Live Attenuated Influenza Vaccine Strains Research Articles

Pathobiology of an NS1-Truncated H3N2 Swine Influenza Virus Strain in Pigs.

ABSTRACTVirus strains in the live attenuated influenza vaccine (LAIV) for swine in the United States that was on the market until 2020 encode a truncated nonstructural protein 1 of 126 amino acids (NS1del126). Their attenuation is believed to be due to an impaired ability to counteract the type I interferon (IFN)-mediated antiviral host response. However, this mechanism has been documented only in vitro for H3N2 strain A/swine/Texas/4199-2/98 NS1del126 (lvTX98), and several cases of clinical respiratory disease in the field were associated with the LAIV strains. We therefore further examined the pathobiology, including type I IFN induction, of lvTX98 in pigs and compared it with IFN induction in pig kidney-15 (PK-15) cells. lvTX98 induced up to 3-fold-higher type I IFN titers than wild-type TX98 (wtTX98) after inoculation of PK-15 cells at a high multiplicity of infection, while virus replication kinetics were similar. Mean nasal lvTX98 excretion by intranasally inoculated pigs was on average 50 times lower than that for wtTX98 but still reached titers of up to 4.3 log10 50% tissue culture infective doses/mL. After intratracheal inoculation, mean lvTX98 titers in the lower respiratory tract were significantly reduced at 18 to 48 h postinoculation (hpi) but similar to wtTX98 titers at 72 hpi. lvTX98 caused milder clinical signs than wtTX98 but induced comparable levels of microscopic and macroscopic lung lesions, peak neutrophil infiltration, and peak type I IFN. Thus, lvTX98 was partly attenuated in pigs, but this could not be associated with higher type I IFN levels.IMPORTANCE Swine influenza A viruses (swIAVs) with a truncated NS1del126 protein were strongly attenuated in previous laboratory-based safety studies and therefore approved for use as LAIVs for swine in the United States. In the field, however, the LAIV strains were detected in diagnostic samples and could regain a wild-type NS1 via reassortment with endemic swIAVs. This suggests a significant degree of LAIV replication and urges further investigation of the level and mechanism of attenuation of these LAIV strains in vivo. Here, we show that H3N2 LAIV strain lvTX98 is only partly attenuated in pigs and is excreted at significant titers after intranasal vaccination. Attenuation and restricted replication of lvTX98 in vivo seemed to be associated with the loss of NS1 functions other than type I IFN antagonism. Our findings can help to explain the occurrence of clinical respiratory disease and reassortment events associated with NS1del126-based LAIV strains in the field.

Generation and in vitro characterization of engineered cold-adapted influenza A strains with modified NS gene

BACKGROUND: The high variability of influenza strains and the emergence of new variants of viruses lead to the need for constant updating of the composition of influenza vaccines. One of the options for solving this problem is the development of vaccines with enhanced cross-protection against a wide range of influenza strains. Genetically engineered preparations based on live influenza vaccine can be used for targeted stimulation of the cellular immune response. It has been experimentally established that CTL epitopes inserted into the NS gene of the live influenza vaccine strain cause the activation of lymphocytes and the formation of a pool of resident memory T-cells in the lungs of model animals. It is optimal to use experimentally confirmed immunogenic regions for insertion.
 AIM: The aim of this study was to rescue a panel of experimental cold-adapted live attenuated influenza vaccine strains with a modified NS gene using A/Leningrad/134/17/57 backbone and recent influenza strains of H1N1, H3N2 and H7N9 subtypes, and evaluate their properties in vitro.
 MATERIALS AND METHODS: A cassette encoding immunogenic, conserved among a wide range of influenza strains T-cell epitopes of the influenza virus PB1 protein restricted by common HLA-allotypes was inserted into the gene encoding the NS1 protein. The modified NS gene was cloned into the pCIPolISapIT influenza virus reverse genetics vector. Chimeric influenza viruses were rescued by transfection of Vero cells by electroporation using a standard 8-plasmid system. The growth characteristics of viruses were assessed in developing chicken embryos.
 Results: Three strains were successfully obtained based on the live influenza vaccine master donor virus A/Leningrad/ 134/17/57 with a modified NS gene and influenza viruses of the H1N1, H3N2, H7N9 subtypes. Thus, modification of NS gene by insertion of immunogenic PB1 epitopes did not affect the viability and replicative activity of the rescued chimeric live influenza vaccine strains, regardless of the composition of the surface proteins. The strains replicated well at an optimal temperature, had temperature-sensitive phenotype and were able to grow at low temperature.
 CONCLUSIONS: The strains will be further studied as candidates for influenza prophylaxis as an experimental universal influenza vaccine.

