Longitudinal Analysis of Neuraminidase and Hemagglutinin Antibodies to Influenza A Viruses after Immunization with Seasonal Inactivated Influenza Vaccines.

Neuraminidase (NA)-based immunity could reduce the harmful impact of novel antigenic variants of influenza viruses. The detection of neuraminidase-inhibiting (NI) antibodies in parallel with anti-hemagglutinin (HA) antibodies may enhance research on the immunogenicity and duration of antibody responses to influenza vaccines. To assess anti-NA antibodies after vaccination with seasonal inactivated influenza vaccines, we used the enzyme-linked lectin assay, and anti-HA antibodies were detected in the hemagglutination inhibition assay. The dynamics of the anti-NA antibody response differed depending on the virus subtype: antibodies to A/H3N2 virus neuraminidase increased later than antibodies to A/H1N1pdm09 subtype neuraminidase and persisted longer. In contrast to HA antibodies, the fold increase in antibody titers to NA after vaccination poorly depended on the preexisting level. At the same time, NA antibody levels after vaccination directly correlated with titers before vaccination. A difference was found in response to NA antigen between split and subunit-adjuvanted vaccines and in NA functional activity in the vaccine formulations.

Assessing the Intense Influenza A(H1N1)pdm09 Epidemic and Vaccine Effectiveness in the Post-COVID Season in the Russian Federation.

The COVID-19 pandemic had a profound impact on influenza activity worldwide. However, as the pandemic progressed, influenza activity resumed. Here, we describe the influenza epidemic of high intensity of the 2022-2023 season. The epidemic had an early start and peaked in week 51.2022. The extremely high intensity of the epidemic may have been due to a significant decrease in herd immunity. The results of PCR-testing of 220,067 clinical samples revealed that the influenza A(H1N1)pdm09 virus dominated, causing 56.4% of positive cases, while A(H3N2) influenza subtype accounted for only 0.6%, and influenza B of Victoria lineage-for 34.3%. The influenza vaccine was found to be highly effective, with an estimated effectiveness of 92.7% in preventing admission with laboratory-confirmed influenza severe acute respiratory illness (SARI) cases and 54.7% in preventing influenza-like illness/acute respiratory illness (ILI/ARI) cases due to antigenic matching of circulated viruses with influenza vaccine strains for the season. Full genome next-generation sequencing of 1723 influenza A(H1N1)pdm09 viruses showed that all of them fell within clade 6B.1A.5.a2; nine of them possessed H275Y substitution in the NA gene, a genetic marker of oseltamivir resistance. Influenza A(H3N2) viruses belonged to subclade 3C.2a1b.2a.2 with the genetic group 2b being dominant. All 433 influenza B viruses belonged to subclade V1A.3a.2 encoding HA1 substitutions A127T, P144L, and K203R, which could be further divided into two subgroups. None of the influenza A(H3N2) and B viruses sequenced had markers of resistance to NA inhibitors. Thus, despite the continuing circulation of Omicron descendant lineages, influenza activity has resumed in full force, raising concerns about the intensity of fore coming seasonal epidemics.

Characterization of a Panel of Monoclonal Antibodies Targeting the F-Protein of the Respiratory Syncytial Virus (RSV) for the Typing of Contemporary Circulating Strains.

Respiratory syncytial virus (RSV) is the most common cause of upper and lower respiratory tract infections in infants and young children. Virus-specific monoclonal antibodies (mAbs) can be used for diagnosis, prophylaxis, and research of RSV pathogenesis. A panel of 16 anti-RSV mAbs was obtained from mice immunized by RSV strain Long. Half of them had virus-neutralizing activity. According to Western blot all of these mAbs effectively bound native oligomeric (homodimeric and homotrimeric) forms of the RSV fusion (F) protein. Only five of the mAbs interacted with the monomeric form, and only one of these possessed neutralizing activity. None of these mAbs, nor the commercial humanized neutralizing mAb palivizumab, reacted with the denaturated F protein. Thus, interaction of all these mAbs with F protein had clear conformational dependence. Competitive ELISA and neutralization assays allowed the identification of nine antigenic target sites for the interaction of mAb with the F protein. Five partially overlapping sites may represent a complex spatial structure of one antigenic determinant, including one neutralizing and four non-neutralizing epitopes. Four sites (three neutralizing and one non-neutralizing) were found to be distinct. As a result of virus cultivation RSV-A, strain Long, in the presence of a large amount of one of the neutralizing mAbs, an escape mutant with a substitution, N240S, in the F protein, was obtained. Thus, it was shown for the first time that position 240 is critical for the protective effect of an anti-RSV antibody. To assess the ability of these mAbs to interact with modern RSV strains circulating in St. Petersburg (Russia) between 2014 and 2022, 73 RSV-A and 22 RSV-B isolates were analyzed. Six mAbs were directed to conserved epitopes of the F protein as they interacted most efficiently with both RSV subtypes in a fixed cell-ELISA and could be used for diagnostic assays detecting RSV.

Respiratory Syncytial Virus G Protein Sequence Variability among Isolates from St. Petersburg, Russia, during the 2013-2014 Epidemic Season.

