Immunogenicity of Viral Vaccines in the Italian Military.

Military personnel of all armed forces receive multiple vaccinations and have been doing so since long ago, but relatively few studies have investigated the possible negative or positive interference of simultaneous vaccinations. As a contribution to fill this gap, we analyzed the response to the live trivalent measles/mumps/rubella (MMR), the inactivated hepatitis A virus (HAV), the inactivated trivalent polio, and the trivalent subunits influenza vaccines in two cohorts of Italian military personnel. The first cohort was represented by 108 students from military schools and the second by 72 soldiers engaged in a nine-month mission abroad. MMR and HAV vaccines had never been administered before, whereas inactivated polio was administered to adults primed at infancy with a live trivalent oral polio vaccine. Accordingly, nearly all subjects had baseline antibodies to polio types 1 and 3, but unexpectedly, anti-measles/-mumps/-rubella antibodies were present in 82%, 82%, and 73.5% of subjects, respectively (43% for all of the antigens). Finally, anti-HAV antibodies were detectable in 14% and anti-influenza (H1/H3/B) in 18% of the study population. At mine months post-vaccination, 92% of subjects had protective antibody levels for all MMR antigens, 96% for HAV, 69% for the three influenza antigens, and 100% for polio types 1 and 3. An inverse relationship between baseline and post-vaccination antibody levels was noticed with all the vaccines. An excellent vaccine immunogenicity, a calculated long antibody persistence, and apparent lack of vaccine interference were observed.

Co-circulation of the two influenza B lineages during 13 consecutive influenza surveillance seasons in Italy, 2004-2017.

BACKGROUND: Since 1985, two antigenically distinct lineages of influenza B viruses (Victoria-like and Yamagata-like) have circulated globally. Trivalent seasonal influenza vaccines contain two circulating influenza A strains but a single B strain and thus provide limited immunity against circulating B strains of the lineage not included in the vaccine. In this study, we describe the characteristics of influenza B viruses that caused respiratory illness in the population in Italy over 13 consecutive seasons of virological surveillance, and the match between the predominant influenza B lineage and the vaccine B lineage, in each season. METHODS: From 2004 to 2017, 26,886 laboratory-confirmed influenza cases were registered in Italy, of which 18.7% were type B. Among them, the lineage of 2465 strains (49%) was retrieved or characterized in this study by a real-time RT-PCR assay and/or sequencing of the hemagglutinin (HA) gene. RESULTS: Co-circulation of both B lineages was observed each season, although in different proportions every year. Overall, viruses of B/Victoria and B/Yamagata lineages caused 53.3 and 46.7% of influenza B infections, respectively. A higher proportion of infections with both lineages was detected in children, and there was a declining frequency of B/Victoria detections with age. A mismatch between the vaccine and the predominant influenza B lineage occurred in eight out of thirteen influenza seasons under study. Considering the seasons when B accounted for > 20% of all laboratory-confirmed influenza cases, a mismatch was observed in four out of six seasons. Phylogenetic analysis of the HA1 domain confirmed the co-circulation of both lineages and revealed a mixed circulation of distinct evolutionary viral variants, with different levels of match to the vaccine strains. CONCLUSIONS: This study contributes to the understanding of the circulation of influenza B viruses in Italy. We found a continuous co-circulation of both B lineages in the period 2004-2017, and determined that children were particularly vulnerable to Victoria-lineage influenza B virus infections. An influenza B lineage mismatch with the trivalent vaccine occurred in about two-thirds of cases.

Strategies to obtain multiple recombinant modified vaccinia Ankara vectors. Applications to influenza vaccines.

