A high dosage influenza vaccine induced significantly more neuraminidase antibody than standard vaccine among elderly subjects

Antibody to the neuraminidase (NA) antigen of influenza viruses has been shown to correlate with immunity to influenza in humans and animal models. In a previous report, we showed that an inactivated influenza vaccine containing 60 μg of the hemagglutinin (HA) of each strain induced significantly more serum anti-HA antibody among elderly persons than did the standard vaccine containing 15 μg of the HA of each component. We developed a lectin-based assay for anti-NA antibody and used it to measure anti-NA antibody responses among subjects who had participated in that study. The high dosage vaccine contained eight times as much NA activity as the standard vaccine and induced a significantly higher frequency of antibody responses and higher mean postvaccination anti-NA titers to the N1 and N2 of the A/H1N1 and A/H3N2 viruses in the vaccines than did the standard vaccine. Ensuring an increased antibody response to the NA antigen in inactivated influenza virus vaccines should increase the protection against influenza. An increased quantity of the NA antigen in the vaccine will ensure an increased response.     

Stability of neuraminidase in inactivated influenza vaccines

Influenza vaccines are effective in protecting against illness and death caused by this seasonal pathogen. Antibodies that block the function of either hemagglutinin (HA) or neuraminidase (NA) contribute to vaccine efficacy, however vaccine potency is based only on HA content. NA protein content in vaccines varies from season to season due to differences in the relative amounts of HA and NA in influenza A, H1N1 and H3N2, and influenza B viruses that are selected for each manufacturing campaign. This, as well as potential inherent differences in NA immunogenicity, may result in varying responses from year to year. Moreover, the antigenic stability of NA is likely to dictate whether similar antibody responses will be obtained to this antigen throughout the shelf-life of the vaccine. To address this factor, we subjected NAs of influenza A (subtypes N1 and N2) and B viruses to denaturing conditions to evaluate the stability of enzyme activity. Each NA type/subtype had unique sensitivity to denaturing conditions. The N2 enzyme activity was more thermostable than that of N1 or influenza B, while the NA activity of influenza B was most resistant to detergent. N1 enzyme activity was most resistant of the three NAs to freeze–thaw cycling. In these experiments, enzyme activity was indicative of the immunogenicity of NA, but was strain-dependent, with greater neuraminidase inhibiting (NI) antibody titers elicited following immunization with the 2009 H1N1 pandemic virus A/California/7/2009, than the previously circulating seasonal H1N1 strain, A/Brisbane/59/2007. Robust NI antibody titers against both N1 and N2 components were induced following vaccination of mice with a trivalent inactivated influenza vaccine. When stored under recommended conditions, the NA of both N1 and N2 subtypes remained immunogenic well after the vaccine expiry date.     

Intradermally-administered influenza virus vaccine is safe and immunogenic in healthy adults 18–64 years of age

Background

To increase vaccine acceptance, intradermal (ID) influenza vaccine (Fluzone^® Intradermal, Sanofi Pasteur Inc.) may be an attractive alternative to intramuscular (IM) vaccination due to smaller needle and volume injected.

Methods

A multicenter, randomized (2:1 ID vs IM vaccines) study, blinded for ID vaccine lots, was conducted among 4292 adults 18–64 years of age enrolled in October 2008. Three lots of investigational trivalent influenza vaccine containing 9 μg hemagglutinin (HA) per strain in 0.1 mL administered ID with a 30 gauge, 1.5 mm long needle were compared to standard dose vaccine (0.5 mL containing 15 μg HA/strain) given IM.

Results

The post-vaccination antibody geometric mean titers (GMT) for the ID vaccine were similar to the IM vaccine (H1N1: 193.2 vs. 178.3, H3N2: 246.7 vs. 230.7, and B: 102.5 vs. 126.9). Non-inferiority was met for the ID vaccine compared to IM vaccine as assessed by antibody GMT ratios (IM/ID) for all three virus strains (H1N1: 0.92, H3N2: 0.94, and B: 1.24). Seroconversion rates were non-inferior for H1N1 and H3N2, but not for B (ID vs. IM: H1N1: 61.2% vs. 60.5%, H3N2: 75.3% vs. 74.8%, and B: 46.2% vs. 54.2%). Seroprotection (HAI titer ≥1:40) rates were similar between groups (ID vs. IM, H1N1: 91.1% vs. 91.7%, H3N2: 90.7% vs. 91.4%, and B: 87.4% vs. 89.3%). Local injection site reactions overall were more common with ID than IM vaccine (ID vs. IM: 89.2% vs. 60.2%), but were usually grade 1 or 2 and transient. The frequencies of local injection site pain and systemic reactions were similar between vaccine groups, except more myalgia with IM vaccine.