Presence of defective viral genes in H1N1 live attenuated influenza vaccine strains is not associated with reduced human cell fitness or vaccine effectiveness

In the 2013–2014 and 2015–2016 seasons, quadrivalent live attenuated influenza vaccine (LAIV) showed reduced pandemic 2009 H1N1 (A/H1N1pdm09) vaccine effectiveness (VE) in the U.S. Impaired fitness of A/H1N1pdm09 LAIV strains in primary human nasal epithelial cells (hNEC) was subsequently shown to be associated with reduced VE. As defective viral genes (DVG) have been detected in QLAIV, it was hypothesised that these might play a role in reduced A/H1N1pdm09 fitness. By applying RT-PCR based approaches, DVG for PB2, PB1 and PA segments were detected in all influenza A LAIV strains tested. Absolute quantification of PA vRNA as a biomarker, using a novel digital RT-PCR assay (RT-dPCR), showed that DVG were a minority population (between 10.2 and 27.8 % of PA vRNA) in LAIV, irrespective of subtype or VE. Importantly, no difference was observed between the fitter pre-2009 H1N1 A/New Caledonia/20/1999 (A/NC99) and less fit A/H1N1pdm09 A/Bolivia/509/2013 (A/BOL13), containing medians of 16.0 % and 10.2 % PA DVG, respectively. Manipulating propagation conditions and minimising A/BOL13 PA DVG to 5.2 % failed to improve viral replication in hNEC, suggesting DVG were not limiting A/BOL13 fitness. Conversely, DVG were able to reduce A/NC99 replication in hNEC to A/BOL13-like levels, but only by enrichment of PA DVG to 66.5 % of vRNA. Notably, this required LAIV propagation under conditions markedly different to those used for vaccine production. We conclude from these data that abundance of DVG in QLAIV is not associated with variations in influenza A VE and that the reduced fitness of A/BOL13 previously described was not driven by the presence of DVG.

The effect of live attenuated influenza vaccine on pneumococcal colonisation densities among children aged 24–59 months in The Gambia: a phase 4, open label, randomised, controlled trial

SummaryBackgroundInfluenza and other respiratory viruses promote Streptococcus pneumoniae proliferation in the upper respiratory tract. We sought to investigate for what we believe is the first time, the effect of intranasal live attenuated influenza vaccine (LAIV) on nasopharyngeal S pneumoniae density in a low-income to middle-income country population with high pneumococcal carriage rates.MethodsIn an open-label, randomised, controlled trial in The Gambia, 330 healthy children aged 24–59 months were randomly assigned 2:1 to receive one trivalent LAIV dose at enrolment (day 0, intervention) or at the end of active follow-up (day 21, control). The investigator team were initially masked to block size and randomisation sequence to avoid allocation bias. Group allocation was later revealed to the investigator team. The primary outcome was PCR-quantified day 7 and 21 pneumococcal density. Asymptomatic respiratory viral infection at baseline and LAIV strain shedding were included as covariates in generalised mixed-effects models, to assess the effect of LAIV and other variables on pneumococcal densities. The study is registered at ClinicalTrials.gov, NCT02972957, and is closed to recruitment.FindingsBetween Feb 8 and April 12, 2017, and Jan 15 and March 28, 2018, of 343 children assessed for eligibility, 213 in the intervention group and 108 in the control group completed the study and were included in the final analysis. Although no significant differences were seen in pneumococcal carriage or density at each timepoint when comparing groups, changes from baseline were observed in the LAIV group. The baseline S pneumoniae carriage prevalence was high in both LAIV and control groups (75%) and increased by day 21 in the LAIV group (85%, p=0·0037), but not in the control group (79%, p=0·44). An increase in pneumococcal density from day 0 amounts was seen in the LAIV group at day 7 (+0·207 log10 copies per μL, SE 0·105, p=0·050) and day 21 (+0·280 log10 copies per μL, SE 0·105, p=0·0082), but not in the control group. Older age was associated with lower pneumococcal density (−0·015 log10 copies per μL, SE 0·005, p=0·0030), with the presence of asymptomatic respiratory viruses at baseline (+0·259 log10 copies per μL, SE 0·097, p=0·017), and greater LAIV shedding at day 7 (+0·380 log10 copies per μL, SE 0·167, p=0·024) associated with higher pneumococcal density. A significant increase in rhinorrhoea was reported in the LAIV group compared with the control group children during the first 7 days of the study (103 [48%] of 213, compared with 25 [23%] of 108, p<0·0001), and between day 7 and 21 (108 [51%] of 213, compared with 28 [26%] of 108, p<0·0001).InterpretationLAIV was associated with a modest increase in nasopharyngeal pneumococcal carriage and density in the 21 days following vaccination, with the increase in density lower in magnitude than previously described in the UK. This increase was accelerated when LAIV was administered in the presence of pre-existing asymptomatic respiratory viruses, suggesting that nasopharyngeal S pneumoniae proliferation is driven by cumulative mixed-viral co-infections. The effect of LAIV on pneumococcal density is probably similar to other respiratory viral infections in children. Our findings provide reassurance for the use of LAIV to expand influenza vaccine programmes in low-income to middle-income country populations with high pneumococcal carriage.FundingWellcome Trust.

