Safety and immunogenicity of the novel seasonal preservative‐ and adjuvant‐free influenza vaccine: Blind,randomized, and placebo‐controlled trial

The producers of influenza vaccines are not capable today to meet the global demand for an influenza vaccine in case of pandemic, so the World Health Organization recommends to develop the own influenza vaccine production in each country. A domestic preservative‐ and adjuvant‐free trivalent split vaccine against seasonal influenza was developed at the Research Institute for Biological Safety Problems. The paper presents the results of assessing safety and immunogenicity of the influenza split vaccine after single immunization of healthy volunteers aged 18‐50 years in the course of Phase I Clinical Trials. This study was randomized, blind, and placebo‐controlled. The volunteers were intramuscularly vaccinated with a dose of split vaccine or placebo. The study has shown that all local and systemic reactions had low degree of manifestation and short‐term character, so there was no need in medication. Serious side effects were not observed. On day 21 post vaccination the portion of vaccinated persons with fourfold seroconversions to influenza А/H1N1pdm09 virus was 100.0%, to influenza А/H3N2 virus—95.5%, to influenza B virus—81.8%, and in placebo group this index was 0%. Seroprotection rates against influenza А/H1N1pdm09, А/H3N2 and B viruses were 95.5, 86.3, and 72.7%, respectively. Geometric mean titers (GMT) of antibodies by day 21 post vaccination reached 175.7 for influenza А/H1N1pdm09 virus, 64.2 for influenza А/H3N2 virus, and 37.6 for influenza B virus; in placebo group GMT growth was not observed. So, the seasonal influenza split vaccine is well tolerated and fits all immunogenicity criteria for human influenza vaccines.     

Classical swine H1N1 influenza viruses confer cross protection from swine-origin 2009 pandemic H1N1 influenza virus infection in mice and ferrets

The hemagglutinin of the 2009 pandemic H1N1 influenza virus is a derivative of and is antigenically related to classical swine but not to seasonal human H1N1 viruses. We compared the A/California/7/2009 (CA/7/09) virus recommended by the WHO as the reference virus for vaccine development, with two classical swine influenza viruses A/swine/Iowa/31 (sw/IA/31) and A/New Jersey/8/1976 (NJ/76) to establish the extent of immunologic cross-reactivity and cross-protection in animal models. Primary infection with 2009 pandemic or NJ/76 viruses elicited antibodies against the CA/7/09 virus and provided complete protection from challenge with this virus in ferrets; the response in mice was variable and conferred partial protection. Although ferrets infected with sw/IA/31 virus developed low titers of cross-neutralizing antibody, they were protected from pulmonary replication of the CA/7/09 virus. The data suggest that prior exposure to antigenically related H1N1 viruses of swine-origin provide some protective immunity against the 2009 pandemic H1N1 virus.     

Antibody Breadth and Protective Efficacy Are Increased by Vaccination with Computationally Optimized Hemagglutinin but Not with Polyvalent Hemagglutinin-Based H5N1 Virus-Like Particle Vaccines

One of the challenges for developing an H5N1 influenza vaccine is the diversity of antigenically distinct isolates within this subtype. Previously, our group described a novel hemagglutinin (HA) derived from a methodology termed computationally optimized broadly reactive antigen (COBRA). This COBRA HA, when used as an immunogen, elicits a broad antibody response against H5N1 isolates from different clades. In this report, the immune responses elicited by the COBRA HA virus-like particle (VLP) vaccine were compared to responses elicited by a mixture of VLPs expressing representative HA molecules from clade 2.1, 2.2, and 2.3 primary H5N1 isolates (polyvalent). The COBRA HA VLP vaccine elicited higher-titer antibodies to a panel of H5N1 HA proteins than did the other VLPs. Both COBRA and polyvalent vaccines protected vaccinated mice and ferrets from experimental infection with highly lethal H5N1 influenza viruses, but COBRA-vaccinated animals had decreased viral replication, less inflammation in the lungs of mice, and reduced virus recovery in ferret nasal washes. Both vaccines had similar cellular responses postchallenge, indicating that higher-titer serum antibodies likely restrict the duration of viral replication. Furthermore, passively transferred immune serum from the COBRA HA VLP-vaccinated mice protected recipient animals more efficiently than immune serum from polyvalent-vaccinated mice. This is the first report comparing these two vaccine strategies. The single COBRA HA antigen elicited a broader antibody response and reduced morbidity and viral titers more effectively than a polyvalent mixture of primary H5N1 HA antigens.     