Conclusions

The ID vaccine elicited immune responses comparable to IM vaccine except for the seroconversion rate to B virus. With the exception of pain, local injection site reactions were more common with the ID vaccine, but well-tolerated and of short duration.

Trial registration

ClinicalTrials.gov identifier: NCT00772109.     

Serum IgG titres,but not avidity,correlates with neutralizing antibody response after H5N1 vaccination

Background

Influenza H5N1 virus constitutes a pandemic threat and development of effective H5N1 vaccines is a global priority. Anti-influenza antibodies directed towards the haemagglutinin (HA) define a correlate of protection. Both antibody concentration and avidity may be important for virus neutralization and resolving influenza disease.

Methods

We conducted a phase I clinical trial of a virosomal H5N1 vaccine adjuvanted with the immunostimulating complex Matrix M™. Sixty adults were intramuscularly immunized with two vaccine doses (21 days apart) of 30 μg HA alone or 1.5, 7.5 or 30 μg HA adjuvanted with Matrix M™. Serum H5 HA1-specific antibodies and virus neutralization were determined at days 0, 21, 42, 180 and 360 and long-term memory B cells at day 360 post-vaccination. The binding of the HA specific antibodies was measured by avidity NaSCN-elution ELISA and surface plasmon resonance (SPR).

Results

The H5 HA1-specific IgG response peaked after the second dose (day 42), was dominated by IgG1 and IgG3 and was highest in the adjuvanted vaccine groups. IgG titres correlated significantly with virus neutralization at all time points (Spearman r ≥ 0.66, p < 0.0001). By elution ELISA, serum antibody avidity was highest at days 180 and 360 post vaccination and did not correlate with virus neutralization. Long-lasting H5 HA1-specific memory B cells produced high IgG antibody avidity similar to serum IgG.

Conclusions

Maturation of serum antibody avidity continued up to day 360 after influenza H5N1 vaccination. Virus neutralization correlated with serum H5 HA1-specific IgG antibody concentrations and not antibody avidity.     

Immunogenicity,reactogenicity, and safety of inactivated quadrivalent influenza vaccine candidate versus inactivated trivalent influenza vaccine in healthy adults aged ≥18 years: A phase III,randomized trial

Background

Two influenza B lineages have been co-circulating since the 1980s, and because inactivated trivalent influenza vaccine (TIV) contains only one B strain, it provides little/no protection against the alternate B-lineage. We assessed a candidate inactivated quadrivalent influenza vaccine (QIV) containing both B lineages versus TIV in healthy adults.

Methods

Subjects received one dose of QIV (lot 1, 2, or 3) or one of two TIVs (B strain from Victoria or Yamagata lineage); randomization was 2:2:2:1:1. Hemagglutination-inhibition assays were performed 21-days post-vaccination; superiority of QIV versus TIV for the alternate B-lineage was demonstrated if the 95% confidence interval (CI) lower limit for the GMT ratio was ≥1.5, and non-inferiority against the shared strains was demonstrated if the 95% CI upper limit for the GMT ratio was ≤1.5. Reactogenicity and safety were assessed during the post-vaccination period. NCT01196975.

Results

Immunogenicity of QIV lots was consistent, QIV was superior to TIV for the alternate B-lineage strain, and QIV was non-inferior versus TIVs for shared strains (A/H1N1, A/H3N2, B-strain). Reactogenicity and safety profile of the QIV was consistent with seasonal influenza vaccines.

Conclusion

QIV provided superior immunogenicity for the added B strain without affecting the antibody response to the TIV strains, and without compromising safety.     