Universal Live-Attenuated Influenza Vaccine Candidates Expressing Multiple M2e Epitopes Protect Ferrets against a High-Dose Heterologous Virus Challenge.

The development of an influenza vaccine with broad protection and durability remains an attractive idea due to the high mutation rate of the influenza virus. An extracellular domain of Matrix 2 protein (M2e) is among the most attractive target for the universal influenza vaccine owing to its high conservancy rate. Here, we generated two recombinant live attenuated influenza vaccine (LAIV) candidates encoding four M2e epitopes representing consensus sequences of human, avian and swine influenza viruses, and studied them in a preclinical ferret model. Both LAIV+4M2e viruses induced higher levels of M2e-specific antibodies compared to the control LAIV strain, with the LAIV/HA+4M2e candidate being significantly more immunogenic than the LAIV/NS+4M2e counterpart. A high-dose heterosubtypic influenza virus challenge revealed the highest degree of protection after immunization with LAIV/HA+4M2e strain, followed by the NS-modified LAIV and the classical LAIV virus. Furthermore, only the immune sera from the LAIV/HA+4M2e-immunized ferrets protected mice from a panel of lethal influenza viruses encoding M genes of various origins. These data suggest that the improved cross-protection of the LAIV/HA+4M2e universal influenza vaccine candidate was mediated by the M2e-targeted antibodies. Taking into account the safety profile and improved cross-protective potential, the LAIV/HA+4M2e vaccine warrants its further evaluation in a phase I clinical trial.

Development of a Genetically Stable Live Attenuated Influenza Vaccine Strain Using an Engineered High-Fidelity Viral Polymerase.

RNA viruses demonstrate a vast range of variants, called quasispecies, due to error-prone replication by viral RNA-dependent RNA polymerase. Although live attenuated vaccines are effective in preventing RNA virus infection, there is a risk of reversal to virulence after their administration. To test the hypothesis that high-fidelity viral polymerase reduces the diversity of influenza virus quasispecies, resulting in inhibition of reversal of the attenuated phenotype, we first screened for a high-fidelity viral polymerase using serial virus passages under selection with a guanosine analog ribavirin. Consequently, we identified a Leu66-to-Val single amino acid mutation in polymerase basic protein 1 (PB1). The high-fidelity phenotype of PB1-L66V was confirmed using next-generation sequencing analysis and biochemical assays with the purified influenza viral polymerase. As expected, PB1-L66V showed at least two-times-lower mutation rates and decreased misincorporation rates, compared to the wild type (WT). Therefore, we next generated an attenuated PB1-L66V virus with a temperature-sensitive (ts) phenotype based on FluMist, a live attenuated influenza vaccine (LAIV) that can restrict virus propagation by ts mutations, and examined the genetic stability of the attenuated PB1-L66V virus using serial virus passages. The PB1-L66V mutation prevented reversion of the ts phenotype to the WT phenotype, suggesting that the high-fidelity viral polymerase could contribute to generating an LAIV with high genetic stability, which would not revert to the pathogenic virus.IMPORTANCE The LAIV currently in use is prescribed for actively immunizing individuals aged 2 to 49 years. However, it is not approved for infants and elderly individuals, who actually need it the most, because it might prolong virus propagation and cause an apparent infection in these individuals, due to their weak immune systems. Recently, reversion of the ts phenotype of the LAIV strain currently in use to a pathogenic virus was demonstrated in cultured cells. Thus, the generation of mutations associated with enhanced virulence in LAIV should be considered. In this study, we isolated a novel influenza virus strain with a Leu66-to-Val single amino acid mutation in PB1 that displayed a significantly higher fidelity than the WT. We generated a novel LAIV candidate strain harboring this mutation. This strain showed higher genetic stability and no ts phenotype reversion. Thus, our high-fidelity strain might be useful for the development of a safer LAIV.