Immunogenicity of a Monovalent Pandemic Influenza A H1N1 Virus Vaccine with or without Prior Seasonal Influenza Vaccine Administration

The immunogenicity of pandemic influenza A H1N1 virus (A/H1pdm) vaccine might be modified by prior seasonal trivalent influenza vaccine (sTIV) administration. We conducted a retrospective analysis of immunogenicity of 243 health care workers (number of sTIV-positive [sTIV⁺] subjects, 216; number of sTIV⁻ subjects, 27) by hemagglutination inhibition. There was no significant difference in the ratios of antibody titers of ≥40 (41.2% versus 48.1%; P = 0.49) and fold increases in geometric mean titer (3.8 versus 4.5; P = 0.37). sTIV injected 7 to 10 days prior to A/H1pdm vaccine administration did not interfere with the immunogenicity of the latter.     

Increasing the immunogenicity of inactivated chitosan adjuvanted vaccine from A/California/7/09 (H1N1) strain and analyzing the antigenic specificity of this influenza virus strain

Addition of chitosan as an adjuvant to subunit vaccine from the swine origin influenza virus A/California/7/09 (H1N1) increases vaccine immunogenicity by 8-16 times and significantly enhances its protective potency. Single immunization with chitosan adjuvanted vaccine induced similar antibody titers as two immunizations with unadjuvanted vaccine. Chitosan stabilized the immunogenicity of subunit vaccine when stored at 4 degrees C. The antigenic specificity of the A/California/7/09 (H1N1) virus strain did not resemble substantially that of the human influenza strains A/Brisbane/59/07 (H1N1) and A/Solomon Isles/3/06 (H1N1), which are among the 2008/2009 and 2007/2008 seasonal influenza vaccines, respectively, as well as that of the human influenza H1N1 virus strains that circulated about 30 years ago.     

Intranasal immunization of ferrets with commercial trivalent influenza vaccines formulated in a nanoemulsion-based adjuvant

NB-1008 is a surfactant-stabilized soybean oil-in-water nanoemulsion (NE) adjuvant with influenza virus antigen incorporated into the NE by simple mixing. Intranasal administration of the antigen with NE adjuvant efficiently produces both mucosal and serum antibody responses as well as a robust cellular Th1 immune response. To demonstrate the adjuvant effect of the W₈₀5EC NE, a killed commercial influenza vaccine for intramuscular administration (Fluzone or Fluvirin) was mixed with the W₈₀5EC NE adjuvant and administered intranasally to naïve ferrets. After a single intranasal immunization, the adjuvanted influenza vaccine elicited elevated serum hemagglutination inhibition (HAI) geometric mean titers (GMTs) ranging from 196 to 905 for the three hemagglutinin (HA) antigens present in the vaccine, which are approximately 19- to 90-fold higher titers at 1/50 the standard intramuscular commercial nonadjuvanted influenza vaccine dose. Seroconversion rates of 67% to 100% were achieved against each of the three viral strains present. The adjuvanted nasal influenza vaccine also produced significant cross immunity to five other H3N2 influenza virus strains not present in the vaccine and produced sterile immunity after challenge with homologous live virus. No safety issues were observed in 249 ferrets receiving the adjuvanted influenza vaccine. These findings demonstrate the ability of W₈₀5EC NE to adjuvant nasally administered influenza vaccine and provide a basis for studying the intranasal W₈₀5EC-adjuvanted influenza vaccine in humans.     

