Genotyping assay for differentiation of wild-type and vaccine viruses in subjects immunized with live attenuated influenza vaccine.

Live attenuated influenza vaccines (LAIVs) are considered as safe and effective tool to control influenza in different age groups, especially in young children. An important part of the LAIV safety evaluation is the detection of vaccine virus replication in the nasopharynx of the vaccinees, with special attention to a potential virus transmission to the unvaccinated close contacts. Conducting LAIV clinical trials in some geographical regions with year-round circulation of influenza viruses warrants the development of robust and reliable tools for differentiating vaccine viruses from wild-type influenza viruses in nasal pharyngeal wash (NPW) specimens of vaccinated subjects. Here we report the development of genotyping assay for the detection of wild-type and vaccine-type influenza virus genes in NPW specimens of young children immunized with Russian-backbone seasonal trivalent LAIV using Sanger sequencing from newly designed universal primers. The new primer set allowed amplification and sequencing of short fragments of viral genes in NPW specimens and appeared to be more sensitive than conventional real-time RT-PCR protocols routinely used for the detection and typing/subtyping of influenza virus in humans. Furthermore, the new assay is capable of defining the origin of wild-type influenza virus through BLAST search with the generated sequences of viral genes fragments.

Enhancement of Magnetic Resonance Imaging with Metasurfaces.

It is revealed that the unique properties of ultrathin metasurface resonators can improve magnetic resonance imaging dramatically. A metasurface formed when an array of metallic wires is placed inside a scanner under the studied object and a substantial enhancement of the radio-frequency magnetic field is achieved by means of subwavelength manipulation with the metasurface, also allowing improved image resolution.

Genetic stability of live attenuated vaccines against potentially pandemic influenza viruses.

BACKGROUND: Ensuring genetic stability is a prerequisite for live attenuated influenza vaccine (LAIV). This study describes the results of virus shedding and clinical isolates' testing of Phase I clinical trials of Russian LAIVs against potentially pandemic influenza viruses in healthy adults. METHODS: Three live attenuated vaccines against potentially pandemic influenza viruses, H2N2 LAIV, H5N2 LAIV and H7N3 LAIV, generated by classical reassortment in eggs, were studied. For each vaccine tested, subjects were randomly distributed into two groups to receive two doses of either LAIV or placebo at a 3:1 vaccine/placebo ratio. Nasal swabs were examined for vaccine virus shedding by culturing in eggs and by PCR. Vaccine isolates were tested for temperature sensitivity and cold-adaptation (ts/ca phenotypes) and for nucleotide sequence. RESULTS: The majority of nasal wash positive specimens were detected on the first day following vaccination. PCR method demonstrated higher sensitivity than routine virus isolation in eggs. None of the placebo recipients had detectable vaccine virus replication. All viruses isolated from the immunized subjects retained the ts/ca phenotypic characteristics of the master donor virus (MDV) and were shown to preserve all attenuating mutations described for the MDV. These data suggest high level of vaccine virus genetic stability after replication in humans. During manufacture process, no additional mutations occurred in the genome of H2N2 LAIV. In contrast, one amino acid change in the HA of H7N3 LAIV and two additional mutations in the HA of H5N2 LAIV manufactured vaccine lot were detected, however, they did not affect their ts/ca phenotypes. CONCLUSIONS: Our clinical trials revealed phenotypic and genetic stability of the LAIV viruses recovered from the immunized volunteers. In addition, no vaccine virus was detected in the placebo groups indicating the lack of person-to-person transmission. LAIV TRIAL REGISTRATION at ClinicalTrials.gov: H7N3-NCT01511419; H5N2-NCT01719783; H2N2-NCT01982331.

H2N2 live attenuated influenza vaccine is safe and immunogenic for healthy adult volunteers.