Evaluation of manufacturing feasibility and safety of an MDCK cell-based live attenuated influenza vaccine (LAIV) platform

Cell culture based live attenuated influenza vaccines (LAIV) as an alternative to egg-based LAIV have been explored because of lack of easy access to SPF eggs for large scale production. In this study, feasibility of MDCK platform was assessed by including multiple LAIV strains covering both type A (H1 and H3) and type B seasonal strains as well as the candidate pandemic potential strains like A/H5 and A/H7 for the growth in MDCK cells. A risk assessment study was conducted on the cell banks to evaluate safety concerns related to tumorigenicity with a regulatory perspective. Tumorigenic potential of the MDCK cells was evaluated in nude mice (107cells/mouse) model system. The 50% tumor producing dose (TPD50) of MDCK cells was studied in SCID mice to determine the amount of cells required for induction of tumors. Further, we conducted an oncogenicity study in three sensitive rodent species as per the requirements specified in the WHO guidelines.We determined TPD50 value of 1.9 X 104 cells/mice through subcutaneous route. Our results suggest that, the intranasal route of administration of the cell culture based LAIV pose minimal to no risk of tumorigenicity associated with the host cells. Also, non-oncogenic nature of MDCK cells was demonstrated. Host cell DNA in the vaccine formulations was < 10 ng/dose which ensures vaccine safety. Production efficiency and consistency were characterized and the observed titer values of the viral harvest and the processed bulk were comparable to the expansion in embryonated eggs.The present study clearly establishes the suitability of MDCK cells as a substrate for the manufacture of a safe and viable LAIV.

A study to evaluate the immunogenicity and shedding of live attenuated influenza vaccine strains in children 24–

BackgroundQuadrivalent live attenuated influenza vaccine (LAIV4) showed reduced effectiveness against the A/H1N1 component in the 2013–2014 and 2015–2016 influenza seasons. The most likely cause of reduced LAIV effectiveness against A(H1N1)pdm09 strains was poor intranasal replication. ObjectivesTo compare the immunogenicity and shedding of a new A/H1N1 strain (A/Slovenia), to a A/H1N1 strain known to have reduced effectiveness (A/Bolivia). Patients/methodsThis was a randomized, double-blind, multicenter study. Children aged 24–<48 months of age were randomized 1:1:1 to receive two doses of LAIV4 2017–2018 (LAIV4A/Slovenia), or LAIV4 2015–2016 or trivalent LAIV (LAIV3) 2015–2016 formulations (LAIV4A/Bolivia or LAIV3A/Bolivia, respectively) on days 1 and 28. The primary endpoint was strain-specific hemagglutination inhibition (HAI) antibody seroresponse at 28 days post each dose, and secondary endpoints included immunogenicity, shedding, and safety. Solicited symptoms, adverse events (AEs), and serious AEs (SAEs) were recorded. Pre-specified statistical testing was limited to the primary endpoint of HAI antibody responses. ResultsA total of 200 children were randomized (median age 35.3 months; 53% male; 57% had previously received influenza vaccine). Significantly higher HAI antibody responses for the A/Slovenia strain were observed after Dose 1 and Dose 2. Neutralizing antibodies and nasal immunoglobulin A antibody responses were higher for A/Slovenia versus A/Bolivia. More children shed the A/Slovenia vaccine strain than the A/Bolivia strain on Days 4–7 after Dose 1. No deaths, SAEs, or discontinuations from vaccine occurred. ConclusionsThe new A(H1N1)pdm09 A/Slovenia LAIV strain demonstrated improved immunogenicity compared with a previous strain with reduced effectiveness and induced immune responses comparable to a highly efficacious pre-pandemic H1N1 LAIV strain. These results support the use of LAIV4 containing A/Slovenia as a vaccine option in clinical practice.

Effect of a Russian-backbone live-attenuated influenza vaccine with an updated pandemic H1N1 strain on shedding and immunogenicity among children in The Gambia: an open-label, observational, phase 4 study

