The induction of virus-neutralizing antibodies in influenza A-infected mice treated with Oseltamivir phosphate: effect of dosage and scheduling.

Oseltamivir phosphate (OS) is currently the most frequently used influenza antiviral drug. It moderates the course of influenza virus type A (IAV) infection, however, its impact on the induction of virus-neutralizing antibodies (VNAbs) is not understood in details. Here, we examined the influence of low (10 mg/kg) or high (60 mg/kg) doses of OS on the viral titer in lungs of BALB/c mice infected with 0.5 LD50 of IAV and on the level of VNAbs. Prophylactic application of OS (6 h before the infection) delayed the increase of viral titer in lungs with a lower peak in comparison to non-treated control mice. After therapeutic OS application (44 h after the infection), maximum of virus titer did not significantly change. However, the induction of VNAbs strongly decreased, to 16.7%-18.1% of the control, after preventive application of high OS dose. A minimal decrease of VNAbs titers was observed in groups of mice treated with low dose of OS applied therapeutically. They lowered to 91.1% / 14 or to 94.1% / 21 days post infection (p.i.) of VNAbs titers of non-treated control mice. In all other groups, levels of VNAbs titers dropped to 26.5-53.7% of those of non-treated mice. It should be noted that VNAbs titers were in direct proportion to maximal virus titers in mouse lungs of corresponding groups. In summary, after OS application the clinical symptoms of the disease were milder or non-observable in all OS-treated groups, but the lowering of VNAbs titers was dependent on the OS dose and interval between drug app-lication and the start of infection. Keywords: influenza A virus; Oseltamivir; prophylactic treatment; therapeutic treatment; virus-neutralizing antibodies.

The Contribution of Viral Proteins to the Synergy of Influenza and Bacterial Co-Infection.

A severe course of acute respiratory disease caused by influenza A virus (IAV) infection is often linked with subsequent bacterial superinfection, which is difficult to cure. Thus, synergistic influenza-bacterial co-infection represents a serious medical problem. The pathogenic changes in the infected host are accelerated as a consequence of IAV infection, reflecting its impact on the host immune response. IAV infection triggers a complex process linked with the blocking of innate and adaptive immune mechanisms required for effective antiviral defense. Such disbalance of the immune system allows for easier initiation of bacterial superinfection. Therefore, many new studies have emerged that aim to explain why viral-bacterial co-infection can lead to severe respiratory disease with possible fatal outcomes. In this review, we discuss the key role of several IAV proteins-namely, PB1-F2, hemagglutinin (HA), neuraminidase (NA), and NS1-known to play a role in modulating the immune defense of the host, which consequently escalates the development of secondary bacterial infection, most often caused by Streptococcus pneumoniae. Understanding the mechanisms leading to pathological disorders caused by bacterial superinfection after the previous viral infection is important for the development of more effective means of prevention; for example, by vaccination or through therapy using antiviral drugs targeted at critical viral proteins.

Quantification of bacteria by in vivo bioluminescence imaging in comparison with standard spread plate method and reverse transcription quantitative PCR (RT-qPCR).

In vivo bioluminescence imaging (BLI) offers a unique opportunity to analyze ongoing bacterial infections qualitatively and quantitatively in intact animals over time, leading to a reduction in the number of animals needed for a study. Since accurate determination of the bacterial burden plays an essential role in microbiological research, the present study aimed to evaluate the ability to quantify bacteria by non-invasive BLI technique in comparison to standard spread plate method and reverse transcription quantitative PCR (RT-qPCR). For this purpose, BALB/c mice were intranasally infected with 1 × 105 CFU of bioluminescent Streptococcus pneumoniae A66.1. At day 1 post-infection, the presence of S. pneumoniae in lungs was demonstrated by spread plate method and RT-qPCR, but not by in vivo BLI. However, on the second day p.i., the bioluminescent signal was already detectable, and the photon flux values positively correlated with CFU counts and RT-qPCR data within days 2-6. Though in vivo BLI is valuable research tool allowing the continuous monitoring and quantification of pneumococcal infection in living mice, it should be kept in mind that early in the infection, depending on the infective dose, the bioluminescent signal may be below the detection limit.

DNA vaccine targeting the ectodomain of influenza M2 protein to endolysosome pathway enhances anti-M2e protective antibody response in mice.

