A cell culture-derived whole-virus H5N1 vaccine induces long-lasting cross-clade protective immunity in mice which is augmented by a homologous or heterologous booster vaccination

Background

Preparation for an H5N1 influenza pandemic in humans could include priming the population in the pre-pandemic period with a vaccine produced from an existing H5N1 vaccine strain, with the possibility of boosting with a pandemic virus vaccine when it becomes available. We investigated the longevity of the immune response after one or two priming immunizations with a whole-virus H5N1 vaccine and the extent to which this can be boosted by later immunization with either a homologous or heterologous vaccine.

Methods

Mice received one or two priming immunizations with a Vero cell culture-derived, whole-virus clade 1 H5N1 vaccine formulated to contain either 750 ng or 30 ng hemagglutinin. Six months after the first priming immunization, mice received either a booster immunization with the same clade 1 vaccine or a heterologous clade 2.1 vaccine, or buffer. Humoral and cellular immune responses were evaluated before and at regular intervals after immunizations. Three weeks after booster immunization, mice were challenged with a lethal dose of wild-type H5N1 virus from clades 1, 2.1 or 2.2 and survival was monitored for 14 days.

Results

One or two priming immunizations with the 750 ng or 30 ng HA formulations, respectively, induced H5N1-neutralizing antibody titers which were maintained for ≥6 months and provided long-term cross-clade protection against wild-type virus challenge. Both humoral and cellular immune responses were substantially increased by a booster immunization after 6 months. The broadest protective immunity was provided by an immunization regimen consisting of one or two priming immunizations with a clade 1 vaccine and a boosting immunization with a clade 2.1 vaccine.

Conclusions

These data support the concept that pre-pandemic vaccination can provide robust and long-lasting H5N1 immunity which could be effectively boosted by immunization either with another pre-pandemic vaccine or with the pandemic strain vaccine.     

Cell culture (Vero cell) derived whole-virus non-adjuvanted H5N1 influenza vaccine induces long-lasting cross-reactive memory immune response: Homologous or heterologous booster response following two dose or single dose priming

Background

Influenza pandemic preparedness involves priming of the population with pre-pandemic vaccines. Such vaccines should be well tolerated and induce a long-lasting immunological memory that can effectively be boosted with a single dose of pandemic vaccine once available. The presented studies assessed different prime-boost regimens with a Vero cell-derived whole virus non-adjuvanted H5N1 vaccine.

Methods

In one study, 281 healthy adult (18–59 years) and 280 elderly (≥60 years) subjects received two vaccinations, 21 days apart, with Vero cell-derived whole virus non-adjuvanted H5N1 vaccine (7.5 μg HA antigen A/Vietnam/1203/2004) followed by a 6, 12–15, or 24 month booster (7.5 or 3.75 μg A/Indonesia/05/2005 or A/Vietnam/1203/2004). In the other study, 230 healthy adults (18–59 years) received single dose priming (7.5 μg A/Vietnam/1203/2004) followed by a 12 month booster (7.5 or 3.75 μg A/Indonesia/05/2005). Antibody responses were assessed by microneutralization (MN) and single radial hemolysis (SRH) assay. Vaccine safety was assessed throughout.

Results

Two dose priming was equally immunogenic in adults and the elderly: >72% of subjects in each population achieved MN titers ≥1:20 after the second vaccination. Booster vaccinations at 6, 12–15, and 24 months induced substantial antibody increases to both strains: after a 7.5 μg A/Indonesia/05/2005 booster, 93–95% of adults and 72–84% of the elderly achieved MN titers ≥ 1:20 against this strain. Homologous and heterologous booster responses were higher in the 7.5 μg dose group than in the 3.75 μg dose group. Booster responses following single dose priming were similar; a 7.5 μg booster dose induced homologous MN titers ≥1:20 in 93% of subjects.

Conclusions

A Vero cell derived whole virus non-adjuvanted H5N1 influenza vaccine is well tolerated and induces long-lasting cross-clade immunological memory that can be effectively boosted 1–2 years after two dose or single dose priming, supporting its suitability for pre-pandemic vaccination.     