Supplementation of conventional trivalent influenza vaccine with purified viral N1 and N2 neuraminidases induces a balanced immune response without antigenic competition

Influenza viruses neuraminidase (NA) were chromatographically extracted from influenza viruses A/Nanchang/933/95 H3(NC)N2(NC) [R] and A/Johannesburg/82/96 H1(JH)N1(JH) [R] and used to supplement conventional inactivated trivalent influenza vaccine. Immunization of mice with this preparation resulted in high titers of antibodies to both hemagglutinins (HA) and neuraminidases (NA); there were no significant differences in the anti-HA antibody titers between the conventional and the supplemented vaccine preparation. Likewise, there were no significant differences in anti-NA antibody titers between the supplemented vaccine and titers from mice immunized with a neuraminidase vaccine containing a mixture of N1-NA and N2-NA. There was no evidence of a diminution of the immune response to the HA components of the vaccine despite the presence of antigenically equivalent amounts of both N1-NA and N2-NAs. Homotypic and distantly related heterotypic infections for both H1, N1 and H3N2 subtypes were suppressed and greater reduction in pulmonary virus titers (PVT) were observed in the trivalent vaccine supplemented with purified neuraminidase from each subtype, N1 and N2. Effects on the influenza B viral components were not studied. Previous studies on supplementation of conventional influenza vaccine focused only on monovalent H3N2 vaccine preparations; this study demonstrates in a mouse model system that supplementation of trivalent influenza vaccine with both influenza A subtype neuraminidases, N1 and N2 is highly immunogenic for HA and NA of each subtype and efficacious in protecting against influenza from homotypic and heterotypic infectious challenges of either subtype.     

High-dose trivalent influenza vaccine compared to standard dose vaccine in elderly adults: Safety,immunogenicity and relative efficacy during the 2009–2010 season

Background

High-dose trivalent influenza vaccine was developed to improve antibody responses to influenza vaccine in the elderly and hence potentially impact favorably on influenza-associated morbidity and mortality in this population.

Methods

A phase IIIb, multicenter, randomized, double-blind, controlled trial was conducted to compare High-Dose (HD) trivalent inactivated influenza vaccine (60 μg of hemagglutinin [HA] per strain) to standard dose (SD) vaccine (15 μg of HA per strain) in adults ≥65 years of age. Assessments of safety (serious adverse events [SAE]), immunogenicity (hemagglutination inhibition [HAI] titers) and relative efficacy were performed during the 2009–2010 influenza season, which coincided with the H1N1 pandemic.

Results

A total of 9172 participants were enrolled in 99 research centers in the US (6117 and 3055 randomized to the HD and SD groups, respectively). Within 180 days after vaccination, 6.7% and 6.5% of participants in the HD and SD vaccine groups, respectively, experienced at least one SAE, of which 0.4% and 0.3% had a fatal outcome. A total of 0.5% of participants in both groups discontinued the study due to a SAE. Post-vaccination HAI titers and rate of post-vaccination HAI titer ≥1:40 were significantly higher in the HD group. No cases of influenza caused by viral types/subtypes similar to those in the vaccines were observed. All cases genetically or antigenically characterized were classified as similar to influenza A/California/7/2009 (H1N1), the pandemic strain. The vaccine efficacy of HD vaccine relative to SD vaccine against any influenza viral type/subtype was 12.6% (95% CI −140.5; 65.8) in the intent-to-treat analysis.

Conclusion

High-dose trivalent inactivated influenza vaccine is safe and well tolerated and provides superior immune responses compared to standard dose vaccine. Demonstration of a superior vaccine efficacy requires a separate large randomized, controlled trial.     

Live attenuated H7N7 influenza vaccine primes for a vigorous antibody response to inactivated H7N7 influenza vaccine

Background

H7 influenza viruses have emerged as potential pandemic threat. We evaluated the safety and immunogenicity of two candidate H7 pandemic live attenuated influenza vaccines (pLAIV) and their ability to prime for responses to an unadjuvanted H7 pandemic inactivated influenza vaccine (pIIV).

Methods

Healthy seronegative adults received two doses of A/Netherlands/219/03 (H7N7) or one dose of A/chicken/British Columbia/CN-6/04 (H7N3) pLAIV all given as 10^7.5 50% tissue culture infective doses (TCID₅₀) intranasally. A subset of subjects received one 45 μg dose of H7N7 pIIV containing the A/Mallard/Netherlands/12/2000 HA intramuscularly 18–24 months after pLAIV. Viral shedding was assessed by culture and real-time polymerase chain reaction (rRT-PCR), B cell responses following pLAIV were evaluated by ELISPOT and flow cytometry. Serum antibody was assessed by hemagglutination-inhibition (HAI), microneutralization (MN) and ELISA assays after each vaccine.