As a vaccination vector, MVA has been widely investigated both in animal models and humans. The construction of recombinant MVA (rMVA) relies on homologous recombination between an acceptor virus and a donor plasmid in infected/transfected permissive cells. Our construction strategy "Red-to-Green gene swapping" - based on the exchange of two fluorescent markers within the flanking regions of MVA deletion ΔIII, coupled to fluorescence activated cell sorting - is here extended to a second insertion site, within the flanking regions of MVA deletion ΔVI. Exploiting this strategy, both double and triple rMVA were constructed, expressing as transgenes the influenza A proteins HA, NP, M1, and PB1. Upon validation of the harbored transgenes co-expression, double and triple recombinants rMVA(ΔIII)-NP-P2A-M1 and rMVA(ΔIII)-NP-P2A-M1-(ΔVI)-PB1 were assayed for in vivo immunogenicity and protection against lethal challenge. In vivo responses were identical to those obtained with the reported combinations of single recombinants, supporting the feasibility and reliability of the present improvement and the extension of Red-to-Green gene swapping to insertion sites other than ΔIII.

Immunogenicity of modified vaccinia virus Ankara expressing the hemagglutinin stalk domain of pandemic (H1N1) 2009 influenza virus.

BACKGROUND: Vaccination offers protection against influenza, although current vaccines need to be reformulated each year. The development of a broadly protective influenza vaccine would guarantee the induction of heterosubtypic immunity also against emerging influenza viruses of a novel subtype. Vaccine candidates based on the stalk region of the hemagglutinin (HA) have the potential to induce broad and persistent protection against diverse influenza A viruses. METHODS: Modified vaccinia virus Ankara (MVA) expressing a headless HA (hlHA) of A/California/4/09 (CA/09) virus was used as a vaccine to immunize C57BL/6 mice. Specific antibody and cell-mediated immune responses were determined, and challenge experiments were performed by infecting vaccinated mice with CA/09 virus. RESULTS: Immunization of mice with CA/09-derived hlHA, vectored by MVA, was able to elicit influenza-specific broad cross-reactive antibodies and cell-mediated immune responses, but failed to induce neutralizing antibodies and did not protect mice against virus challenge. CONCLUSION: Although highly immunogenic, our vaccine was unable to induce a protective immunity against influenza. A misfolded and unstable conformation of the hlHA molecule may have affected its capacity of inducing neutralizing antiviral, conformational antibodies. Design of stable hlHA-based immunogens and their delivery by recombinant MVA-based vectors has the potential of improving this promising approach for a universal influenza vaccine.

Protective immunity against influenza in HLA-A2 transgenic mice by modified vaccinia virus Ankara vectored vaccines containing internal influenza proteins.

BACKGROUND: The emergence of novel strains of influenza A viruses with hemagglutinins (HAs) that are antigenically distinct from those circulating in humans, and thus have pandemic potential, pose concerns and call for the development of more broadly protective influenza vaccines. In the present study, modified vaccinia virus Ankara (MVA) encoding internal influenza antigens were evaluated for their immunogenicity and ability to protect HLA-A2.1 transgenic (AAD) mice from infection with influenza viruses. METHODS: MVAs expressing NP (MVA-NP), M1 (MVA-M1) or polymerase PB1 (MVA-PB1) of A/California/4/09 (CA/09) virus were generated and used to immunize AAD mice. Antibodies and CD8+T cell responses were assessed by ELISA and ELISPOT, respectively, and challenge experiments were performed by infecting vaccinated mice with CA/09 virus. RESULTS: CD8+T cells specific to immunodominant and subdominant epitopes on the internal influenza proteins were elicited by MVA-based vectors in AAD mice, whereas influenza-specific antibodies were detected only in MVA-NP-immunized mice. Both M1- and NP-based MVA vaccines, regardless of whether they were applied individually or in combination, conferred protection against lethal influenza virus challenge. CONCLUSION: Our data further emphasize the promising potential of MVA vector expressing internal antigens toward the development of a universal influenza vaccine.

Human-Animal Interface: The Case for Influenza Interspecies Transmission.