Safety of pandemic H1N1 vaccines in children and adolescents

During the 2009 influenza A (H1N1) pandemic several pandemic H1N1 vaccines were licensed using fast track procedures, with relatively limited data on the safety in children and adolescents. Different extensive safety monitoring efforts were put in place to ensure timely detection of adverse events following immunization. These combined efforts have generated large amounts of data on the safety of the different pandemic H1N1 vaccines, also in children and adolescents. In this overview we shortly summarize the safety experience with seasonal influenza vaccines as a background and focus on the clinical and post marketing safety data of the pandemic H1N1 vaccines in children.We identified 25 different clinical studies including 10,505 children and adolescents, both healthy and with underlying medical conditions, between the ages of 6 months and 23 years. In addition, large monitoring efforts have resulted in large amounts of data, with almost 13,000 individual case reports in children and adolescents to the WHO. However, the diversity in methods and data presentation in clinical study publications and publications of spontaneous reports hampered the analysis of safety of the different vaccines.As a result, relatively little has been learned on the comparative safety of these pandemic H1N1 vaccines - particularly in children. It should be a collective effort to give added value to the enormous work going into the individual studies by adhering to available guidelines for the collection, analysis, and presentation of vaccine safety data in clinical studies and to guidance for the clinical investigation of medicinal products in the pediatric population. Importantly the pandemic has brought us the beginning of an infrastructure for collaborative vaccine safety studies in the EU, USA and globally.     

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.     

A cell culture-derived whole-virus H9N2 vaccine induces high titer antibodies against hemagglutinin and neuraminidase and protects mice from severe lung pathology and weight loss after challenge with a highly virulent H9N2 isolate

Background

Influenza viruses of subtype A/H9N2 are enzootic in poultry across Asia and the Middle East and are considered to have pandemic potential. The development of new vaccine manufacturing technologies is a cornerstone of influenza pandemic preparedness.

Methods

A non-adjuvanted whole-virus H9N2 vaccine was developed using Vero cell culture manufacturing technology. The induction of hemagglutination inhibition (HI) and virus-neutralizing antibodies was assessed in CD1 mice and guinea pigs. A highly sensitive enzyme-linked lectin assay was used to investigate the induction of antibodies capable of inhibiting the enzymatic activity of the H9N2 neuraminidase. Protective efficacy against virus replication in the lung after challenge with the homologous virus was evaluated in BALB/c mice by a TCID₅₀ assay, and prevention of virus replication in the lung and associated pathology were evaluated by histology and immunohistochemistry. To investigate the ability of the vaccine to prevent severe disease, BALB/c mice were challenged with a highly virulent mouse-adapted H9N2 isolate which was generated by multiple lung-to-lung passage of wild-type virus.

Results

The vaccine elicited high titers of functional H9N2-specific HA antibodies in both mice and guinea pigs, as determined by HI and virus neutralization assays. High titer H9N2-specific neuraminidase inhibiting (NAi) antibodies were also induced in both species. Vaccinated mice were protected from lung virus replication in a dose-dependent manner after challenge with the homologous H9N2 virus. Immunohistochemical analyses confirmed the lack of virus replication in the lung and an associated substantial reduction in lung pathology. Dose-dependent protection from severe weight loss was also provided after challenge with the highly virulent mouse-adapted H9N2 virus.

Conclusions

The induction of high titers of H9N2-specific HI, virus-neutralizing and NAi antibodies and dose-dependent protection from virus replication and severe disease in animal models suggest that the Vero cell culture-derived whole-virus vaccine will provide an effective intervention in the event of a H9N2 pandemic situation.     

Poor immune response to a standard single dose non-adjuvanted vaccination against 2009 pandemic H1N1 influenza virus A in the adult and elder hemodialysis patients

Background

Hemodialysis patients have higher risk of mortality and morbidity when infected with 2009 pandemic H1N1 (pH1N1/09) virus. Depending on different methodologies and criteria, previous studies reported variable response rates to adjuvanted vaccines against pH1N1/09 virus in hemodialysis patients, however, the efficacy of non-adjuvanted vaccines, which are currently used in many countries such as the USA and Asian areas, has not been comprehensively evaluated in hemodialysis population before.

Methods

We evaluated the efficacy of a standard single15 μg-dose of non-adjuvanted monovalent pH1N1/09 vaccine (AdimFlu-S) in vaccine-naïve 110 hemodialysis and 173 healthy participants. When enrolling, all participants had not any clinical symptom or sign suggesting pH1N1/09 infection since the index case was identified in Taiwan. Sera from all participants were tested by hemagglutination inhibition (HI) and micro-neutralization-ELISA (microNT-ELISA) tests before and 21 days after vaccination. The outcome parameters were seroconversion rate (≥4-fold in HI titer with titer ≥1:40), seroprotection rate (HI titers ≥1:40), seroresponse rate (≥4-fold increase in HI or microNT-ELISA titer), fold of increase in geometric mean (GM) titers, and adverse effects.