Results

Serum HAI or MN responses were not detected in any subject following one or two doses of either H7 pLAIV, although some subjects had detectable H7 specific B cells after vaccination. However, 10/13 subjects primed with two doses of H7N7 pLAIV responded to a subsequent dose of the homologous H7N7 pIIV with high titer HAI and MN antibody that cross-reacted with both North American and Eurasian lineage H7 viruses, including H7N9. In contrast, naïve subjects and recipients of a single dose of the mismatched H7N3 pLAIV did not develop HAI or MN antibody after pIIV.

Conclusions

While pLAIVs did not elicit detectable serum MN or HAI antibody, strain-specific pLAIV priming established long term immune memory that was cross-reactive with other H7 influenza strains. Understanding the mechanisms underlying priming by pLAIV may aid in pandemic vaccine development.     

Pre-vaccination immunity and immune responses to a cell culture-derived whole-virus H1N1 vaccine are similar to a seasonal influenza vaccine

Background

Immune responses to novel pandemic influenza vaccines may be influenced by previous exposure to antigenically similar seasonal strains.

Methods

An open-label, randomized, phase I/II study was conducted to assess the immunogenicity and safety of a non-adjuvanted, inactivated whole-virus H1N1 A/California/07/2009 vaccine. 408 subjects were stratified by age (18–59 and >60 years) and randomized 1:1 to receive two vaccinations with either 3.75 or 7.5 μg hemagglutinin antigen 21 days apart. Safety, immunogenicity and the influence of seasonal influenza vaccination and antibody cross-reactivity with a seasonal H1N1 strain was assessed.

Results

A single vaccination with either dose induced substantial increases in H1N1 A/California/07/2009 hemagglutination inhibition (HI) and neutralizing (MN) antibody titers in both adult and elderly subjects. A single 7.5 μg dose induced seroprotection rates of 86.9% in adults and 75.2% in elderly subjects. Two 7.5 μg vaccinations induced seroprotection rates in adult and elderly subjects of 90.9% and 89.1%, respectively. The robust immune response to vaccination was confirmed by analyses of neutralizing antibody titers. Both HI and MN antibodies persisted for ≥6 months post-vaccination. Between 34% and 49% of subjects had seroprotective levels of H1N1 A/California/07/2009 antibodies at baseline. Higher baseline HI titers were associated with receipt of the 2008–09 or 2009–10 seasonal influenza vaccine. High baseline A/California/07/2009 neutralizing antibody titers were also associated with high baseline titers against A/New Caledonia/20/99, a seasonal H1N1 strain which circulated and was included in the seasonal vaccine from 2000–01 to 2006–07. Pre-adsorption with A/H1N1/New Caledonia/20/99 antigen reduced A/H1N1/California/07/2009 baseline titers in 55% of tested sera. The vaccine was well tolerated with low rates of fever.

Conclusions

A whole-virus H1N1 A/California/07/2009 vaccine was safe and well tolerated and a single dose induced substantial immune responses similar to seasonal influenza vaccines, probably due to immunological priming by previous seasonal influenza vaccines or infections.     

An alternative method for preparation of pandemic influenza strain-specific antibody for vaccine potency determination

