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Highlights: [•] FFF-MALS is a simple and reliable method that can determine the concentration of both live and inactivated influenza virus. [•] Simplified quantification is desirable as a quality control step throughput vaccine production. [•] FFF-MALS is a potentially useful technique that can increase speed and efficiency during the vaccine production pipeline. [ABSTRACT FROM AUTHOR]

Abstract: Sendai virus (SeV) and human parainfluenza virus type I (hPIV1) are highly homologous but have distinct host ranges, murine versus human. To identify the factors that affect the host specificity of parainfluenza viruses, we determined the infectivity and anti-IFN activities of SeV and hPIV1 in human and murine culture cells. SeV infected normal human lung MRC-5 and murine lung MM14.Lu or MLg2908 cells efficiently. Infection with SeV induced the release of IFN-β into culture medium in MRC-5 cells at similar levels with that of cells infected with hPIV1. SeV or hPIV1 infections, as well as expression of SeV or hPIV1 C proteins, inhibited the nuclear localization of STAT1 induced by IFN-β, suggesting that both SeV and hPIV1 C proteins block the IFN Jak/STAT pathway in MRC-5 cells. Pretreatment of MRC-5 cells with IFN suppressed replication of SeV and hPIV1 at an early stage of infection. However, hPIV1 overcame this suppression while SeV did not. SeV replication was restored in IFN-β pretreated murine MM14.Lu cells, suggesting SeV anti-IFN activity is species specific. These results suggest that SeV is less effective than hPIV1 in overcoming antiviral activity in human cells, which could be one of the factors that restrict the host range of SeV. [Copyright &y& Elsevier]

The paramyxovirus hemagglutinin-neuraminidase (HN) is a multifunctional protein responsible for attachment to receptors containing sialic acid, neuraminidase (NA) activity, and the promotion of membrane fusion, which is induced by the fusion protein. Analysis of the three-dimensional structure of Newcastle disease virus (NDV) HN protein revealed the presence of a large pocket, which mediates both receptor binding and NA activities. Recently, a second sialic acid binding site on HN was revealed by cocrystallization of the HN with a thiosialoside Neu5Ac-2-S-α(2,6)Gal1OMe, suggesting that NDV HN contains an additional sialic acid binding site. To evaluate the role of the second binding site on the life cycle of NDV, we rescued mutant viruses whose HNs were mutated at Arg516, a key residue that is involved in the second binding site. Loss of the second binding site on mutant HNs was confirmed by the hemagglutination inhibition test, which uses an inhibitor designed to block the NA active site. Characterization of the biological activities of HN showed that the mutation at Arg516 had no effect on NA activity. However, the fusion promotion activity of HN was substantially reduced by the mutation. Furthermore, the mutations at Arg516 slowed the growth rate of virus in tissue culture cells. These results suggest that the second binding site facilitates virus infection and growth by enhancing the fusion promotion activity of the HN. [ABSTRACT FROM AUTHOR]

The paramyxovirus hemagglutinin-neuraminidase (HN) is a multifunctional protein mediating hemagglutination (HA), neuraminidase (NA), and fusion promotion activities. It has been a matter of debate whether HN contains combined or separate sites for HA and NA activities. To clear the issue, we determined the presence of the second binding site on human parainfluenza virus (hPIV) type 1, 2, and 3 and Sendai virus (SeV) HN proteins. Results of virus elution from erythrocytes at an elevated temperature and HA inhibition by NA inhibitor BCX-2798 suggest that all hPIVs bind to the receptor only through the NA catalytic site, while SeV HN has an additional receptor binding site. Comparison of SeV and hPIV1 HN sequences revealed two amino acid differences at residues 521 and 523 in the region close to the second binding site identified in Newcastle disease virus HN. We mutated hPIV1 HN at position 523 from Asn to the residue of SeV HN, Asp, and rescued a recombinant SeV that carries the mutated hPIV1 HN by a reverse genetics system. The hPIV1 HN with Asp at position 523 hemagglutinated in the presence of BCX-2798, suggesting that the amino acid difference at position 523 is critical for the formation of a second binding site. Creation of the second binding site on hPIV1 HN, however, did not significantly affect the growth or fusion activity of the recombinant virus. Our study indicates that the presence and requirement of a second binding site vary among paramyxoviruses. [ABSTRACT FROM AUTHOR]