Results

In method A analyses, we included all participants’ data in final analyses, and the seroconversion rates and the fold increase of GM titer after vaccination were 25.4% and 1.8 in adult (18–60-year olds) hemodialysis subgroup, and 23.4% and 1.8 in elder (>60-year olds) hemodialysis subgroup based on HI titers, which were all significantly lower than those of the corresponding healthy control subgroups. Similar trends were observed based on microNT-ELISA titers, further validating the results. Multivariable analysis revealed hemoglobin and cholesterol levels were significant predictors for seroresponse in hemodialysis patients, suggesting the possible impacts of nutrition status and anemia. In method B analyses, we excluded participants with pre-vaccination seroprotection (based on HI or microNT-ELISA criteria) in final analyses. The response rates in various subgroups from method B analyses were also similar as those from method A analyses. No severe adverse effect was noted.

Conclusions

According to the European and U.S. criteria, a single 15 μg-dose of non-adjuvanted pH1N1/09 vaccination is safe but ineffective in both adult and elder hemodialysis patients. Further studies using multiple doses or higher antigen amount are warrant to define the most appropriate regimen.     

AF03-adjuvanted and non-adjuvanted pandemic influenza A (H1N1) 2009 vaccines induce strong antibody responses in seasonal influenza vaccine-primed and unprimed mice

Pandemic influenza vaccines have been manufactured using the A/California/07/2009 (H1N1) strain as recommended by the World Health Organization. We evaluated in mice the immunogenicity of pandemic (H1N1) 2009 vaccine and the impact of prior vaccination against seasonal trivalent influenza vaccines (TIV) on antibody responses against pandemic (H1N1) 2009. In naïve mice, a single dose of unadjuvanted H1N1 vaccine (3 μg of HA) was shown to elicit hemagglutination inhibition (HI) antibody titers >40, a titer associated with protection in humans against seasonal influenza. A second vaccine dose of pandemic (H1N1) 2009 vaccine strongly increased these titers, which were consistently higher in mice previously primed with TIV than in naïve mice. At a low immunization dose (0.3 μg of HA), the AF03-adjuvanted vaccine elicited higher HI antibody titers than the corresponding unadjuvanted vaccines in both naïve and TIV-primed animals, suggesting a potential for antigen dose-sparing. These results are in accordance with the use in humans of a split-virion inactivated pandemic (H1N1) 2009 vaccine formulated with or without AF03 adjuvant to protect children and young adults against influenza A (H1N1) 2009 infection.     

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.     

Lessons from pandemic influenza A(H1N1): the research-based vaccine industry's perspective

As A(H1N1) influenza enters the post-pandemic phase, health authorities around the world are reviewing the response to the pandemic. To ensure this process enhances future preparations, it is essential that perspectives are included from all relevant stakeholders, including vaccine manufacturers. This paper outlines the contribution of R&D-based influenza vaccine producers to the pandemic response, and explores lessons that can be learned to improve future preparedness.The emergence of 2009 A(H1N1) influenza led to unprecedented collaboration between global health authorities, scientists and manufacturers, resulting in the most comprehensive pandemic response ever undertaken, with a number of vaccines approved for use three months after the pandemic declaration. This response was only possible because of the extensive preparations undertaken during the last decade.During this period, manufacturers greatly increased influenza vaccine production capacity, and estimates suggest a further doubling of capacity by 2014. Producers also introduced cell-culture technology, while adjuvant and whole virion technologies significantly reduced pandemic vaccine antigen content. This substantially increased pandemic vaccine production capacity, which in July 2009 WHO estimated reached 4.9 billion doses per annum. Manufacturers also worked with health authorities to establish risk management plans for robust vaccine surveillance during the pandemic. Individual producers pledged significant donations of vaccine doses and tiered-pricing approaches for developing country supply.Based on the pandemic experience, a number of improvements would strengthen future preparedness. Technical improvements to rapidly select optimal vaccine viruses, and processes to speed up vaccine standardization, could accelerate and extend vaccine availability. Establishing vaccine supply agreements beforehand would avoid the need for complex discussions during a period of intense time pressure.Enhancing international regulatory co-operation and mutual recognition of approvals could accelerate vaccine supply, while maintaining safety standards. Strengthening communications with the public and healthcare workers using new approaches and new channels could help improve vaccine uptake. Finally, increasing seasonal vaccine coverage will be particularly important to extend and sustain pandemic vaccine production capacity.     

