Anti-Influenza Effect of Nanosilver in a Mouse Model.

The present study assesses copper metabolism of the host organism as a target of antiviral strategy, basing on the "virocell" concept. Silver nanoparticles (AgNPs) were used as a specific active agent because they reduce the level of holo-ceruloplasmin, the main extracellular cuproenzyme. The mouse model of influenza virus A infection was used with two doses: 1 LD50 and 10 LD50. Three treatment regimens were used: Scheme 1-mice were pretreated 4 days before infection and then every day during infection development; Scheme 2-mice were pretreated four days before infection and on the day of virus infection; Scheme 3-virus infection and AgNP treatment started simultaneously, and mice were injected with AgNPs until the end of the experiment. The mice treated by Scheme 1 demonstrated significantly lower mortality, the protection index reached 60-70% at the end of the experiment, and mean lifespan was prolonged. In addition, the treatment of the animals with AgNPs resulted in normalization of the weight dynamics. Despite the amelioration of the infection, AgNP treatment did not influence influenza virus replication. The possibility of using nanosilver as an effective indirectly-acting antiviral drug is discussed.

Safety, immunogenicity and protection of A(H3N2) live attenuated influenza vaccines containing wild-type nucleoprotein in a ferret model.

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.

Prevention of Influenza A(H7N9) and Bacterial Infections in Mice Using Intranasal Immunization With Live Influenza Vaccine and the Group B Streptococcus Recombinant Polypeptides.

We investigate the protective effect of combined vaccination based on live attenuated influenza vaccine (LAIV) and group B streptococcus (GBS) recombinant polypeptides against potential pandemic H7N9 influenza infection followed by GBS burden. Mice were intranasally immunized using 107 50% egg infectious dose (EID50) of H7N3 LAIV, the mix of the 4 GBS peptides (group B streptococcus vaccine [GBSV]), or combined LAIV + GBSV vaccine. The LAIV raised serum hemagglutination-inhibition antibodies against H7N9 in higher titers than against H7N3. Combined vaccination provided advantageous protection against infections with A/Shanghai/2/2013(H7N9)CDC-RG influenza and serotype II GBS. Combined vaccine significantly improved bacterial clearance from the lungs after infection compared with other vaccine groups. The smallest lung lesions due to combined LAIV + GBSV vaccination were associated with a prevalence of lung interferon-γ messenger RNA expression. Thus, combined viral and bacterial intranasal immunization using H7N3 LAIV and recombinant bacterial polypeptides induced balanced adaptive immune response, providing protection against potential pandemic influenza H7N9 and bacterial complications.

Evaluation in Mouse Model of Combined Virus-bacterial Vaccine Based on Attenuated Influenza A(H7N3) Virus and the Group B Streptococcus Recombinant Polypeptides.

BACKGROUND: Secondary bacterial influenza complications are a common cause of excesses morbidity and mortality, which determines the need to develop means for specific prophylaxis. Group B streptococcal infection is especially common cause of pneumonia among children and the elderly with underlying conditions. Here we investigate in a mouse model the effects of combined intranasal immunization using live attenuated influenza vaccine and recombinant polypeptides based on group B Streptococcus surface proteins. METHODS: Groups of outbred mice received two doses of the following preparations: 1) the reassortant A/17/Mallard/Netherlands/00/95 (H7N3) influenza virus; 2) a mixture of P6, ScaAB, ScpB1 and Stv recombinant GBS proteins (20 µg total); 3) the A(H7N3) influenza vaccine pooled with the four bacterial peptide preparation; 4) control animals were treated with PBS. RESULTS: Intranasal vaccination using LAIV in combination with GBS polypeptides provided advantageous protection against infections with homologous A/Mallard/Netherlands/12/00 (H7N3) wild type virus or heterologous A/Puerto Rico/8/34 (H1N1) followed by serotype II GBS infection. Also, combined vaccination improved bacterial clearance from the lungs of mice. CONCLUSION: Intranasal immunization with LAIV+GBSV was safe and enabled to induce the antibody response to each of vaccine components. Thus, the combined vaccine increased the protective effect against influenza and its bacterial complications in mice compared to LAIV-only.

Serum strain-specific or cross-reactive neuraminidase inhibiting antibodies against pandemic А/California/07/2009(H1N1) influenza in healthy volunteers.