SummaryBackgroundThe efficacy and effectiveness of the pandemic H1N1 (pH1N1) component in live attenuated influenza vaccine (LAIV) is poor. The reasons for this paucity are unclear but could be due to impaired replicative fitness of pH1N1 A/California/07/2009-like (Cal09) strains. We assessed whether an updated pH1N1 strain in the Russian-backbone trivalent LAIV resulted in greater shedding and immunogenicity compared with LAIV with Cal09.MethodsWe did an open-label, prospective, observational, phase 4 study in Sukuta, a periurban area in The Gambia. We enrolled children aged 24–59 months who were clinically well. Children received one dose of the WHO prequalified Russian-backbone trivalent LAIV containing either A/17/California/2009/38 (Cal09) or A/17/New York/15/5364 (NY15) based on their year of enrolment. Primary outcomes were the percentage of children with LAIV strain shedding at day 2 and day 7, haemagglutinin inhibition seroconversion, and an increase in influenza haemagglutinin-specific IgA and T-cell responses at day 21 after LAIV. This study is nested within a randomised controlled trial investigating LAIV–microbiome interactions (NCT02972957).FindingsBetween Feb 8, 2017, and April 12, 2017, 118 children were enrolled and received one dose of the Cal09 LAIV from 2016–17. Between Jan 15, 2018, and March 28, 2018, a separate cohort of 135 children were enrolled and received one dose of the NY15 LAIV from 2017–18, of whom 126 children completed the study. Cal09 showed impaired pH1N1 nasopharyngeal shedding (16 of 118 children [14%, 95% CI 8·0–21·1] with shedding at day 2 after administration of LAIV) compared with H3N2 (54 of 118 [46%, 36·6–55·2]; p<0·0001) and influenza B (95 of 118 [81%, 72·2–87·2]; p<0·0001), along with suboptimal serum antibody (seroconversion in six of 118 [5%, 1·9–10·7]) and T-cell responses (CD4+ interferon γ-positive and/or CD4+ interleukin 2-positive responses in 45 of 111 [41%, 31·3–50·3]). After the switch to NY15, a significant increase in pH1N1 shedding was seen (80 of 126 children [63%, 95% CI 54·4–71·9]; p<0·0001 compared with Cal09), along with improvements in seroconversion (24 of 126 [19%, 13·2–26·8]; p=0·011) and influenza-specific CD4+ T-cell responses (73 of 111 [66%, 60·0–75·6; p=0·00028]). The improvement in pH1N1 seroconversion with NY15 was even greater in children who were seronegative at baseline (24 of 64 children [38%, 95% CI 26·7–49·8] vs six of 79 children with Cal09 [8%, 2·8–15·8]; p<0·0001). Persistent shedding to day 7 was independently associated with both seroconversion (odds ratio 12·69, 95% CI 4·1–43·6; p<0·0001) and CD4+ T-cell responses (odds ratio 7·83, 95% CI 2·99–23·5; p<0·0001) by multivariable logistic regression.InterpretationThe pH1N1 component switch that took place between 2016 and 2018 might have overcome the poor efficacy and effectiveness reported with previous LAIV formulations. LAIV effectiveness against pH1N1 should, therefore, improve in upcoming influenza seasons. Our data highlight the importance of assessing replicative fitness, in addition to antigenicity, when selecting annual LAIV components.FundingThe Wellcome Trust.

Successes and Failures of the Live-attenuated Influenza Vaccine: Can We Do Better?

BackgroundThe effectiveness of the live-attenuated influenza vaccine (LAIV) can vary widely, ranging from 0% to 50%. The reasons for these discrepancies remain largely unclear.MethodsWe use mathematical models to explore how the efficacy of LAIV is affected by the degree of mismatch with the currently circulating influenza strain and interference with pre-existing immunity. The models incorporate 3 key antigenic distances: the distances between the vaccine strain, pre-existing immunity, and the challenge strain.ResultsOur models show that an LAIV that is matched with the currently circulating strain is likely to have only modest efficacy. Our results suggest that the efficacy of the vaccine would be increased (optimized) if, rather than being matched to the circulating strain, it is antigenically slightly further from pre-existing immunity than the circulating strain. The models also suggest 2 regimes in which LAIV that is matched to circulating strains may be protective: in children before they have built immunity to circulating strains and in response to novel strains (such as antigenic shifts) which are at substantial antigenic distance from previously circulating strains. We provide an explanation for the variation in vaccine effectiveness between studies and countries of vaccine effectiveness observed during the 2014–2015 influenza season.ConclusionsLAIV is offered to children across the world; however, its effectiveness significantly varies between studies. Here, we propose a mechanistic explanation to understand these differences. We further propose a way to select the LAIV strain that would have a higher chance of being protective.

Amino Acid Substitutions N123D and N149D in Hemagglutinin Molecule Enhance Immunigenicity of Live Attenuated Influenza H7N9 Vaccine Strain in Experiment.

We compared three cold-adapted live attenuated influenza vaccine strains prepared by reverse genetics methods on the basis of master donor virus A/Leningrad/134/17/57 and influenza H7N9 strains A/Anhui/1/2013 and A/Shanghai/1/2013. Two strains based on A/Anhui/1/2013 differed by amino acid positions 123 and 149 in HA1 (123N/149N; 123D/149D). All strains efficiently replicated in developing chicken embryos; A/Shanghai/1/2013-based strain and A/Anhui/1/2013-123N/149N variant were characterized by reduced replication in MDCK cells. Strains based on A/Anhui/1/2013 virus agglutinated erythrocytes with α2,3- and α2,6-linked sialic acid residues, whereas strain A/Shanghai/1/2013 only α2,3. In experiments with BALB/c mice, Anhui-123D/149D strain was most immunogenic and induced high crossreactive humoral immune response, therefore it can be recommended as the model virus for the construction of recombinant vector vaccines based on live attenuated influenza vaccine.