A promising candidate for developing the universal influenza vaccine is the ectodomain of the M2 protein (M2e). We designed and prepared an experimental DNA vaccine with an improved potential to induce anti-M2e immune response. The sequence for truncated NS1 protein followed by 4xM2e was inserted into the expression vector pTriEx-4 (pEx). M2e repeats were fused to the transmembrane domain and cytoplasmic tail of lysosome-associated membrane glycoprotein  2 isoform A (LAMP-2a) to target the M2e to the endo-lysosome pathway, facilitating increased antigen presentation by MHC II. Using confocal microscope immunofluorescence analysis, we confirmed a strong colocalization of pEx 4M2e-LAMP-2a with early endosomes and a weaker colocalization with late endosomes. BALB/c mice immunized with three doses of pEx 4M2e-LAMP-2a DNA vaccine and challenged with 2LD50 mouse-adapted influenza virus developed significantly (up to 16 times) higher anti-M2e antibody response in comparison to mice immunized with pEx 4M2e vaccine using the same immunization protocol. This was in correlation with the increased survival rate (near to 67% vs 50%) observed in animals immunized with pEx 4M2e-LAMP-2a DNA in comparison to mice immunized with pEx 4M2e. Keywords: influenza A; matrix protein 2 ectodomain; NS1; LAMP-2a; DNA vaccine.

Comparison of infectious influenza A virus quantification methods employing immuno-staining.

Infections caused by highly variable influenza A viruses (IAVs) pose perpetual threat to humans as well as to animals. Their surveillance requires reliable methods for their qualitative and quantitative analysis. The most frequently utilized quantification method is the titration by plaque assay or 50% tissue culture infectious dose estimation by TCID50. However, both methods are time-consuming. Moreover, some IAV strains form hardly visible plaques, and the evaluation of TCID50 is subjective. Employment of immuno-staining into the classic protocols for plaque assay or TCID50 assay enables to avoid these problems and moreover, shorten the time needed for reliable infectious virus quantification. Results obtained by these two alternatives of classic virus titration methods were compared to the newer rapid culture assay (RCA), where titration endpoint of infectious virus was estimated microscopically based on the immuno-staining of infected cells. In our analysis of compared methods, five different IAV strains of H1, H3 and H5 subtypes were used and results were statistically evaluated. We conclude that the RCA proved to be at least as reliable in assessment of infectious viral titer as plaque assay and TCID50, considering the employed immuno-staining.

Stereoisomers of oseltamivir - synthesis, in silico prediction and biological evaluation.

Oseltamivir is an important antiviral drug, which possess three chirality centers in its structure. From eight possible stereoisomers, only two have been synthesized and evaluated so far. We describe herein the stereoselective synthesis, computational activity prediction and biological testing of another three diastereoisomers of oseltamivir. These isomers have been synthesized using stereoselective organocatalytic Michael addition, cyclization and reduction. Their binding to viral neuraminidase N1 of influenza A virus was evaluated by quantum-chemical calculations and their anti-influenza activities were tested by an in vitro virus-inhibition assay. All three isomers displayed antiviral activity lower than that of oseltamivir, however, one of the stereoisomers, (3S,4R,5S)-isomer, of oseltamivir showed in vitro potency towards the Tamiflu-sensitive influenza viral strain A/Perth/265/2009(H5N1) comparable to Tamiflu.

Perspective of Use of Antiviral Peptides against Influenza Virus.

The threat of a worldwide influenza pandemic has greatly increased over the past decade with the emergence of highly virulent avian influenza strains. The increased frequency of drug-resistant influenza strains against currently available antiviral drugs requires urgent development of new strategies for antiviral therapy, too. The research in the field of therapeutic peptides began to develop extensively in the second half of the 20(th) century. Since then, the mechanisms of action for several peptides and their antiviral prospect received large attention due to the global threat posed by viruses. Here, we discussed the therapeutic properties of peptides used in influenza treatment. Peptides with antiviral activity against influenza can be divided into three main groups. First, entry blocker peptides such as a Flupep that interact with influenza hemagglutinin, block its binding to host cells and prevent viral fusion. Second, several peptides display virucidal activity, disrupting viral envelopes, e.g., Melittin. Finally, a third set of peptides interacts with the viral polymerase complex and act as viral replication inhibitors such as PB1 derived peptides. Here, we present a review of the current literature describing the antiviral activity, mechanism and future therapeutic potential of these influenza antiviral peptides.

The ubiquitination of the influenza A virus PB1-F2 protein is crucial for its biological function.