The traditional assay used to measure potency of inactivated influenza vaccines is a single-radial immunodiffusion (SRID) assay that utilizes an influenza strain-specific antibody to measure the content of virus hemagglutinin (HA) in the vaccine in comparison to a homologous HA reference antigen. Since timely preparation of potency reagents by regulatory authorities is challenging and always a potential bottleneck in influenza vaccine production, it is extremely important that additional approaches for reagent development be available, particularly in the event of an emerging pandemic influenza virus. An alternative method for preparation of strain-specific antibody that can be used for SRID potency assay is described. The approach does not require the presence or purification of influenza virus, and furthermore, is not limited by the success of the traditional technique of bromelain digestion and purification of virus HA. Multiple mammalian expression vectors, including plasmid and modified vaccinia virus Ankara (MVA) vectors expressing the HAs of two H5N1 influenza viruses and the HA of the recently emerging pandemic H1N1 (2009) virus, were developed. An immunization scheme was designed for the sequential immunization of animals by direct vector injection followed by protein booster immunization using influenza HA produced in vitro from MVA vector infection of cells in culture. Each HA antibody was highly specific as shown by hemagglutination inhibition assay and the ability to serve as a capture antibody in ELISA. Importantly, each H5N1 antibody and the pandemic H1N1 (2009) antibody preparation were suitable for use in SRID assays for determining the potency of pandemic influenza virus vaccines. The results demonstrate a feasible approach for addressing one of the potential bottlenecks in inactivated pandemic influenza vaccine production and are particularly important in light of the difficulties in preparation of potency reagent antibody for pandemic H1N1 (2009) virus vaccines.     

An observer-blind,randomized, multi-center trial assessing long-term safety and immunogenicity of AS03-adjuvanted or unadjuvanted H1N1/2009 influenza vaccines in children 10–17 years of age

Background

Vaccination is an effective strategy to prevent influenza. This observer-blind, randomized study in children 10–17 years of age assessed whether the hemagglutination inhibition (HI) antibody responses elicited by H1N1/2009 vaccines adjuvanted with AS03 (an adjuvant system containing α-tocopherol and squalene in an oil-in-water emulsion) or without adjuvant, met the European regulatory immunogenicity criteria at Days 21 and 182.

Methods

Three hundred and ten healthy children were randomized (3:3:3:5) to receive one dose of 3.75 μg hemagglutinin (HA) AS03_A-adjuvanted vaccine, one or two doses of 1.9 μg HA AS03_B-adjuvanted vaccine, or one dose of 15 μg HA pandemic vaccine. All children received a booster dose of the allocated vaccine at Day 182. Serum samples were tested for HI antibody response at Days 21, 42, 182 and 189.

Results

All vaccination regimens elicited HI antibody responses that met the European regulatory criteria at Days 21 and 42. HI antibody responses fulfilling European regulatory criteria were still observed six months after the first vaccine dose in all study vaccines groups. Two doses of 1.9 μg HA AS03_B-adjuvanted vaccine elicited the strongest HI antibody response throughout the study. The non-adjuvanted 15 μg HA vaccine elicited a lower HI antibody response than the AS03-adjuvanted vaccines. At Day 189, the European regulatory criteria were met for all vaccines with baseline HI antibody titers as reference. An anamnestic response for all vaccines was suggested at Day 189, based on the rapid increase in HI antibody geometric mean titers (1.5–2.5-fold increase). Injection site reactogenicity was higher following the AS03-adjuvanted vaccines compared with the non-adjuvanted vaccine. No safety concerns were identified for any study vaccine.

Conclusion

All study vaccines elicited HI antibody responses that persisted at purported protective levels through six months after vaccination and fulfilled the European regulatory criteria.     

Safety and efficacy of a novel microneedle device for dose sparing intradermal influenza vaccination in healthy adults

Background

Intradermal vaccine delivery has been shown to induce good immune responses with low vaccine doses. Technologies for drug-delivery which specifically target the skin may render intradermal vaccination more accessible.

Methods

We conducted a prospective, randomized trial in 180 intended-to-treat healthy adults. Study objectives were to evaluate the safety and immunogenicity of low-dose intradermal (ID) influenza vaccines delivered using a novel microneedle device (MicronJet). This device replaces a conventional needle, and is designed specifically for intradermal delivery.Subjects were randomly assigned to receive either the full-dose standard flu shot (containing 15 μg hemagglutinin per strain) delivered intramuscularly using a conventional needle (IM group), a medium dose intradermal injection (6 μg hemagglutinin per strain) delivered with the MicronJet (ID2 group), or a low-dose intradermal injection (3 μg hemagglutinin per strain) delivered with the MicronJet (ID1 group). A marketed influenza vaccine for the 2006/2007 influenza season (α-RIX^® by GSK Biologicals) was used for all injections.Adverse events were recorded over a 42-day period. Immunogenicity was evaluated by changes in hemagglutination inhibition (HAI) antibody titer, and by comparing geometric mean titers (GMTs), seroconversion, and seroprotection rates between the study groups.