The Effect of Preexisting Immunity on Virus Detection and Immune Responses in a Phase II, Randomized Trial of a Russian-Backbone, Live, Attenuated Influenza Vaccine in Bangladeshi Children

BackgroundIn a 2012 Phase II clinical trial, 300 Bangladeshi children aged 24 to 59 months with no prior influenza vaccine exposure were randomized to receive a single intranasally-administered dose of either trivalent, Russian-backbone, live, attenuated influenza vaccine (LAIV) or placebo. Protocol-defined analyses, presented in the companion manuscript, demonstrate decreased viral detection and immunogenicity for A/H1N1pdm09, relative to the A/H3N2 and B strains. This post hoc analysis of the trial data aims to investigate the LAIV strain differences by testing the hypothesis that preexisting humoral and mucosal immunity may influence viral recovery and immune responses after LAIV receipt.MethodsWe used logistic regressions to evaluate the relations between markers of preexisting immunity (ie, hemagglutination inhibition [HAI], microneutralization, and immunoglobulin G and immunoglobulin A (both serum and mucosal antibodies) and LAIV viral recovery in the week post-vaccination. We then tested for potential effect modification by baseline HAI titers (ie, <10 versus ≥10) and week 1 viral recovery on the LAIV-induced serum and mucosal immune responses, measured between days 0 and 21 post-vaccination.ResultsHigher levels of preexisting immunity to influenza A/H3N2 and B were strongly associated with strain-specific prevention of viral shedding upon LAIV receipt. While evidence of LAIV immunogenicity was observed for all 3 strains, the magnitudes of immune responses were most pronounced in children with no evidence of preexisting HAI and in those with detectable virus.ConclusionsThe results provide evidence for a bidirectional association between viral replication and immunity, and underscore the importance of accounting for preexisting immunity when evaluating virologic and immunologic responses to LAIVs.Clinical Trials RegistrationNCT01625689.

Live-Attenuated Influenza Vaccine Effectiveness in Children From 2009 to 2015-2016: A Systematic Review and Meta-Analysis.

BackgroundThis systematic review and meta-analysis describes and consolidates findings from all studies that assessed the effectiveness of live-attenuated influenza vaccine (LAIV) against laboratory-confirmed influenza since the 2009 pandemic in children and young adults.MethodsA MEDLINE search was conducted for articles published from January 1, 2010 to November 30, 2016. All original publications reporting an effectiveness estimate of LAIV against cases of influenza confirmed by reverse-transcription polymerase chain reaction or culture were retained for analysis. Effectiveness estimates were categorized by LAIV formulation (monovalent, trivalent, and quadrivalent) and strain (any influenza strain, A(H1N1)pdm09, A(H3N2), and B strains). Consolidated estimates were obtained with a random-effects model.ResultsA total of 24 publications presenting 29 observational studies were retained for meta-analysis. Live-attenuated influenza vaccine was not shown to be effective against A(H1N1)pdm09 strains as a monovalent formulation in 2009–2010 or as a trivalent formulation from 2010–2011 to 2013–2014, but consolidated sample sizes were small. It was effective as a quadrivalent formulation but less effective than inactivated influenza vaccine (IIV). Live-attenuated influenza vaccine was consistently effective against B strains and matched A(H3N2) strains but was not shown to provide significant protection against mismatched A(H3N2) strains in 2014–2015.ConclusionsThese findings confirm that effectiveness of LAIV against A(H1N1)pdm09 strains has been lower than IIV. A systematic investigation has been initiated to determine the root cause of the difference in effectiveness between pre- and postpandemic A(H1N1) vaccine strains and to identify a more consistently effective A(H1N1)pdm09 vaccine strain.