Since the 1990s, the threat of influenza viruses to veterinary and human public health has increased. This coincides with the larger global populations of poultry, pigs, and people and with changing ecological factors. These factors include the redistribution of the human population to cities, rapid mass transportation of people and infectious agents, increased global land use, climate change, and possible changes in viral ecology that perpetuate highly pathogenic influenza viruses in the aquatic bird reservoir. The emergence of H5N1, H7N9, and H9N2 subtypes of influenza A virus and the increased genetic exchange among influenza viruses in wild aquatic birds, domestic poultry, swine, and humans pose a continuing threat to humanity. Here we consider the fundamental and practical knowledge of influenza A viruses at the human-animal interfaces to facilitate the development of novel control strategies and modified agricultural practices that will reduce or prevent interspecies transmission.

A heat-inactivated H7N3 vaccine induces cross-reactive cellular immunity in HLA-A2.1 transgenic mice.

BACKGROUND: Cross-reactive immunity against heterologous strains of influenza virus has the potential to provide partial protection in individuals that lack the proper neutralizing antibodies. In particular, the boosting of memory CD8+ T cell responses to conserved viral proteins can attenuate disease severity caused by influenza virus antigenic variants or pandemic strains. However, little is yet known about which of these conserved internal antigens would better induce and/or recall memory CD8+ T cells after in vivo administration of an inactivated whole virus vaccine. METHODS: We explored the CD8 + T cell responses to selected epitopes of the internal proteins of an H7N3 influenza virus that were cross-reactive with A/PR/8/34 virus in HLA-A2.1 transgenic (AAD) mice. RESULTS: CD8+ T cells against dominant and subdominant epitopes were detected upon infection of mice with live H7N3 virus, whereas immunization with non-replicating virus elicited CD8+ T cell responses against mostly immunodominant epitopes, which were rapidly recalled following infection with A/PR/8/34 virus. These vaccine-induced T cell responses were able to reduce the lung viral load in mice challenged intranasally with the heterologous influenza virus. CONCLUSIONS: A single immunization with non-replicating influenza virus vaccines may be able to elicit or recall cross-reactive CD8+ T cell responses to conserved immunodominant epitopes and, to some extent, counteract an infection by heterologous virus.

Generation of switched memory B cells in response to vaccination in Down syndrome children and their siblings.

BACKGROUND: Immunodeficiency is an integral aspect of Down syndrome, as demonstrated by the increased susceptibility to infection of affected. Mortality is still higher than in general population, with respiratory infections among the major causes of death. As more people with Down syndrome are living today than ever before, it is indispensable to develop strategies to prevent and cure the associated disorders. Vaccination is the most successful instrument of preventive medicine. Special seasonal influenza and pneumococcal vaccination strategies have been designed for individuals with risk conditions of all ages. Down syndrome individuals are not included in the high-risk categories. METHODS: We enrolled in our study 15 children with Down syndrome and their siblings, vaccinated for the first time with seasonal influenza vaccine and receiving a booster dose of a glyco-conjugated pneumococcal vaccine. We compared the immunological features and response to vaccination measuring serum antibody titers and frequency of specific memory B cells. RESULTS: We confirm that a severe reduction of switched memory B cells is always associated to Down syndrome. After primary vaccination Down syndrome children generate significantly less specific switched memory B cells than their siblings. The response to a booster dose of vaccine is instead comparable in both groups. The production of specific antibodies was equally effective in Down syndrome and controls both after primary and secondary immunization. CONCLUSIONS: Down syndrome individuals should be considered a high risk group, because of their increased susceptibility to infection and reduced number of switched memory B cells. Tailored vaccination protocols are needed in order to reduce their burden of infections throughout life.

Human infection with highly pathogenic A(H7N7) avian influenza virus, Italy, 2013.

During an influenza A(H7N7) virus outbreak among poultry in Italy during August-September 2013, infection with a highly pathogenic A(H7N7) avian influenza virus was diagnosed for 3 poultry workers with conjunctivitis. Genetic analyses revealed that the viruses from the humans were closely related to those from chickens on affected farms.

Prostaglandin A1 inhibits avian influenza virus replication at a postentry level: Effect on virus protein synthesis and NF-κB activity.