Results

Local reactions were significantly more frequent following intradermal vaccination, but were mild and transient in nature. At 21 days after injection, GMT fold increase was 22, 18 and 22 in the ID1, ID2 and IM groups respectively for the H1N1 strain; 9, 9 and 16 for the H3N2 strain and 9, 13 and 11 for strain B. The CPMP criteria for re-licensure of seasonal influenza vaccines were met in full for all study groups.

Conclusions

Low-dose influenza vaccines delivered intradermally using microneedles elicited immunogenic responses similar to those elicited by the full-dose intramuscular vaccination. The microneedle injection device used in this study was found to be effective, safe, and reliable.     

VaxArray for hemagglutinin and neuraminidase potency testing of influenza vaccines

Practical methods to measure the potency of influenza vaccines are needed as alternatives for the standard single radial immunodiffusion (SRID) assay. VaxArray assays for influenza hemagglutinin (HA) and neuraminidase (NA) have been developed to address this need. In this report, we evaluate the use of these assays to assess the potency of HA and NA of an A/H3N2 subunit vaccine by determining the correlation between the amounts measured by VaxArray and the immunogenicity in mice. The antibody response after one and two doses of five formulations of the vaccine ranging from 5 µg/mL to 80 µg/mL of HA, was measured by hemagglutination inhibition (HAI) and neuraminidase inhibition (NAI) assays. For hemagglutinin, vaccine potency determined by VaxArray was equivalent to potency measured SRID and these amounts were predictive of immunogenicity, with excellent correlation between potency measured by VaxArray and the HAI geometric mean titers (GMT). Likewise, the amount of NA measured by VaxArray was predictive of the NAI GMT. The VaxArray NA assay reported non-detectable levels of intact NA for a sample that had been heat degraded at 56 °C for 20 h, demonstrating that the assay measures the native, active form of NA. Similarly, the HA potency measured by VaxArray in this heat-treated sample was very low when a monoclonal antibody was used to detect the amount of antigen bound. Importantly, the force degraded sample induced low HAI titers and the NAI titers were not measurable, supporting the conclusion that the VaxArray HA and NA assays measure the immunogenic forms of these A/H3N2 antigens. This study indicates that VaxArray assays can be used to assess the potency of HA and NA components in influenza vaccines as a proxy for immunogenicity.     

Effect of two injections of non-adjuvanted influenza A H1N1pdm2009 vaccine in renal transplant recipients: INSERM C09-32 TRANSFLUVAC trial

Background

Enhancing vaccine immunogenicity in kidney transplant recipients, particularly against influenza, is required since the immunosuppression used to prevent graft rejection limits vaccine immunogenicity. We therefore investigated the immunogenicity and safety of a double dose non-adjuvanted vaccination regimen against influenza H1N1pdm2009 in kidney transplant adult recipients.

Methods

A prospective single-arm study was conducted including 121 renal transplant recipients under triple immunosuppressive regimen. Patients received 2 injections (day 0, day 21) of an inactivated, non-adjuvanted H1N1pdm2009 vaccine. Immunogenicity (hemagglutination-inhibition [HI] antibodies and anti-hemagglutin [HA] specific T cells) was evaluated after one and two injections (day 21, day 42) and at 6 months (day 182).

Results

The seroprotection rate (HI antibody titer ≥ 1/40) was 19% at day 0 (n = 119), 53% at day 21 (n = 118), 60% at day 42 (n = 116) (p = 0.013; day 42 vs. day 21) and 56% at day 182 (n = 113). The seroconversion rate was 24% and 32%, the geometric mean fold rise was 3.7 and 4.6 after the first and second injections, respectively. T-cell immunity to the H1N1pdm2009 vaccine showed a two-fold increase from baseline, though not statistically significant, in H1N1pdm2009-HA-specific CD4+ and CD8+ T cells in 34% and 48% of cases, respectively. No rejection episodes related to vaccination were observed while the donor-specific antibodies and creatinine clearance remained unchanged throughout the study.

Conclusion

Administration of two doses of the non-adjuvanted influenza H1N1pdm2009 vaccine in renal transplant patients is safe and induces a significant seroprotection, not strong enough yet to meet European or US requirements for adults below 60 years, but comparable to seroprotection levels usually observed in the non immunosuppressed elderly population or conferred by a single dose of adjuvanted vaccine in solid organ transplant recipients. These results provide useful indications for future strategies required to improve immunogenicity of vaccines against influenza in transplanted patients.     