Association of vaccine handling conditions with effectiveness of live attenuated influenza vaccine against H1N1pdm09 viruses in the United States

PurposeThis analysis examined potential causes of the lack of vaccine effectiveness (VE) of live attenuated influenza vaccine (LAIV) against A/H1N1pdm09 viruses in the United States (US) during the 2013–2014 season. Laboratory studies have demonstrated reduced thermal stability of A/California/07/2009, the A/H1N1pdm09 strain utilized in LAIV from 2009 through 2013–2014. MethodsPost hoc analyses of a 2013–2014 test-negative case-control (TNCC) effectiveness study investigated associations between vaccine shipping conditions and LAIV lot effectiveness. Investigational sites provided the LAIV lot numbers administered to each LAIV recipient enrolled in the study, and the vaccine distributor used by the site for commercially purchased vaccine. Additionally, a review was conducted of 2009–2014 pediatric observational TNCC effectiveness studies of LAIV, summarizing effectiveness by type/subtype, season, and geographic location. ResultsFrom the 2013 to 2014 TNCC study, the proportion of LAIV recipients who tested positive for H1N1pdm09 was significantly higher among children who received a lot released between August 1 and September 15, 2013, compared with a lot shipped either earlier or later (21% versus 4%; P<0.01). A linear relationship was observed between the proportion of subjects testing positive for H1N1pdm09 and outdoor temperatures during truck unloading at distributors’ central locations. The review of LAIV VE studies showed that in the 2010–2011 and 2013–2014 influenza seasons, no significant effectiveness of LAIV against H1N1pdm09 was demonstrated for the trivalent or quadrivalent formulations of LAIV in the US, respectively, in contrast to significant effectiveness against A/H3N2 and B strains during 2010–2014. ConclusionsThis study showed that the lack of VE observed with LAIV in the US against H1N1pdm09 viruses was associated with exposure of some LAIV lots to temperatures above recommended storage conditions during US distribution, and is likely explained by the increased susceptibility of the A/California/7/2009 (H1N1pdm09) LAIV strain to thermal degradation.Clinical trial registry: NCT01997450

Comparison of influenza vaccine regimens in pediatric patients:immunity and efficacy in 2014–15

Abstract Currently, there are two types of influenza vaccines available to children and adolescents, Influenza Inactivated Vaccine (IIV) which is administered intramuscularly, and Live Attenuated Influenza Vaccine (LAIV) which is administered intranasally. The present study was designed to compare the efficacy of the two vaccines in pediatric patients in eliciting immune responses and protecting against infection in the 2014–15 influenza season. To conduct the study, patients ranging from ages 3 to 17 years were recruited for vaccination and evaluation. Patients were segregated by age (3 to 8 vs. 9 to17) and vaccine (IIV vs. LAIV). Peripheral blood samples were collected between the months of August and December, 2014 and processed for collection and storage of serum, plasma and peripheral blood mononuclear cells (PBMC). Each child who agreed to participate in the study was prescreened, consented and provided blood samples at three time points: D0 (blood drawn prior to vaccination), D7 and D21 following influenza vaccination. At the end of the vaccination season and once all enrolled patient samples had been collected, antibody responses were evaluated for reactivity to the vaccine strains as well as against additional circulating or heterogonous strains of influenza. Early in the 2014–15 season, it became apparent that the LAIV strain in the vaccine was not well matched to the circulating strain of influenza, resulting in less-than-effective immunity. Therefore, our analyses focused on IIV only. Our results showed that all participants in the study demonstrated higher titers and increased breadth of reactivity to diverse influenza strains when vaccinated with IIV, regardless of prior vaccination history.

Factors affecting the immunogenicity of the live attenuated influenza vaccine produced in continuous cell line

ABSTRACT The biological basis for the restricted immunogenicity of some live attenuated influenza vaccine strains generated on the backbone of the cold adapted (ca) A/Singapore/1/1957/ca (H2N2) influenza A virus master strain and produced in the Vero cells was investigated. According to our previous results the vaccine candidate made from A/Hong Kong/1035/1998 (H1N1) Vero-derived virus did not provoke a measurable antibody titers following the intranasal immunization of humans. We report here that the hemagglutinin (HA) of A/Hong Kong/1035/1998 virus contained the mutation 10Ile→Val in the HA2 subunit, that increased the pH threshold of HA conformational change (pH of activation) by 0.3 pH units and therefore might be responsible for the lack of immune response in humans. Similar effect was shown for the reassortant made from the Vero-derived A/Switzerland/5389/1995 (H1N1) (5389wt) virus which had the HA2 mutation 3Phe→Leu leading to the lack of immune response in mice. Another factor compromising the immunogenicity of a vaccine candidate is the incompatibility of epidemic virus HA with the M gene of the master strain. In mice the 6/2 A/Switzerland/5389/1995 reassortant induced antibodies that were directed predominantly to the HA2 subunit and were detectable by ELISA but not by a hemagglutination inhibition (HAI) test. In contrast, the 5/3 reassortant, bearing the HA, neuraminidase (NA), and M genes from the epidemic virus induced an equivalent amount of antibodies against the HA1 and HA2 subunits detected by HAI and ELISA. By comparing the sensitivity of the viruses to amantadine, we showed that the M2 ion channel of the master strain had lower activity than that of the A/Switzerland/5389/1995. These data suggest that M2 of the master strain was not sufficiently active to keep the pH of the trans- Golgi network high enough to prevent the conformational change of the acid sensitive HA to the low pH form. Overall, the adaptation mutations in the HA of the vaccine candidate that increase the pH of HA activation as well as the incompatibility of HA and M genes must be taken into consideration when constructing the reassortant strains for the live attenuated vaccine.

