic HA cleavage site at an invariable level. The other genes of these experimental strains were from the H2N2 cold-adapted master strain A/Leningrad/134/17/57 (VN-Len and Ku-Len) or from the apathogenic H6N2 virus A/gull/Moscow/3100/2006 (VN-Gull and Ku-Gull). A single immunization of mice with all tested strains elicited a high level of serum antibodies and provided complete protection against the challenge with the lethal dose of A/chicken/Kurgan/3/05. The pathogenicity indexes of the Ku-at and the other strains for chicken were virtually zero, whereas the index of the parent H5N1 virus A/chicken/Kurgan/3/2005 was 2.98. Intravenous, intranasal, and aerosol routes of vaccination were compared. It was shown that the strain dk/4182 was totally apathogenic for one-day-old chicken and provided complete protection against the highly pathogenic H5N1 virus.

[Effect of Point Mutations in the Polymerase Genes of the Influenza A/PR/8/34 (H1N1) Virus on the Immune Response in a Mouse Model].

The vaccine strains for live attenuated influenza vaccines (LAIVs) have cold-adapted, temperature-sensitive, and attenuated phenotypes, which are guaranteed by the presence of specific mutations from the master donor virus in their internal genes. In this study, we used mutant viruses of the pathogenic A/Puerto Rico/8/34 (H1N1) that contained ts-mutations in PB1 (K265N, V591I), PB2 (V478L), and PA (L28P, V341L) genes along and/or in different combinations to evaluate the impact of these mutations in the immune responses. Sequential addition of tested mutations resulted in the stepwise decrease in virus-specific serum and, to a lesser extent, mucosal antibody levels. We demonstrated strong positive correlation between virus attenuation (virus titer in lung) and antibody titers. The ts-mutations in PB1, PB2, and PA genes are mostly involved in the modulation of the humoral immunity, but also have a moderate effect on the cellular adaptive immune response.

[Contribution of neuraminidase of influenza viruses to the sensitivity to the serum inhibitors and reassortment efficiency].

The live attenuated influenza vaccine (LAIV) consists of reassortant viruses with hemagglutinin (HA) and neuraminidase (NA) gene segments inherited from the circulating wild-type (WT) parental and the 6 internal protein-encoding gene segments from the cold-adapted attenuated master donor viruses (genome composition 6:2). In this study, we describe the obstacles to developing LAIV vaccine strains depending on the phenotypic peculiarities of the WT viruses used for reassortment. The genomic composition analysis of 849 reassortants revealed that over 80% of the reassortants based on the inhibitor-resistant WT viruses inherited WT NA as compared to 26% of reassortants based on the inhibitor-sensitive WT viruses. In addition, the highest percentage of the vaccine genotype reassortants was achieved when WT parental viruses were resistant to the non-specific serum inhibitors. We demonstrate that NA may play a role in the influenza virus sensitivity to the non-specific serum inhibitors. Replacing NA of the inhibitor-sensitive WT virus with the NA of the inhibitor-resistant master donor virus significantly decreased the sensitivity of the resulting reassortant virus to the non-specific inhibitors.

[Codon optimization of hemagglutinin as a promising tool for improving of the influenza vaccines immunogenicity].

Modification of the codon bias of sequences is a promising tool of the gene expression control. The theoretical basis of the codon optimization is reviewed, data on experiments in changing the viral gene codon bias for purposes of vaccine development are discussed. Research into the field of the influenza vaccine immunogenicity improvement with codon optimization method is reviewed. Prospects of the use of the codon optimization technique for influenza vaccine development are considered.

[Local antibody immune responses in influenza patients and persons vaccinated with seasonal, pre-pandemic, and pandemic live attenuated influenza vaccines].

Mucosal immunity is one of the most important factors of human anti-influenza defense. The data about local immune responses in influenza A (H3N2) patients and in persons vaccinated within 2000-2009 with different seasonal LAIVs, A (H1N1)pdm2009 LAIV, and A (H5N2) LAIV are discussed. The influenza infection resulted in the larger quantities of local IgA and IgG conversions than seasonal LAIV vaccination. 56% of young (18-21 y.o.) persons had high titers (> or = 1:64) of IgA to A (H1N1)pdm2009 virus before its circulation. 19% of persons had anti A (H5N2) IgA before vaccination. Two-fold vaccination with A (H1N1) pdm2009 and A (H5N2) LAIVs resulted in local antibody conversions in 54% and 27% of volunteers, respectively. Both these vaccines increased local IgA avidity. The number of antibody conversions after vaccination with seasonal LAIVs was in inverse dependence on their titers before vaccination. These results make it possible to conclude that the intensity of local antibody immune response to any LAIV depends on the state of local immunological memory, particularly on the presence of the crossreactive antibody-secreting B cells.