H2N2 influenza viruses have not circulated in the human population since 1968, but they are still being regularly detected in the animal reservoir, suggesting their high pandemic potential. To prepare for a possible H2N2 pandemic, a number of H2N2 vaccine candidates have been generated and tested in preclinical and clinical studies. Here we describe the results of a randomized, double-blind placebo-controlled phase 1 clinical trial of an H2N2 live attenuated influenza vaccine (LAIV) candidate prepared from a human influenza virus isolated in 1966. The vaccine candidate was safe and well-tolerated by healthy adults, and did not cause serious adverse events or an increased rate of moderate or severe reactogenicities. The H2N2 vaccine virus was infectious for Humans. It was shed by 78.6% and 74.1% volunteers after the first and second dose, respectively, most probably due to the human origin of the virus. Importantly, no vaccine virus transmission to unvaccinated subjects was detected during the study. We employed multiple immunological tests to ensure the adequate assessment of the H2N2 pandemic LAIV candidate and demonstrated that the majority (92.6%) of the vaccinated subjects responded to the H2N2 LAIV in one or more immunological tests, including 85.2% of subjects with antibody responses and 55.6% volunteers with cell-mediated immune responses. In addition, we observed strong correlation between the H2N2 LAIV virus replication in the upper respiratory tract and the development of antibody responses.

Contribution of neuraminidase of influenza viruses to the sensitivity to sera inhibitors and reassortment efficiency.

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

Assessment of human immune responses to H7 avian influenza virus of pandemic potential: results from a placebo-controlled, randomized double-blind phase I study of live attenuated H7N3 influenza vaccine.

INTRODUCTION: Live attenuated influenza vaccines (LAIVs) are being developed to protect humans against future epidemics and pandemics. This study describes the results of a double-blinded randomized placebo-controlled phase I clinical trial of cold-adapted and temperature sensitive H7N3 live attenuated influenza vaccine candidate in healthy seronegative adults. OBJECTIVE: The goal of the study was to evaluate the safety, tolerability, immunogenicity and potential shedding and transmission of H7N3 LAIV against H7 avian influenza virus of pandemic potential. METHODS AND FINDINGS: Two doses of H7N3 LAIV or placebo were administered to 40 randomly divided subjects (30 received vaccine and 10 placebo). The presence of influenza A virus RNA in nasal swabs was detected in 60.0% and 51.7% of subjects after the first and second vaccination, respectively. In addition, vaccine virus was not detected among placebo recipients demonstrating the absence of person-to-person transmission. The H7N3 live attenuated influenza vaccine demonstrated a good safety profile and was well tolerated. The two-dose immunization resulted in measurable serum and local antibody production and in generation of antigen-specific CD4⁺ and CD8⁺ memory T cells. Composite analysis of the immune response which included hemagglutinin inhibition assay, microneutralization tests, and measures of IgG and IgA and virus-specific T cells showed that the majority (86.2%) of vaccine recipients developed serum and/or local antibodies responses and generated CD4⁺ and CD8⁺ memory T cells. CONCLUSIONS: The H7N3 LAIV was safe and well tolerated, immunogenic in healthy seronegative adults and elicited production of antibodies broadly reactive against the newly emerged H7N9 avian influenza virus. TRIAL REGISTRATION: ClinicalTrials.gov NCT01511419.

Possible outcomes of reassortment in vivo between wild type and live attenuated influenza vaccine strains.

Reassortment of influenza viruses in nature has been well documented. Genetic reassortment plays a key role in emergence of new influenza A strains, including pandemic viruses. Permissive host can be simultaneously coinfected with multiple influenza viruses. During genetic reassortment gene segments are exchanged between parental viruses that may lead to some enhancement of virulence of reassortant progeny. At present, vaccination with live attenuated cold-adapted (ca) reassortant vaccine (LAIV) is used as an effective public health measure for influenza prophylaxis. However, there are concerns about a potential of simultaneous infection of human host with ca and wild type (wt) influenza viruses which might produce progeny that contain novel, more virulent genotypes. The aim of this study was to investigate potential consequences of reassortment of wt with LAIV strains in vivo. We demonstrated that reassortment of wt viruses with ca strains in guinea pigs have resulted in progeny virus which caused reduced macroscopic lesions of chicken embryos. According to phenotypical data 95% (19 out of 20) isolated reassortants were restricted in replication at elevated temperature of 40°C. None of reassortants were more virulent than wt parents, or revealed significantly higher macroscopic lesions than wt parental viruses. Our results suggest that genetic reassortment between wt and vaccine strain is unlikely to lead to virulent reassortant progeny. These findings provide additional support of LAIV safety data.