Inactivated trivalent seasonal influenza vaccine induces limited cross-reactive neutralizing antibody responses against 2009 pandemic and 1934 PR8 H1N1 strains

Background

In June 2009, we conducted a prospective study in Singapore on 51 individuals to determine their serologic responses before and following receipt of the 2009 Southern Hemisphere seasonal influenza vaccine.

Materials and methods

Paired serum samples were obtained before and 3–4 weeks after vaccination. Virus microneutralization assays were performed to quantify antibodies against A/Brisbane/59/2007 vaccine, pandemic H1N1-2009 and A/Puerto Rico/08/34 H1N1 strains.

Results

Post-vaccination, 43%, 12% and 24% of subjects displayed a 4-fold or greater rise in neutralizing antibody titers against the three strains, respectively. There was a positive correlation among individuals who showed increased titers to both pandemic H1N1-2009 and A/Puerto Rico/08/34 (p < 0.001). However, this correlation was not observed for A/Brisbane/59/2007 with either strain. The relative conservation and accessibility of predicted B-cell epitopes may explain the limited cross-reactivity of the antibodies directed against common H1N1 epitopes.

Conclusions

These results suggest that seasonal influenza vaccination confers a certain degree of cross-protection to other H1N1 strains. The correlation in cross-reactive antibody titers to A/Puerto Rico/08/34 and pandemic H1N1-2009 implies that previous exposure to pre-1957 H1N1 strains may confer some protection against the 2009 pandemic strain.     

Immunogenicity and safety of a monovalent vaccine for the 2009 pandemic influenza virus A (H1N1) in children and adolescents

The 2009 pandemic influenza A (H1N1) has caused significant morbidity and mortality around the world. Safety and immunogenicity studies of 2009 pandemic influenza A (H1N1) virus in children and adolescents are limited. In this prospective, open-label study, 2 doses of a monovalent, unadjuvanted, inactivated, split-virus 2009 pandemic influenza virus A (H1N1) vaccine (AdimFlu-S) were administered to 183 healthy children and adolescents aged 1–17 years. Adverse reactions were assessed, and hemagglutination inhibition antibody titers were determined. Three weeks after the first dose, 36.2% of children aged 1–2 years, 52.5% of children aged 3–5 years, 56.7% of children aged 6–9 years, and 90.3% of adolescents aged 10–17 years generated protective antibodies. A second vaccination given 3 weeks later induced protective antibodies in 89.4% of all age groups. No severe adverse effects were found 6 weeks after vaccination.     

A highly immunogenic vaccine against A/H7N9 influenza virus

Since the first case of human infection in March 2013, continued reports of H7N9 cases highlight a potential pandemic threat. Highly immunogenic vaccines to this virus are urgently needed to protect vulnerable populations who lack protective immunity. In this study, an egg- and adjuvant-independent adenoviral vector-based, hemagglutinin H7 subtype influenza vaccine (HAd-H7HA) demonstrated enhanced cell-mediated immunity as well as serum antibody responses in a mouse model. Most importantly, this vaccine provided complete protection against homologous A/H7N9 viral challenge suggesting its potential utility as a pandemic vaccine.     

Anaphylaxis following H1N1 pandemic vaccines: safety data in perspective

We present here a detailed analysis of anaphylaxis cases reported to GlaxoSmithKline safety database following vaccination with its H1N1 pandemic influenza vaccines, Pandemrix™ and Arepanrix™. Cases were assessed according to the Brighton Collaboration Case Definition (BCCD) as either confirmed diagnosis (97/395, 24.6%), insufficient information to fulfil the minimal criteria of the case definition (117/395, 29.6%) or anaphylaxis excluded (181/395, 45.8%). There was no evidence that the rate of anaphylaxis following vaccination with Pandemrix™ or Arepanrix™ is increased with respect to the rates of anaphylaxis for other vaccines. Our analysis also highlighted the challenges of reliably determining the rate of anaphylaxis as an adverse event in the postmarketing setting following mass vaccination, as anaphylaxis was excluded in 45.8% of reported cases.     