BACKGROUND: Pre-existing antibodies to influenza virus neuraminidase may provide protection against infection influenza viruses containing novel hemagglutinin (HA). The aim of our study was to evaluate serum neuraminidase-inhibiting (NI) antibodies against А/California/07/2009(H1N1) [H1N1/2009pdm] and А/New Caledonia/20/1999(H1N1) [H1N1/1999] influenza viruses in relation with the age of participants and hemagglutination-inhibition (HI) antibody levels. Anti-H1N1/2009pdm neuraminidase and anti-H1N1/1999 neuraminidase antibody levels were measured in total 219 serum samples from Russian healthy peoples of various ages examined before and a year after pandemic strain appearance. We adjusted peroxidase-linked lectin micro-procedure to measure NI antibody titers using the reassortant A/H7N1 influenza viruses based on A/equine/Prague/1/56(H7N7). Also, HI antibody titers were estimated against H1N1/2009pdm, H1N1/1999 and a panel of seasonal A/H1N1 influenza viruses. RESULTS: In sera samples collected during the fall of 2010, mean titers of specific HI and NI antibodies to H1N1/2009pdm were 2-2.1 times lower than antibody levels against H1N1/1999. Of the 163 individuals examined, 58 (35.6%) had NI anti-H1N1/2009pdm antibody titers > 1:20, compared to 93 (57.1%) who had NI anti-H1N1/1999 antibody titers > 1:20. There were low correlations between HI and NI antibody levels against either H1N1/1999 or H1N1/2009pdm in the same serum samples. The 24 adults born between 1957 and 1977 expressed very low levels of NI antibodies to A/H1N1 influenza viruses. Persons with low HI anti-H1N1/2009pdm titers but positive to seasonal A/H1N1 demonstrated significantly higher NI anti-A/H1N1 antibody titers than unexposed subjects. In 2005 cross-reactive NI anti-H1N1/2009pdm antibody titers > 1:20 were detected among 7.1% of young people. CONCLUSIONS: Our study confirmed that contact with seasonal influenza viruses may have contributed to generating the cross-reacting anti-H1N1/2009pdm NI antibodies which were detected in the sera of 18-20 years old people examined before the pandemic virus active circulation. The lowest levels of antibodies to the neuraminidase of N1 subtype were in the group of participants born during the circulation of influenza A/H2N2 or A/H3N2 viruses. The low correlation between HI and NI antibody titers suggests that NI antibody detection can be used as an additional test to evaluate the immune response after influenza infections or immunizations.

Comparative study of influenza virus replication in MDCK cells and in primary cells derived from adenoids and airway epithelium.

Although clinical trials with human subjects are essential for determination of safety, infectivity, and immunogenicity, it is desirable to know in advance the infectiousness of potential candidate live attenuated influenza vaccine strains for human use. We compared the replication kinetics of wild-type and live attenuated influenza viruses, including H1N1, H3N2, H9N2, and B strains, in Madin-Darby canine kidney (MDCK) cells, primary epithelial cells derived from human adenoids, and human bronchial epithelium (NHBE cells). Our data showed that despite the fact that all tissue culture models lack a functional adaptive immune system, differentiated cultures of human epithelium exhibited the greatest restriction for all H1N1, H3N2, and B vaccine viruses studied among three cell types tested and the best correlation with their levels of attenuation seen in clinical trials with humans. In contrast, the data obtained with MDCK cells were the least predictive of restricted viral replication of live attenuated vaccine viruses in humans. We were able to detect a statistically significant difference between the replication abilities of the U.S. (A/Ann Arbor/6/60) and Russian (A/Leningrad/134/17/57) cold-adapted vaccine donor strains in NHBE cultures. Since live attenuated pandemic influenza vaccines may potentially express a hemagglutinin and neuraminidase from a non-human influenza virus, we assessed which of the three cell cultures could be used to optimally evaluate the infectivity and cellular tropism of viruses derived from different hosts. Among the three cell types tested, NHBE cultures most adequately reflected the infectivity and cellular tropism of influenza virus strains with different receptor specificities. NHBE cultures could be considered for use as a screening step for evaluating the restricted replication of influenza vaccine candidates.

Comparative safety, immunogenicity, and efficacy of several anti-H5N1 influenza experimental vaccines in a mouse and chicken models (Testing of killed and live H5 vaccine).