Abstract Live attenuated influenza vaccines (LAIVs) are promising tools for the induction of broad protection from influenza due to their ability to stimulate cross-reactive T cells against influenza pathogens. One of the major targets for cytotoxic T-cell immunity is viral nucleoprotein (NP), which is relatively conserved among antigenically distant influenza viruses. Nevertheless, a diversity of epitope composition has been found in the NP protein of different lineages of influenza A viruses. The H2N2 master donor virus which is currently used as a backbone for the LAIV and donor of the six genomic segments encoding the internal proteins, A/Leningrad/134/17/57 (MDV Len/17), was isolated 60 years ago. As such, NP-specific T-cell immunity induced upon vaccination with classical LAIVs with a 6:2 genome composition containing this older NP might be suboptimal against currently circulating influenza viruses. In this study, a panel of H3N2 LAIV candidates with wild-type NP genes derived from circulating viruses were generated by reverse genetics (5:3 genome composition). These viruses displayed the cold adaptation and temperature sensitivity phenotypes of MDV Len/17 in vitro. LAIVs with both 6:2 and 5:3 genome compositions were attenuated and replicated to a similar extent in the upper respiratory tract of ferrets. LAIVs were immunogenic as high neutralizing and hemagglutination inhibition serum antibody titers were detected 21 days after infection. All vaccinated animals were protected against infection with heterologous H3N2 influenza A viruses. Thus, LAIV with a 5:3 genome composition is safe, immunogenic and can induce cross-protective immunity. Highlights • Classical LAIVs might induce suboptimal NP-specific T-cell responses against recent influenza viruses; • A panel of H3N2 LAIV candidates with wild-type NP genes were compared to classical 6:2 LAIVs in ferrets; • Wild-type NP gene does not affect the safety profile of H3N2 LAIVs; • LAIVs with wild-type NP genes induced cross-reactive antibody immune responses similar to classical LAIVs; [ABSTRACT FROM AUTHOR]

Cold adapted influenza virus A/Leningrad/134/17/57 (H2N2) is a reliable master donor virus (Len/17-MDV) for preparing live attenuated influenza vaccines (LAIV). LAIVs are 6:2 reasortants that contain 6 segments of Len/17-MDV and the hemagglutinin (HA) and neuraminidase (NA) of contemporary circulating influenza A viruses. The problem with the classical reassortment procedure used to generate LAIVs is that there is limited selection pressure against NA of the Len/17-MDV resulting in 7:1 reassortants with desired HA only, which are not suitable LAIVs. The monoclonal antibodies (mAb) directed against the N2 of Len/17-MDV were generated. 10C4–8E7 mAb inhibits cell-to-cell spread of viruses containing the Len/17-MDV N2, but not viruses with the related N2 from contemporary H3N2 viruses. 10C4–8E7 antibody specifically inhibited the Len/17-MDV replication in vitro and in ovo but didn’t inhibit replication of H3N2 or H1N1pdm09 reassortants. Our data demonstrate that addition of 10C4–8E7 in the classical reassortment improves efficiency of LAIV production. [ABSTRACT FROM AUTHOR]

Since conserved viral proteins of influenza virus, such as nucleoprotein (NP) and matrix 1 protein, are the main targets for virus-specific CD8+ cytotoxic T-lymphocytes (CTLs), we hypothesized that introduction of the NP gene of wild-type virus into the genome of vaccine reassortants could lead to better immunogenicity and afford better protection. This paper describes in vitro and in vivo preclinical studies of two new reassortants of pandemic H1N1 live attenuated influenza vaccine (LAIV) candidates. One had the hemagglutinin (HA) and neuraminidase (NA) genes from A/South Africa/3626/2013 H1N1 wild-type virus on the A/Leningrad/134/17/57 master donor virus backbone (6 : 2 formulation) while the second had the HA, NA, and NP genes of the wild-type virus on the same backbone (5 : 3 formulation). Although both LAIVs induced similar antibody immune responses, the 5 : 3 LAIV provoked greater production of virus-specific CTLs than the 6 : 2 variant. Furthermore, the 5 : 3 LAIV-induced CTLs had higher in vivo cytotoxic activity, compared to 6 : 2 LAIV. Finally, the 5 : 3 LAIV candidate afforded greater protection against infection and severe illness than the 6 : 2 LAIV. Inclusion in LAIV of the NP gene from wild-type influenza virus is a new approach to inducing cross-reactive cell-mediated immune responses and cross protection against pandemic influenza. [ABSTRACT FROM AUTHOR]

This study sought to improve an existing live attenuated influenza vaccine (LAIV) by including nucleoprotein (NP) from wild-type virus rather than master donor virus (MDV). H7N9 LAIV reassortants with 6:2 (NP from MDV) and 5:3 (NP from wild-type virus) genome compositions were compared with regard to their growth characteristics, induction of humoral and cellular immune responses in mice, and ability to protect mice against homologous and heterologous challenge viruses. Although, in general, the 6:2 reassortant induced greater cellmediated immunity in C57BL6 mice than the 5:3 vaccine, mice immunized with the 5:3 LAIV were better protected against heterologous challenge. The 5:3 LAIV-induced CTLs also had better in vivo killing activity against target cells loaded with the NP366 epitope of recent influenza viruses. Modification of the genome of reassortant vaccine viruses by incorporating the NP gene from wild-type viruses represents a simple strategy to improve the immunogenicity and cross-protection of influenza vaccines. [ABSTRACT FROM AUTHOR]