The aim of the present study was to identify what influences the short half-life of the influenza A virus PB1-F2 protein and whether a prolonged half-life affects the properties of this molecule. We hypothesized that the short half-life of PB1-F2 could conceal the phenotype of the protein. Because proteasome degradation might be involved in PB1-F2 degradation, we focused on ubiquitination, a common label for proteasome targeting. A cluster of lysine residues was demonstrated as an ubiquitination acceptor site in evolutionary and functionally distinct proteins. The PB1-F2 sequence alignment revealed a cluster of lysines on the carboxy terminal end of PB1-F2 in almost all of the GenBank sequences available to date. Using a proximity ligation assay, we identified ubiquitination as a novel posttranslational modification of PB1-F2. Changing the lysines at positions 73, 78, and 85 to arginines suppressed the ubiquitination of A/Puerto Rico/8/1934 (H1N1)-derived PB1-F2. The mutation of the C-terminal lysine residue cluster positively affected the overall expression levels of avian A/Honk Kong/156/1997 (H5N1)- and mammalian A/Puerto Rico/8/1934 (H1N1)-derived PB1-F2. Moreover, increased PB1-F2 copy numbers strengthened the functions of this virus in the infected cells. The results of a minigenome luciferase reporter assay revealed an enhancement of viral RNA-dependent RNA polymerase activity in the presence of stabilized PB1-F2, regardless of viral origin. IFNß antagonism was enhanced in 293T cells transfected with a plasmid expressing stabilized K→R mutant variants of PB1-F2. Compared with PB1-F2 wt, the loss of ubiquitination enhanced the antibody response after DNA vaccination. In summary, we revealed that PB1-F2 is an ubiquitinated IAV protein, and this posttranslational modification plays a central role in the regulation of the biological functions of this protein.

Heterosubtypic protection against influenza A induced by adenylate cyclase toxoids delivering conserved HA2 subunit of hemagglutinin.

The protective efficacy of currently available influenza vaccines is restricted to vaccine strains and their close antigenic variants. A new strategy to obtain cross-protection against influenza is based on conserved antigens of influenza A viruses (IAV), which are able to elicit a protective immune response. Here we describe a vaccination approach involving the conserved stem part of hemagglutinin, the HA2 subunit, shared by different HA subtypes of IAV. To increase its immunogenicity, a novel strategy of antigen delivery to antigen presenting cells (APCs) has been used. The HA2 segment (residues 23-185) was inserted into a genetically detoxified adenylate cyclase toxoid (CyaA-E5) which specifically targets and penetrates CD11b-expressing dendritic cells. The CyaA-E5-HA2 toxoid induced HA2(93-102), HA2(96-104) and HA2(170-178)-specific and Th1 polarized T-cell responses, and also elicited strong broadly cross-reactive HA2-specific antibody response. BALB/c mice immunized with three doses of purified CyaA-E5-HA2 without any adjuvant recovered from influenza infection 2days earlier than the control mock-immunized mice. More importantly, immunized mice were protected against a lethal challenge with 2LD(50) dose of a homologous virus (H3 subtype), as well as against the infection with a heterologous (H7 subtype) influenza A virus. This is the first report on heterosubtypic protection against influenza A infection mediated by an HA2-based vaccine that can induce both humoral and cellular immune responses without the need of adjuvant.

Epitope specificity of anti-HA2 antibodies induced in humans during influenza infection.

BACKGROUND: The conserved, fusion-active HA2 glycopolypeptide (HA2) subunit of influenza A hemagglutinin comprises four distinct antigenic sites. Monoclonal antibodies (MAbs) recognizing three of these sites are broadly cross-reactive and protective. OBJECTIVES: This study aimed to establish whether antibodies specific to these three antigenic sites were elicited during a natural influenza infection or by vaccination of humans. METHODS: Forty-five paired acute and convalescent sera from individuals with a confirmed influenza A (subtype H3) infection were examined for the presence of HA2-specific antibodies. The fraction of antibodies specific to three particular antigenic sites (designated IIF4, FC12, and CF2 here) was investigated using competitive enzyme immunoassay. RESULTS: Increased levels of antibodies specific to an ectodomain of HA2 (EHA2: N-terminal residues 23-185 of HA2) were detected in 73% of tested convalescent sera (33/45), while an increased level of antibodies specific to the HA2 fusion peptide (N-terminal residues 1-38) was induced in just 15/45 individuals (33%). Competitive assays confirmed that antibodies specific to the IIF4 epitope (within HA2 residues 125-175) prevailed in 86% (13/15) over those specific to the other two epitopes during infection. However, only a negligible increase in HA2-specific antibodies was detectable following vaccination with a current subunit vaccine. CONCLUSIONS: We observed that the antigenic site localized within N-terminal HA2 residues 125-175 was more immunogenic than that within residues 1-38 (HA2 fusion protein), although both are weak natural immunogens. We suggest that new anti-influenza vaccines should include HA2 (or specific epitopes localized within this glycopolypeptide) to enhance their cross-protective efficacy.