Effectiveness of live attenuated influenza vaccine and inactivated influenza vaccine in children 2–17 years of age in 2013–2014 in the United States

BackgroundA postmarketing observational study was initiated to evaluate quadrivalent live attenuated influenza vaccine (LAIV) effectiveness in children aged 2–17 years in the United States. MethodsChildren and adolescents aged 2–17 years seeking outpatient care for febrile acute respiratory illness <5 days duration were enrolled at 4 geographically diverse sites during the 2013–2014 influenza season. Nasal swabs were tested for influenza using reverse transcription polymerase chain reaction. Vaccination status was documented from medical records or immunization registries. Children who received ≥1 dose of influenza vaccine ≥14 days before study visit were considered vaccinated. Vaccine effectiveness (VE) was estimated as 100×(1−adjusted odds ratio), where the odds of interest are the odds of vaccine exposure among influenza cases and test-negative controls. ResultsIn total, 1033 children and adolescents were included in the analysis. Influenza was detected in 14% (145/1033) of all children, with 74% (108/145) of the influenza cases due to A/H1N1pdm09 strains, 21% (31) to influenza B, and 4% (6) to influenza H3N2. LAIV did not show significant effectiveness against A/H1N1pdm09 (VE 13% [95% CI: −55 to 51]) but was effective against B/Yamagata strains (82% [95% CI: 12–96]). Inactivated influenza vaccine was effective against A/H1N1pdm09 (74% [95% CI: 50–86]) and B/Yamagata (70% [95% CI: 18–89]). ConclusionsLAIV provided significant protection against B/Yamagata influenza but not against A/H1N1pdm09 in children aged 2–17 years in 2013–2014, resulting in a proposed change of the 2015–2016 formulation with a new and more heat-stable A/H1N1pdm09 LAIV strain.

Analysis of the genetic stability of the master virus seeds for live attenuated influenza vaccine production

Objective To analyze the genetic stability of master virus seed lots of live attenuated influenza vaccine A/17/California/2009/38(H1N1) and A/17/Perth/09/87(H3N2) strains. Methods The master virus seed lots were inoculated into chicken eggs for subculture. The complete genome of the 2nd, 3rd, 5th and 10th generations of viruses were amplified and sequenced. The genes encoding hemagglutinin (HA) and neuraminidase (NA) were compared with those of the WHO recommended circulating wild-type virus strains used for vaccine production in northern hemisphere during 2011-2012 influenza season. Six internal genes (PB2, PB1, PA, NP, M and NS) of each virus generation were compared with their master donor virus strain (A/Leningrad/134/17/57) for the evaluation of the genetic stability. Results The mutation rates of H1N1 and H3N2 strains after 10 passages were 0.035% and 0.022%, respectively. No mutations were found at the critical sites for controling thecold adapted (ca), temperature sensitive (ts) and attenuated (att) phenotypes. Conclusion The live attenuated influenza vaccine strains possessed high genetic stability as their tenth generations still shared 99% of homology with the original seed lots. All of the working virus seed lots met the requirements of Pharmacopoeia of the People′s Republic of China (2010 edition). Key words: Live attenuated influenza virus vaccine; Genetic stability; PCR

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.

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

Live attenuated influenza vaccine (LAIV) consists of reassortant viruses with hemagglutinin (HA) and neuraminidase (NA) gene segments inherited from the circulating wild-type (WT) parental viruses and six internal protein-encoding gene segments from cold-adapted attenuated master donor viruses (genome composition 6 : 2). In this study, we describe the difficulties in development of LAIV strains depending on the phenotypic peculiarities of the WT viruses used for reassortment. Genomic-composition analysis of 849 reassortants revealed that over 80% of reassortants based on the inhibitor-resistant WT viruses inherited WT NA as compared to 26% of reassortants based on the inhibitor-sensitive WT viruses. In addition, the highest percentage of vaccine genotype reassortants was achieved when WT parental viruses were resistant to nonspecific serum inhibitors. We show that NA may play a role in the influenza virus’ sensitivity to a nonspecific serum inhibitors. Replacing the NA of the inhibitor-sensitive WT virus with the NA of the inhibitor-resistant master donor virus significantly decreased the sensitivity of the resulting reassortant virus to nonspecific inhibitors.