Long-Term Immunogenicity of an Inactivated Split-Virion 2009 Pandemic Influenza A H1N1 Virus Vaccine with or without Aluminum Adjuvant in Mice

In 2009, a global epidemic of influenza A(H1N1) virus caused the death of tens of thousands of people. Vaccination is the most effective means of controlling an epidemic of influenza and reducing the mortality rate. In this study, the long-term immunogenicity of influenza A/California/7/2009 (H1N1) split vaccine was observed as long as 15 months (450 days) after immunization in a mouse model. Female BALB/c mice were immunized intraperitoneally with different doses of aluminum-adjuvanted vaccine. The mice were challenged with a lethal dose (10× 50% lethal dose [LD₅₀]) of homologous virus 450 days after immunization. The results showed that the supplemented aluminum adjuvant not only effectively enhanced the protective effect of the vaccine but also reduced the immunizing dose of the vaccine. In addition, the aluminum adjuvant enhanced the IgG antibody level of mice immunized with the H1N1 split vaccine. The IgG level was correlated to the survival rate of the mice. Aluminum-adjuvanted inactivated split-virion 2009 pandemic influenza A H1N1 vaccine has good immunogenicity and provided long-term protection against lethal influenza virus challenge in mice.     

Humans and Ferrets with Prior H1N1 Influenza Virus Infections Do Not Exhibit Evidence of Original Antigenic Sin after Infection or Vaccination with the 2009 Pandemic H1N1 Influenza Virus

The hypothesis of original antigenic sin (OAS) states that the imprint established by an individual''s first influenza virus infection governs the antibody response thereafter. Subsequent influenza virus infection results in an antibody response against the original infecting virus and an impaired immune response against the newer influenza virus. The purpose of our study was to seek evidence of OAS after infection or vaccination with the 2009 pandemic H1N1 (2009 pH1N1) virus in ferrets and humans previously infected with H1N1 viruses with various antigenic distances from the 2009 pH1N1 virus, including viruses from 1935 through 1999. In ferrets, seasonal H1N1 priming did not diminish the antibody response to infection or vaccination with the 2009 pH1N1 virus, nor did it diminish the T-cell response, indicating the absence of OAS in seasonal H1N1 virus-primed ferrets. Analysis of paired samples of human serum taken before and after vaccination with a monovalent inactivated 2009 pH1N1 vaccine showed a significantly greater-fold rise in the titer of antibody against the 2009 pH1N1 virus than against H1N1 viruses that circulated during the childhood of each subject. Thus, prior experience with H1N1 viruses did not result in an impairment of the antibody response against the 2009 pH1N1 vaccine. Our data from ferrets and humans suggest that prior exposure to H1N1 viruses did not impair the immune response against the 2009 pH1N1 virus.     

Immunization with a low dose of hemagglutinin-encoding plasmid protects against 2009 H1N1 pandemic influenza virus in mice

A vaccine against the novel pandemic influenza virus (2009 H1N1) is available, but several problems in preparation of vaccines against the new emerging influenza viruses need to be overcome. DNA vaccines represent a novel and powerful alternative to conventional vaccine approaches. To evaluate the ability of a DNA vaccine encoding the hemagglutinin (HA) of 2009 H1N1 to generate humoral responses and protective immunity, BALB/c mice were immunized with various doses of 2009 H1N1 HA-encoding plasmid and anti-HA total IgG, hemagglutination inhibition antibodies and neutralizing antibodies were assayed. The total IgG titers against HA correlated positively with the doses of DNA vaccine, but immunization with either a low dose (10 μg) or a higher dose (25-200 μg) of HA plasmid resulted in similar titers of hemagglutination inhibition and neutralizing antibodies, following a single booster. Further, 10 μg plasmid conferred effective protection against lethal virus challenge. These results suggested that the DNA vaccine encoding the HA of 2009 H1N1 virus is highly effective for inducing neutralizing antibodies and protective immunity. DNA vaccines are a promising new strategy for the rapid development of efficient vaccines to control new emerging pandemic influenza viruses.     