Conserved epitopes of influenza A virus inducing protective immunity and their prospects for universal vaccine development.

Influenza A viruses belong to the best studied viruses, however no effective prevention against influenza infection has been developed. The emerging of still new escape variants of influenza A viruses causing epidemics and periodic worldwide pandemics represents a threat for human population. Therefore, current, hot task of influenza virus research is to look for a way how to get us closer to a universal vaccine. Combination of chosen conserved antigens inducing cross-protective antibody response with epitopes activating also cross-protective cytotoxic T-cells would offer an attractive strategy for improving protection against drift variants of seasonal influenza viruses and reduces the impact of future pandemic strains. Antigenically conserved fusion-active subunit of hemagglutinin (HA2 gp) and ectodomain of matrix protein 2 (eM2) are promising candidates for preparation of broadly protective HA2- or eM2-based vaccine that may aid in pandemic preparedness. Overall protective effect could be achieved by contribution of epitopes recognized by cytotoxic T-lymphocytes (CTL) that have been studied extensively to reach much broader control of influenza infection. In this review we present the state-of-art in this field. We describe known adaptive immune mechanisms mediated by influenza specific B- and T-cells involved in the anti-influenza immune defense together with the contribution of innate immunity. We discuss the mechanisms of neutralization of influenza infection mediated by antibodies, the role of CTL in viral elimination and new approaches to develop epitope based vaccine inducing cross-protective influenza virus-specific immune response.

Single residue deletions along the length of the influenza HA fusion peptide lead to inhibition of membrane fusion function.

A panel of eight single amino acid deletion mutants was generated within the first 24 residues of the fusion peptide domain of the of the hemagglutinin (HA) of A/Aichi/2/68 influenza A virus (H3N2 subtype). The mutant HAs were analyzed for folding, cell surface transport, cleavage activation, capacity to undergo acid-induced conformational changes, and membrane fusion activity. We found that the mutant DeltaF24, at the C-terminal end of the fusion peptide, was expressed in a non-native conformation, whereas all other deletion mutants were transported to the cell surface and could be cleaved into HA1 and HA2 to activate membrane fusion potential. Furthermore, upon acidification these cleaved HAs were able to undergo the characteristic structural rearrangements that are required for fusion. Despite this, all mutants were inhibited for fusion activity based on two separate assays. The results indicate that the mutant fusion peptide domains associate with target membranes in a non-functional fashion, and suggest that structural features along the length of the fusion peptide are likely to be relevant for optimal membrane fusion activity.

HA2-specific monoclonal antibodies as tools for differential recognition of influenza A virus antigenic subtypes.

Antigenic reactivity of a set of monoclonal antibodies (MAb) raised against the HA2 subunit of hemagglutinin of H3 subtype was characterized in a rapid culture assay. MAbs FC12 and FE1, known to recognize the same antigenic site (IV), cross-reacted with influenza viruses of H3 and H4 subtypes, regardless of their host origin. No cross-reactivity was detected with other antigenic subtypes tested (H1-H13). The involvement of conserved residues D160, N168, and F171 in the differential recognition of H3 and H4 subtypes is proposed. In contrast, MAb IIF4 that recognizes antigenic site II exhibited a broader inter-subtype reactivity including subtypes H3, H4, H5, H8 and some viruses of H2, H6 and H13 subtypes. The ability of HA2-specific antibodies to differentially react with distinct antigenic subtypes can be utilized in development of diagnostics and in the influenza virus surveillance.

Evaluation of the subtype specificity of monoclonal antibodies raised against H1 and H3 subtypes of human influenza A virus hemagglutinins.

Three previously described monoclonal antibodies (MAbs) specific for influenza A(H1) hemagglutinins (HA) revealed high sensitivity (98.2 to 99.1%) and specificity (100%) when tested against 245 strains of different subtypes. One of them was included in the World Health Organization's influenza reagent kit for 2001 to 2002. In contrast, two MAbs raised against human influenza A(H3) HA revealed cross-reactivity with viruses of other subtypes.