OBJECTIVE: Parallel testing of inactivated (split and whole virion) and live vaccine was conducted to compare the immunogenicity and protective efficacy against homologous and heterosubtypic challenge by H5N1 highly pathogenic avian influenza virus. METHOD: Four experimental live vaccines based on two H5N1 influenza virus strains were tested; two of them had hemagglutinin (HA) of A/Vietnam/1203/04 strain lacking the polybasic HA cleavage site, and two others had hemagglutinins from attenuated H5N1 virus A/Chicken/Kurgan/3/05, with amino acid substitutions of Asp54/Asn and Lys222/Thr in HA1 and Val48/Ile and Lys131/Thr in HA2 while maintaining the polybasic HA cleavage site. The neuraminidase and non-glycoprotein genes of the experimental live vaccines were from 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). Inactivated H5N1 and H1N1 and live H1N1 vaccine were used for comparison. All vaccines were applied in a single dose. Safety, immunogenicity, and protectivity against the challenge with HPAI H5N1 virus A/Chicken/Kurgan/3/05 were estimated. RESULTS: All experimental live H5 vaccines tested were apathogenic as determined by weight loss and conferred more than 90% protection against lethal challenge with A/Chicken/Kurgan/3/05 infection. Inactivated H1N1 vaccine in mice offered no protection against challenge with H5N1 virus, while live cold-adapted H1N1 vaccine reduced the mortality near to zero level. CONCLUSIONS: The high yield, safety, and protectivity of VN-Len and Ku-Len made them promising strains for the production of inactivated and live vaccines against H5N1 viruses.

Master donor viruses A/Leningrad/134/17/57 (H2N2) and B/USSR/60/69 and derived reassortants used in live attenuated influenza vaccine (LAIV) do not display neurovirulent properties in a mouse model.

Demonstration of the absence of neurovirulent properties of reassortant viruses contained in live attenuated influenza vaccine (LAIV) is a regulatory requirement. A mouse model was used to detect neurovirulent properties of the cold-adapted, temperature-sensitive and attenuated influenza master donor viruses (MDVs) A/Leningrad/134/17/57 (H2N2) and B/USSR/60/69 and derived reassortant influenza viruses. A/NWS/33 (H1N1), which is known to be neurovirulent in mice, was used as a positive control. Under conditions where the positive control virus induced symptoms of disease and showed viral replication in the upper respiratory tract as well as in the brain, replication of the influenza master donor viruses and reassortant influenza A and B viruses was limited to the upper respiratory tract where they were administered. None of the mice inoculated with MDVs or reassortant influenza viruses suffered from disease, and no virus or viral replication was observed in the brains of these mice. The results demonstrate the absence of neurovirulent properties of the MDVs and reassortant influenza viruses derived therefrom used in LAIV.

PB2 and PA genes control the expression of the temperature-sensitive phenotype of cold-adapted B/USSR/60/69 influenza master donor virus.

The cold-adapted (ca) and temperature-sensitive (ts) influenza master donor virus (MDV) B/USSR/60/69 was derived from its wild-type parental virus after successive passages in eggs at 32 degrees C and 25 degrees C. This strain is currently in use for preparing reassortant influenza B vaccine viruses which are used in the Russian trivalent live attenuated influenza vaccine. Vaccine viruses are obtained by classical reassortment of MDV and a currently circulating wild-type virus. The phenotypic properties cold adaptation and temperature sensitivity are inherited from the six genes encoding the internal proteins of the MDV. However, the role of the individual gene segments in temperature sensitivity and thus attenuation is not known. In this study, 35 reassortant viruses of B/USSR/60/69 MDV with current wild-type non-ts influenza B viruses were generated in eggs or MDCK cells and studied in order to identify the genes responsible for their ts phenotype. For each virus the exact genome composition was determined as well as its ts phenotype. The results demonstrated that the polymerase PB2 and PA gene segments of B/USSR/60/69 MDV independently controlled expression of the ts phenotype of B/USSR/60/69 MDV-based reassortant viruses. The other genes coding for internal proteins played no role in this respect. This suggests that mutations in the polymerase genes PB2 and PA play an essential role in attenuation of B/USSR/60/69 MDV-based reassortant influenza B vaccine viruses.

Cross-protective immunity in mice induced by live-attenuated or inactivated vaccines against highly pathogenic influenza A (H5N1) viruses.

Because of the time required to identify and produce an antigenically well-matched pandemic vaccine, vaccines that offer broader cross-reactive immunity and protection are desirable. We have compared a live attenuated influenza vaccine (LAIV) and inactivated influenza vaccine (IIV) based on a related H5 hemagglutinin (HA) from a nonpathogenic avian influenza virus, A/Duck/Pottsdam/1042-6/86 (H5N2), for the ability to induce cross-reactive immunity and/or cross-protective efficacy against a contemporary highly pathogenic H5N1 viruses. Both LAIV and IIV provided cross-protection from systemic infection, severe disease, and death following lethal challenges with antigenically distinct A/Vietnam/1203/2004 (VN/1203) virus. Substantial levels of serum anti-VN/1203 HA IgG were detected in mice that received either IIV or LAIV, while nasal wash anti-VN/1203 HA IgA was detected in mice that received LAIV. Formulation of IIV with alum adjuvant augmented neutralizing antibody responses and protective efficacy. These results demonstrated that vaccination of mice with H5 IIV or LAIV induced a high degree of cross-protection from illness and death following lethal challenges with a heterologous H5N1 virus.