Generation of reassortant influenza vaccines by reverse genetics that allows utilization of a DIVA (Differentiating Infected from Vaccinated Animals) strategy for the control of avian influenza

Vaccination of poultry with inactivated influenza vaccine can be an effective tool in the control of avian influenza (AI). One major concern of using inactivated vaccine is vaccine-induced antibody interference with serologic surveillance and epidemiology. In the United States, low pathogenicity H5 and H7 subtype AI viruses have caused serious economic losses in the poultry industry. Most of these viruses also have the accompanying N2 subtype and no H5N1 or H7N8 subtype AI viruses have been identified in poultry in the US. In order to allow the Differentiation of Infected from Vaccinated Animals (DIVA) while maintaining maximum efficacy of the vaccine, we generated reassortant viruses by reverse genetics that contained the same H5 and H7 hemagglutinin (HA) gene as the challenge virus, but a heterologous N1 or N8 neuraminidase (NA) gene. In vaccination-challenge experiments in 2-week-old specific pathogen free chickens, reassortant influenza vaccines (rH5N1 and rH7N8) demonstrated similar antibody profiles and comparable protection rates as vaccines prepared with parent H5N2 and H7N2 viruses. Further, we were able to differentiate the sera from infected and vaccinated birds by neuraminidase inhibition test and indirect immunofluorescent antibody assay on the basis of different antibodies elicited by their NA proteins. These results demonstrate the usefulness of a reverse genetics system for the rapid generation of reassortant AI virus that allows utilization of the DIVA strategy for the control of AI infections in poultry.     

The impact of pandemic A(H1N1)pdm09 influenza and vaccine-associated adverse events on parental attitudes and influenza vaccine uptake in young children

Introduction

Parental attitudes towards vaccination significantly influence vaccine uptake. The A(H1N1)pdm09 influenza pandemic was followed in 2010 by an unprecedented increase in febrile reactions in children receiving trivalent inactivated influenza vaccine manufactured by bioCSL. Uptake of TIV in children <5 years in Western Australia (WA) decreased in 2010 and has remained low. The impact of pandemic A(H1N1)pdm09 and adverse-events on parental attitudes towards vaccination is uncertain.

Materials and Methods

A parental attitudes survey towards influenza illness and vaccination was conducted as part of the West Australian Influenza Vaccine Effectiveness study. Vaccination status was assessed by parental interview and confirmed by the national register and/or vaccine providers. Parental attitudes from vaccinated and unvaccinated children and attitudes in 2008–2009 and 2010–2012 were compared. Principal Component Analysis was conducted to determine core attitudes that influenced vaccine uptake.

Results

Vaccination history and parental attitude surveys were available from 2576 children. Parents of fully vaccinated children less frequently stated that influenza was a mild disease, more frequently stated that influenza vaccine was safe and were less frequently worried about vaccine side effects.Uptake of influenza vaccine decreased significantly from 2010 onwards. From 2010, parents were less concerned about severe influenza, but more concerned about vaccine side effects and safety. Despite this significant shift in attitudes towards influenza vaccine, parental acceptance of vaccines on the national immunisation program did not change. Principal Component Analysis revealed that attitudes around vaccine safety and efficacy were the most important attitudes impacting on vaccine uptake.

Conclusions

Parental attitudes to influenza vaccine changed from 2010. Confidence in the WA preschool influenza vaccination program remains low yet appeared unchanged for other vaccines. Restoring public confidence in childhood influenza vaccination is needed before uptake can be improved.     