Cold-adapted influenza strains A/Leningrad/134/17/57 (H2N2) and B/USSR/60/69, originally developed in Russia, have been reliable master donors of attenuation for preparing live attenuated influenza vaccines (LAIV). The classical strategy for generating LAIV reassortants is robust, but has some disadvantages. The generation of reassortants requires at least 3 passages under selective conditions after co-infection; each of these selective passages takes six days. Screening the reassortants for a genomic composition traditionally starts after a second limiting dilution cloning procedure, and the number of suitable reassortants is limited. We developed a new approach to shorten process of preparing LAIV seed viruses. Introducing the genotyping of reassortants by pyrosequencing and monitoring sequence integrity of surface antigens starting at the first selective passage allowed specific selection of suitable reassortants for the next cloning procedure and also eliminate one of the group selective passage in vaccine candidate generation. Homogeneity analysis confirmed that reducing the number of selective passages didn’t affect the quality of LAIV seed viruses. Finally, the two-way hemagglutination inhibition test, implemented for all the final seed viruses, confirmed that any amino acid substitutions acquired by reassortants during egg propagation didn’t affect antigenicity of the vaccine. Our new strategy reduces the time required to generate a vaccine and was used to generate seasonal LAIVs candidates for the 2012/2013, 2014/2015, and 2015/2016 seasons more rapidly. [ABSTRACT FROM AUTHOR]

Background: Avian influenza A (H7N9) virus has emerged recently and continues to cause severe disease with a high mortality rate in humans prompting the development of candidate vaccine viruses. Live attenuated influenza vaccines (LAIV) are 6:2 reassortant viruses containing the HA and NA gene segments from wild type influenza viruses to induce protective immune responses and the six internal genes from Master Donor Viruses (MDV) to provide temperature sensitive, cold-adapted and attenuated phenotypes. Methodology/Principal Findings: LAIV candidate A/Anhui/1/2013(H7N9)-CDC-LV7A (abbreviated as CDC-LV7A), based on the Russian MDV, A/Leningrad/134/17/57 (H2N2), was generated by classical reassortment in eggs and retained MDV temperature-sensitive and cold-adapted phenotypes. CDC-LV7A had two amino acid substitutions N123D and N149D (H7 numbering) in HA and one substitution T10I in NA. To evaluate the role of these mutations on the replication capacity of the reassortants in eggs, the recombinant viruses A(H7N9)RG-LV1 and A(H7N9)RG-LV2 were generated by reverse genetics. These changes did not alter virus antigenicity as ferret antiserum to CDC-LV7A vaccine candidate inhibited hemagglutination by homologous A(H7N9) virus efficiently. Safety studies in ferrets confirmed that CDC-LV7A was attenuated compared to wild-type A/Anhui/1/2013. In addition, the genetic stability of this vaccine candidate was examined in eggs and ferrets by monitoring sequence changes acquired during virus replication in the two host models. No changes in the viral genome were detected after five passages in eggs. However, after ten passages additional mutations were detected in the HA gene. The vaccine candidate was shown to be stable in the ferret model; post-vaccination sequence data analysis showed no changes in viruses collected in nasal washes present at day 5 or day 7. Conclusions/Significance: Our data indicate that the A/Anhui/1/2013(H7N9)-CDC-LV7A reassortant virus is a safe and genetically stable candidate vaccine virus that is now available for distribution by WHO to vaccine manufacturers. [ABSTRACT FROM AUTHOR]

Background: Live attenuated influenza vaccine viruses (LAIVs) can be generated by classical reassortment of gene segments between a cold adapted, temperature sensitive and attenuated Master Donor Virus (MDV) and a seasonal wild-type (wt) virus. The vaccine candidates contain hemagglutinin (HA) and neuraminidase (NA) genes derived from the circulating wt viruses and the remaining six genes derived from the MDV strains. Rapid, efficient selection of the viruses with 6∶2 genome compositions from the large number of genetically different viruses generated during reassortment is essential for the biannual production schedule of vaccine viruses. Methodology/Principal Findings: This manuscript describes a new approach for the genotypic analysis of LAIV reassortant virus clones based on pyrosequencing. LAIV candidate viruses were created by classical reassortment of seasonal influenza A (H3N2) (A/Victoria/361/2011, A/Ohio/02/2012, A/Texas/50/2012) or influenza A (H7N9) (A/Anhui/1/2013) wt viruses with the MDV A/Leningrad/134/17/57(H2N2). Using strain-specific pyrosequencing assays, mixed gene variations were detected in the allantoic progenies during the cloning procedure. The pyrosequencing analysis also allowed for estimation of the relative abundance of segment variants in mixed populations. This semi-quantitative approach was used for selecting specific clones for the subsequent cloning procedures. Conclusions/Significance: The present study demonstrates that pyrosequencing analysis is a useful technique for rapid and reliable genotyping of reassortants and intermediate clones during the preparation of LAIV candidates, and can expedite the selection of vaccine virus candidates. [ABSTRACT FROM AUTHOR]

Highlights: [•] Sensitive and strain-specific rRT-PCR assays were developed for each gene segment of influenza viruses used to prepare live attenuated influenza vaccine (LAIV) reassortant viruses. [•] rRT-PCR assays proved to be adequate and sufficient to provide the evidence for the homogeneity of the LAIV reassortant viruses. [•] Complete genome sequencing of all viruses confirmed the stability of the LAIV reassortant genome. [ABSTRACT FROM AUTHOR]