Protection against a lethal H5N1 influenza challenge by intranasal immunization with virus-like particles containing 2009 pandemic H1N1 neuraminidase in mice

Highly pathogenic H5N1 influenza shares the same neuraminidase (NA) subtype with the 2009 pandemic (H1N1pdm09), and cross-reactive NA immunity might protect against or mitigate lethal H5N1 infection. In this study, mice were either infected with a sublethal dose of H1N1pdm09 or were vaccinated and boosted with virus-like particles (VLP) consisting of the NA and matrix proteins, standardized by NA activity and administered intranasally, and were then challenged with a lethal dose of HPAI H5N1 virus. Mice previously infected with H1N1pdm09 survived H5N1 challenge with no detectable virus or respiratory tract pathology on day 4. Mice immunized with H5N1 or H1N1pdm09 NA VLPs were also fully protected from death, with a 100-fold and 10-fold reduction in infectious virus, respectively, and reduced pathology in the lungs. Human influenza vaccines that elicit not only HA, but also NA immunity may provide enhanced protection against the emergence of seasonal and pandemic viruses.     

A pandemic H1N1 influenza virus-like particle vaccine induces cross-protection in mice

Influenza virus-like particles (VLPs) represent promising alternative vaccines. However, it is necessary to demonstrate that influenza VLPs confer cross-protection against antigenically distinct viruses. In this study, a VLP vaccine comprising hemagglutinin (HA) and M1 from the A/California/04/2009 (H1N1) were used and its ability to induce cross-protective efficacy against heterologous viruses A/PR/8/34 (H1N1) and A/New Caledonia/20/99 (H1N1) in mice was assessed. Vaccination with 2009 H1 VLPs induced significantly higher levels of IgG cross-reactive with these heterologous viruses after the second boost compared to after the prime or first boost. Lung virus titers also decreased significantly and the lung cross-reactive IgG response after lethal virus challenge was significantly greater in immunized mice compared to naïve mice. Vaccinated mice showed 100% protection against A/PR/8/34 and A/Caledonia/20/99 viruses with only moderate body weight loss and induction of cross-reactive recall, IgG antibody-secreting cell responses. The variations in HA amino acid sequences and antigenic sites were determined and correlated with induction of cross-protective immunity. These results indicate that VLPs can be used as an effective vaccine that confers cross-protection against antigenically distinct viruses.     

Pandemic H1N1 influenza virus causes a stronger inflammatory response than seasonal H1N1 influenza virus in ferrets

A 2009 H1N1 influenza virus pandemic, which had its origin in swine, caused severe illness and mortality in humans. Inflammatory responses may be responsible for pathogenesis caused by infection with influenza viruses. To better understand the pathogenic mechanism, clinical signs and inflammatory responses in ferrets infected with the pandemic H1N1 were compared with those caused by seasonal H1N1 influenza virus. Ferrets infected with the 2009 pandemic H1N1 virus displayed higher body temperatures, greater reduction in body weight, and higher viral titers in the tracheae and lungs. Levels of inflammatory cytokines, including interleukin-6, interferon-alpha, and tumor necrosis factor-alpha, were higher in the lungs of ferrets infected with the 2009 pandemic H1N1. The data support the idea that increased pathogenesis caused by the 2009 pandemic H1N1 influenza virus may have been partially mediated by a higher induction of pro-inflammatory cytokines in the lungs of affected humans or animals.     

Molecular characterization of the influenza A(H1N1)pdm09 isolates collected in the 2015-2016 season and comparison of HA mutations detected in Turkey since 2009

Influenza A(H1N1)pdm09 pandemic virus causing the 2009 global outbreak moved into the post-pandemic period, but its variants continued to be the prevailing subtype in the 2015-2016 influenza season in Europe and Asia. To determine the molecular characteristics of influenza A(H1N1)pdm09 isolates circulating during the 2015-2016 season in Turkey, we identified mutations in the hemagglutinin (HA) genes and investigated the presence of H275Y alteration in the neuraminidase genes in the randomly selected isolates. The comparison of the HA nucleotide sequences revealed a very high homology (>99.5%) among the studied influenza A(H1N1)pdm09 isolates, while a relatively low homology (96.6%-97.2%), was observed between Turkish isolates and the A/California/07/2009 vaccine virus. Overall 14 common mutations were detected in HA sequences of all 2015-2016 influenza A(H1N1)pdm09 isolates with respect to the A/California/07/2009 virus, four of which located in three different antigenic sites. Eleven rare mutations in 12 HA sequences were also detected. Phylogenetic analysis revealed that all characterized influenza A(H1N1)pdm09 isolates formed a single genetic cluster, belonging to the genetic subclade 6B.1, defined by HA amino acid substitutions S84N, S162N, and I216T. Furthermore, all isolates showed an oseltamivir-sensitive genotype, suggesting that Tamiflu (Oseltamivir) could still be the drug of choice in Turkey.     