Influenza virus-like particles elicit broader immune responses than whole virion inactivated influenza virus or recombinant hemagglutinin

Influenza virus is a highly infectious respiratory pathogen that results in severe morbidity and mortality. The current licensed trivalent vaccine formulations in the U.S. are made from virus grown in allantoic fluid from infected hen eggs that is then chemically inactivated and split into subunit components. These vaccines elicit antibodies, primarily to the viral hemagglutinin (HA), which are efficacious in healthy adults, but are limited in protecting high risk individuals, such as the elderly and immunocompromised. To address the need for improved influenza vaccines and the limitations of egg-based manufacturing, we have engineered an influenza virus-like particle (VLP) as a new generation of non-egg or non-mammalian cell culture-based candidate vaccine against influenza infection. VLPs, based on the A/Fujian/411/2002 (H3N2) isolate, were purified from the supernatants of Spodoptera frugiperda Sf9 insect cells following infection of baculovirus vectors encoding an expression cassette comprised of only three influenza virus structural proteins, hemagglutinin (HA), neuraminidase (NA), and matrix (M1). Mice or ferrets were vaccinated intramuscularly with VLPs in a dose sparing experiment, based on HA concentration (3 microg-24 ng), and the immune responses were compared to responses elicited in animals vaccinated with recombinant HA (rHA) or inactivated whole influenza virions (WIV). All vaccinated animals had high titer anti-HA antibodies regardless of the vaccine immunogen and animals vaccinated with the highest doses of VLPs (3 microg and 600 ng) also had antibodies against NA. Purified rHA elicited primarily IgG1 antibodies, which is indicative of a T helper (Th) type 2 response, whereas mice vaccinated with the VLPs or WIV were associated with a dominant Th1 immune response (IgG2a and IgG2b). Interestingly, VLPs elicited antibodies that recognized a broader panel of antigenically distinct H3N2 viral isolates compared to rHA or WIV in a hemagglutination-inhibition (HAI) assay.     

Immune response after one or two doses of pandemic influenza A (H1N1) monovalent,AS03-adjuvanted vaccine in HIV infected adults

Introduction

Continued research is needed to evaluate and improve the immunogenicity of influenza vaccines in HIV infected patients. We aimed to determine the antibody responses after one or two doses of the AS03-adjuvanted pandemic influenza A (H1N1) vaccine in HIV infected patients.

Method

Following the influenza season 2009/2010, 219 HIV infected patients were included and divided into three groups depending on whether they received none (n = 60), one (n = 31) or two (n = 128) doses of pandemic influenza A (H1N1) vaccine. At inclusion, antibody titers for all patients were analyzed and compared to pre-pandemic antibody titers analyzed from serum samples in a local storage facility.

Results

4–9 months after a single immunization, we found a seroprotection rate of 77.4% and seroconversion rate of 67.7%. After two immunizations the rates increased significantly to seroprotection rate of 97.7% and seroconversion rate of 86.7%.

Conclusion

A single dose of AS03-adjuvanted pandemic influenza A (H1N1) vaccine created an adequate immune response in HIV infected patients lasting as long as 4–9 months. Two doses improved the immunogenicity further.     

An open label Phase I trial of a live attenuated H6N1 influenza virus vaccine in healthy adults

Background

We describe the results of an open label Phase I trial of a live attenuated H6N1 influenza virus vaccine (ClinicalTrials.gov Identifier: NCT00734175).

Methods and findings

We evaluated the safety, infectivity, and immunogenicity of two doses of 10⁷ TCID₅₀ of the H6N1 Teal HK 97/AA ca vaccine, a cold-adapted and temperature sensitive live, attenuated influenza vaccine (LAIV) in healthy seronegative adults.Twenty-two participants received the first dose of the vaccine, and 18 received the second dose of vaccine 4 weeks later. The vaccine had a safety profile similar to that of other investigational LAIVs bearing avian hemagglutinin (HA) and neuraminidase (NA) genes. The vaccine was highly restricted in replication: two participants had virus detectable by rRT-PCR beyond day 1 after each dose. Antibody responses to the vaccine were also restricted: 43% of participants developed a serum antibody response as measured by any assay: 5% by hemagglutination-inhibition assay, 5% by microneutralization assay, 29% by ELISA for H6 HA-specific IgG and 24% by ELISA for H6 HA specific IgA after either 1 or 2 doses. Following the second dose, vaccine specific IgG and IgA secreting cells as measured by ELISPOT increased from a mean of 0.6 to 9.2/10⁶ PBMCs and from 0.2 to 2.2/10⁶ PBMCs, respectively.

Conclusion

The H6N1 LAIV had a safety profile similar to that of LAIV bearing other HA and NA genes, but was highly restricted in replication in healthy seronegative adults. The H6N1 LAIV was also not as immunogenic as the seasonal LAIV.