Human respiratory syncytial virus (RSV) is the most common cause of upper and lower respiratory tract infections in infants and young children. It is actively evolving under environmental and herd immunity influences. This work presents, for the first time, sequence variability analysis of RSV G gene and G protein using St. Petersburg (Russia) isolates. Viruses were isolated in a cell culture from the clinical samples of 61 children hospitalized (January-April 2014) with laboratory-confirmed RSV infection. Real-time RT-PCR data showed that 56 isolates (91.8%) belonged to RSV-A and 5 isolates (8.2%) belonged to RSV-B. The G genes were sequenced for 27 RSV-A isolates and all of them belonged to genotype ON1/GA2. Of these RSV-A, 77.8% belonged to the ON1(1.1) genetic sub-cluster, and 14.8% belonged to the ON1(1.2) sub-cluster. The ON1(1.3) sub-cluster constituted a minor group (3.7%). Many single-amino acid substitutions were identified in the G proteins of St. Petersburg isolates, compared with the Canadian ON1/GA2 reference virus (ON67-1210A). Most of the amino acid replacements were found in immunodominant B- and T-cell antigenic determinants of G protein. These may affect the antigenic characteristics of RSV and influence the host antiviral immune response to currently circulating viruses.

Cross-Protective Efficacy of Monovalent Live Influenza B Vaccines against Genetically Different Lineages of B/Victoria and B/Yamagata in Ferrets.

BACKGROUND: Currently, two genetic lineages of influenza B virus, B/Victoria and B/Yamagata, are cocirculating in humans in various countries. This situation has raised a question regarding the possibility of cross-protection between B components of live attenuated influenza vaccine (LAIV) belonging to different lineages. This study aimed to assess in naïve ferrets the potential protective activity of monovalent B-LAIVs against challenge with homologous and heterologous wild-type (WT) influenza B viruses. METHODS: Groups of seronegative female ferrets 5-6 months of age were given one dose of monovalent LAIV based on B/Victoria or B/Yamagata lineage virus. Ferrets were challenged 21 days later with B/Victoria or B/Yamagata WT virus. Ferrets were monitored closely for clinical signs and morbidity outcomes including febrile response, body weight loss, nasal symptoms, and level of activity one week prior to vaccination and for three days following vaccination/challenge. Nasal washes were collected three days after vaccination/challenge. Samples of lung tissue were taken three days after challenge. All samples were analyzed for the presence of challenge virus by culturing in embryonated chicken eggs and real-time polymerase chain reaction. Antibody response to vaccination was assessed by routine hemagglutination inhibition assay and microneutralization test. RESULTS: Vaccination led to intensive production of specific neutralizing and antihemagglutinating antibodies to vaccine virus, protected ferrets from homologous challenge infection, and significantly reduced clinical signs and replication of homologous challenge virus. In contrast, cross-lineage serum antibodies were not detected. However, ferrets vaccinated with monovalent B-LAIV had a significantly lower level of heterologous challenge virus in the respiratory tract than those given challenge virus only. CONCLUSIONS: Monovalent B-LAIV has the potential to be cross-protective against infection with genetically different influenza lineages. Further studies are required to confirm this effect.

Clinical testing of pre-pandemic live attenuated A/H5N2 influenza candidate vaccine in adult volunteers: results from a placebo-controlled, randomized double-blind phase I study.

BACKGROUND: This study describes a double-blinded randomized placebo-controlled phase I clinical trial of A/H5N2 live attenuated influenza vaccine in healthy volunteers. METHODS: Two doses of vaccine or placebo were administered intranasally to 30 and 10 subjects, respectively. Nasal swabs were examined for vaccine shedding and local antibody responses; serum samples were tested for binding, hemagglutinating and neutralizing antibodies and peripheral blood mononuclear cells were tested for cell-mediated immune responses. RESULTS: The vaccine was well tolerated and not associated with increased rates of adverse events or the occurrence of serious adverse events. Influenza virus was detected in nasal swabs on the first day in the majority of volunteers (93%), while 17% of volunteers tested positive on the second, none on the third day or later following the first vaccination; lower frequency of shedding was observed after the second vaccination. The vaccine was immunogenic as assessed four weeks after the second dose, with 37.9% and 48.3% of subjects seroconverting by hemagglutination inhibition and neutralization assays, respectively. An immune response was observed in 96.6% subjects that received A/H5N2 LAIV in at least one of the assays conducted. None of the placebo recipients exhibited a response in any of the assays. CONCLUSION: The A/H5N2 vaccine was safe, well tolerated, and immunogenic in healthy adults. TRIAL REGISTRATION: ClinicalTrials.gov NCT01719783.

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.

An inactivated, adjuvanted whole virion clade 2.2 H5N1 (A/Chicken/Astana/6/05) influenza vaccine is safe and immunogenic in a single dose in humans.

In this study, we assessed in humans the immunogenicity and safety of one dose (7.5 or 15 µg of hemagglutinin [HA]) of a whole-virion inactivated prepandemic influenza vaccine adjuvanted with aluminum hydroxide. The vaccine strain was made by reverse genetics from the highly pathogenic avian A/Chicken/Astana/6/05 (H5N1) clade 2.2 strain isolated from a dead bird in Kazakhstan. The humoral immune response was evaluated after a single vaccination by hemagglutination inhibition (HI) and microneutralization (MN) assays. The vaccine was safe and immunogenic, inducing seroconversion in 55% of the evaluated patients, with a geometric mean titer (GMT) of 17.1 and a geometric mean increase (GMI) of 3.42 after a dose of 7.5 µg in the HI test against the vaccine strain. The rate of seroconversion increased up to 70% when the dose of 15 µg was used. The percentages of individuals achieving anti-HA titers of ≥1:40 were 52.5% and 57.5% for the 7.5- and 15-µg dose groups, respectively. Similar results were obtained when antibodies were analyzed in an MN test. Substantial cross-neutralization titers (seroconversion in 35% and 52.5% of subjects in the two dose groups, respectively) were detected against heterologous clade 1 strain NIBRG14 (H5N1). Thus, one dose of this whole-virion prepandemic vaccine adjuvanted with aluminum has the potential to be effective against H5N1 viruses of different clades.