A novel monoclonal antibody effective against lethal challenge with swine-lineage and 2009 pandemic H1N1 influenza viruses in mice

The HA protein of the 2009 pandemic H1N1 viruses (H1N1pdm) is antigenically closely related to the HA of classical North American swine H1N1 influenza viruses (cH1N1). Since 1998, through mutation and reassortment of HA genes from human H3N2 and H1N1 influenza viruses, swine influenza strains are undergoing substantial antigenic drift and shift. In this report we describe the development of a novel monoclonal antibody (S-OIV-3B2) that shows high hemagglutination inhibition (HI) and neutralization titers not only against H1N1pdm, but also against representatives of the α, β, and γ clusters of swine-lineage H1 influenza viruses. Mice that received a single intranasal dose of S-OIV-3B2 were protected against lethal challenge with either H1N1pdm or cH1N1 virus. These studies highlight the potential use of S-OIV-3B2 as effective intranasal prophylactic or therapeutic antiviral treatment for swine-lineage H1 influenza virus infections.     

Influenza virus infections from 0 to 2 years of age: A birth cohort study

Background/purposeInfluenza vaccine has been recommended in Finland since 2007 for all children of 6–35 months of age and in 2009 for those ≥6 months against pandemic influenza. We investigated the incidence of influenza and vaccine effectiveness in a birth cohort of children in 2008–2011.MethodsWe followed 923 children from birth to 2 years of age for respiratory tract infections. A nasal swab sample for PCR for influenza A and B viruses was taken at the onset of acute respiratory infections. Samples were collected either at the study clinic or at home by parents. Vaccination data was retrieved from the health registries.ResultsVaccination coverage of children aged 6–23 months was 22–47% against seasonal influenza and 80% against the A(H1N1)pdm09 virus in the pandemic season 2009–2010. During 3 influenza seasons, 1607 nasal swab samples were collected. Influenza was confirmed in 56 (6.1%) of 923 children (16 A(H1N1), 14 A(H3N2), and 26 B viruses). The incidence of influenza was 5.1% in 2008–2009, 2.7% in 2009–2010, and 5.0% in 2010–2011. Effectiveness of the adjuvanted vaccine against the pandemic influenza A(H1N1)pdm09 was 97% (95% confidence interval, 76–100%). Three children with influenza were hospitalized.ConclusionThe yearly incidence of seasonal influenza was 5% in this cohort of very young children with variable influenza vaccine coverage. Adjuvanted vaccine against the pandemic influenza was highly effective. Both seasonal and pandemic influenza cases were mostly non-severe.     

Development of two types of rapid diagnostic test kits to detect the hemagglutinin or nucleoprotein of the swine-origin pandemic influenza A virus H1N1

Since its emergence in April 2009, pandemic influenza A virus H1N1 (H1N1 pdm), a new type of influenza A virus with a triple-reassortant genome, has spread throughout the world. Initial attempts to diagnose the infection in patients using immunochromatography (IC) relied on test kits developed for seasonal influenza A and B viruses, many of which proved significantly less sensitive to H1N1 pdm. Here, we prepared monoclonal antibodies that react with H1N1 pdm but not seasonal influenza A (H1N1 and H3N2) or B viruses. Using two of these antibodies, one recognizing viral hemagglutinin (HA) and the other recognizing nucleoprotein (NP), we developed kits for the specific detection of H1N1 pdm and tested them using clinical specimens of nasal wash fluid or nasopharyngeal fluid from patients with influenza-like illnesses. The specificities of both IC test kits were very high (93% for the HA kit, 100% for the NP kit). The test sensitivities for detection of H1N1 pdm were 85.5% with the anti-NP antibody, 49.4% with the anti-HA antibody, and 79.5% with a commercially available influenza A virus detection assay. Use of the anti-NP antibody could allow the rapid and accurate diagnosis of H1N1 pdm infections.     