Vaccination for 2009 pandemic H1N1 influenza A did not induce conserved epitope-specific memory CD8 T cell responses in HIV+ northern Thai children

The influenza virus causes severe illness in susceptible populations, including children and people living with human immunodeficiency virus (HIV). Here, we investigated cell-mediated immune responses (CMI) against influenza CD8 T cell conserved epitopes in HIV-infected (HIV+) northern Thai children following the 2009 pandemic H1N1 influenza A vaccination. Sixty HIV+ children were vaccinated with two doses of the 2009 pandemic influenza vaccine and their CD8 T cell responses were assessed. We found no significant differences in the increase of cytokines-producing and CD107a-expressing CD8+ T cells or CD8+ memory T cells in response to pooled conserved epitopes stimulation in vitro between children with different serologic responses to the vaccine at all time points of the study. Our results suggest that the 2009 pandemic H1N1 vaccine did not induce the conserved epitope-specific immune responses in HIV+ children. Vaccine design and vaccination strategy against influenza in these populations warrant further studies.     

Age-related changes in memory and effector T cells responding to influenza A/H3N2 and pandemic A/H1N1 strains in humans

Age-dependent changes in the cellular immune response have been mainly described in CD8+ T cells, with relative sparing of CD4+ T cells. We show that in older compared to young adults, effector memory and effector CD8+ T-cell subsets responding to influenza A/H3N2 challenge have diminished cytolytic activity. In contrast, effector CD4+ T-cell subsets in older adults share similar phenotypic and functional characteristics with those from young adults. Further, we observed a diminished cytolytic T-cell response to both seasonal influenza A/H3N2 and pandemic H1N1 (pH1N1) strains in older compared to young adults who had received seasonal influenza vaccine. These results are consistent with the observed rates of serious complications from seasonal and pandemic influenza infections in different age groups, and suggest that CD4+ T cells may provide a compensatory response to influenza infection when CD8+ T cells become compromised during the aging process.     

Non-adjuvanted 2009 influenza A (H1N1)v vaccine in pregnant women: the results of a French prospective descriptive study

In 2009, during the influenza A (H1N1)v pandemic, the French Health authorities recommended influenza immunisation for pregnant women because of the higher risk of serious influenza outcomes in that population. Thus, the non-adjuvanted inactivated influenza vaccine Panenza^® was administered to French women from the second trimester of pregnancy. Several studies suggest that inactivated seasonal influenza vaccines are safe during pregnancy but there are few data about the effects of new A (H1N1)vaccines (new antigen) on pregnant women.

Objective

The aim of the present prospective study was to describe pregnancy outcomes among women vaccinated with non-adjuvanted influenza vaccines in South Western France.

Methods

the study ran from November 2009 to February 2010 and included, on a voluntary basis, pregnant women who were vaccinated against A (H1N1) influenza in vaccination clinics or maternity wards.

Results

569 pregnant women were monitored until delivery. Compared with the general population, the risks of maternal conditions, malformations and neonatal conditions were not statistically different.

Conclusion

This study does not reveal any sign of safety concerns regarding the effects of the vaccine on pregnancy outcomes.     

Pandemic influenza A/H1N1 vaccine administered sequentially or simultaneously with seasonal influenza vaccine to HIV-infected children and adolescents

In order to evaluate the immunogenicity, safety and tolerability of the 2009 A/H1N1 MF59-adjuvanted influenza vaccine administered sequentially or simultaneously with seasonal virosomal-adjuvanted influenza vaccine to HIV-infected children and adolescents, 36 HIV-infected children and adolescents, and 36 age- and gender-matched healthy controls were randomised 1:1 to receive the pandemic vaccine upon enrolment and the seasonal vaccine one month later, or to receive the pandemic and seasonal vaccines simultaneously upon enrolment. Seroconversion and seroprotection rates against the pandemic influenza A/H1N1 virus were 100% two months after vaccine administration in both groups, regardless of the sequence of administration. Geometric mean titres against pandemic and seasonal antigens were significantly higher when the seasonal and pandemic vaccines were administered simultaneously than when the seasonal vaccine was administered alone. Local and systemic reactions were mild and not increased by simultaneous administration. In conclusion, the 2009 pandemic influenza A/H1N1 MF59-adjuvanted vaccine is as immunogenic, safe and well tolerated in HIV-infected children and adolescents as in healthy controls. Its simultaneous administration with virosomal-adjuvanted seasonal antigens seems to increase immune response to both pandemic and seasonal viruses with the same safety profile as that of the pandemic vaccine alone. However, because this finding cannot be clearly explained by an immunological viewpoint, further studies are needed to clarify the reasons of its occurrence.