唐山市甲型H1N1流感病毒血凝素基因序列分析

目的 分析2010—2016年唐山市甲型H1N1流感病毒血凝素(hemagglutinin,HA)基因序列进化特征.方法 选取唐山市3家哨点医院流感样病例分离到的24株甲型H1N1病毒,通过RT-PCR和测序方法获得HA基因的全长序列,运用分子生物学软件和统计学软件对序列进行拼接、比对和分析.结果 同源进化分析显示,24株甲型H1N1流感病毒HA基因与疫苗株A/California/7/2009的核苷酸和氨基酸的同源性分别为97.0%~99.0%和97.0%~98.5%.进化分析显示,2010—2016年唐山地区流行的甲型H1N1流感病毒属于1、7、6三个基因分支,其中6分支毒株分为6C、6B、6B.1和6B.2亚支.氨基酸位点分析显示,不同毒株与疫苗株比较存在8~16处氨基酸位点改变,其中7个变异涉及3个抗原表位:H138Q/Y和S203T突变位于Ca区,N125S、K153E、S162N、K163T/Q突变位于Sa区,S185T突变位于Sb区同时也位于受体结合部位;2015—2016流行季6B.1分支毒株抗原位点S162N突变增加了新的潜在糖基化位点.结论 与疫苗株比较,随着时间推移唐山地区甲型H1N1流感病毒发生了抗原漂变,未来仍应关注6B分支流行株的变化.     

Epidemiology and full genome sequence analysis of H1N1pdm09 from Northeast China

Pandemic influenza A (H1N1) 2009 virus (H1N1pdm09) was a novel tri-assortment virus that emerged in Mexico and North America in 2009 and caused the first influenza pandemic in the 21st century. This study investigated the prevalence pattern and molecular characteristics of H1N1pdm09 in three continuous years from April 2009 to March 2012 in populations of Tianjin, Northeast China. Totally, 3,068 influenza viruses (25.4 %) were detected from 12,089 respiratory specimens. Among them, 41.4 % (1,269/3,068) were H1N1pdm09 positive. 15.1 % (192/1,269) severe respiratory infection cases were H1N1pdm09 positive. H1N1pdm09 was the predominant prevalence subtype in October 2009–March 2010 (69.1 %, 930/1,346) and October 2010–March 2011 (42.1 %, 220/523). Eight isolated H1N1pdm09 viruses from severe infection/death cases in three different years were selected to sequence the whole genome through splicing the sequences following 46 PCRs. HA sequences of seven H1N1pdm09 isolates from mild infection cases were detected. Phylogenetic analysis showed that HA, NA, M, NP and NS genes of H1N1pdm09 viruses gathered together with swine influenza A (H1N1), whereas PB2 and PA genes originated from avian influenza virus, and PB1 gene originated from human seasonal influenza virus. Identity analysis indicated that all the genes were highly conserved. Compared with vaccine strain A/California/07/2009(H1N1), the maximal mutation gene was HA (0.7–2.6 %), then NA (0.6–1.7 %), last one was M (mutation rate 0–0.6 %). More site substitutions were observed in 2011 isolates than in 2009 and 2010 isolates of HA (p = 0.002), NA (p = 0.003) and PA (p = 0.001) proteins. The amino acid substitution rates were varied among eight gene segments, ranging from 7.39 × 10^?4 for PB2 to 7.40 × 10^?3 for NA. The higher d _N / d _S rates were observed in HA, PA and NS segments in H1N1pdm09 in Tianjin. Three HA amino acid site substitutions occurred at the HA receptor-binding sites and antigenic determinant, including S179N and K180T (located at antigenic site Sa) in A/Tianjinhedong/SWL44/2011(H1) and A/Tianjinjinnan/SWL41/2011(H1), and D239N (located at antigenic site Ca) in A/Tianjinninghe/SWL49/2009(H1). Antigenic drift may have occurred in H1N1pdm09 with time. No oseltamivir-resistance site substitution was observed at 275 and 295 sites. Amino acid residue site at 31 in M2 protein was N in all 8 isolates, which suggested that H1N1pdm09 was resistant to amantadine.