Influenza A viruses (IAV) have the potential to cause devastating pandemics. In recent years, the emergence of new avian strains able to infect humans represents a serious threat to global human health. The increase in drug-resistant IAV strains underscores the need for novel approaches to anti-influenza chemotherapy. Herein we show that prostaglandin-A1 (PGA1) possesses antiviral activity against avian IAV, including H5N9, H7N1 and H1N1 strains, acting at a level different from the currently available anti-influenza drugs. PGA1 acts at postentry level, causing dysregulation of viral protein synthesis and preventing virus-induced disassembly of host microtubular network and activation of pro-inflammatory factor NF-κB. The antiviral activity is dependent on the presence of a cyclopentenone ring structure and is associated with activation of a cytoprotective heat shock response in infected cells. The results suggest that cyclopentenone prostanoids or prostanoids-derived molecules may represent a new tool to combat avian influenza virus infection.

B-sides serologic markers of immunogenicity in kidney transplanted patients: report from 2012-2013 flu vaccination experience.

BACKGROUND: Safety and immunogenicity data of seasonal influenza vaccination in transplanted patients (Tps) are controversial. Preexisting cross-reactive antibodies generated by repeated vaccination with drift variant strains could bias interpretation of immunogenicity data in Tp. METHODS: The unadjuvanted 2012-2013 seasonal influenza vaccine was administered to 81 kidney Tps being routinely vaccinated against influenza and 23 healthy controls (HCs). Immunogenicity was evaluated by both strain-specific antibody responses with standard hemagglutination inhibition assay and by memory B-cell enzyme-linked immunosorbent spot. Safety was also evaluated by measuring anti-human leukocyte antigen (HLA) antibodies. RESULTS: The majority of Tps were seroprotected before vaccination (81.5%, 81.5%, and 43.2% vs. 47.8%, 34.8%, and 30.4% in HC for H1N1, H3N2, and B strain, respectively) resulting into lower seroconversion rates (P≤0.01) as compared with HC (40.7%, 39.5%, and 54.3% vs. 73.9%, 82.6%, and 65.2% for H1N1, H3N2, and B strain, respectively). An inverse correlation was found between seroconversion rates and number of previous vaccinations in Tps. On the contrary, similar increase in the frequencies of strain-specific memory B cells were detected by B-cell enzyme-linked immunosorbent spot in both Tps and HCs after vaccination. No serious adverse events have been reported. Donor-specific HLA antibodies increased in two patients after vaccination, and de novo anti-HLA antibodies were identified in two additional patients (non-donor-specific HLA antibodies). CONCLUSION: This report on safety and immunogenicity of the seasonal unadjuvanted 2012-2013 flu vaccination suggests that evaluating immunogenicity of influenza vaccination exclusively by hemagglutination inhibition assay may be misleading in individuals receiving yearly seasonal vaccines. Further investigations are required to understand the relation between vaccination and anti-HLA antibody development.

Antibody responses to intradermal or intramuscular MF59-adjuvanted influenza vaccines as evaluated in elderly institutionalized volunteers during a season of partial mismatching between vaccine and circulating A(H3N2) strains.

BACKGROUND: The age-related weakening of the immune system makes elderly subjects less responsive to influenza vaccination. In the last years, two "enhanced vaccines" were licensed for individuals aged ≥65 years, one being a subunit vaccine (Fluad®) containing the MF59 adjuvant administered intramuscularly (IM-MF59) and the other one a split non-adjuvanted vaccine administered intradermally (Intanza® 15mcg) (ID). In the present study, we evaluated and compared the antibody responses against the three vaccine antigens and heterovariant A(H3N2) circulating viruses induced by IM-MF59 and ID influenza vaccines in 80 elderly institutionalized volunteers (40 per group) during the Winter season 2011-2012. RESULTS: Hemagglutination inhibiting (HI) antibody titers were assessed in blood samples collected before, 1 and 6 months after vaccination. One month after vaccination both the IM-MF59 and ID vaccines induced increases in HI titers against all the three vaccine strains. The results in the two groups were similar against the A(H3N2) and A(H1N1) strains. Responses against the B strain typically tended to be higher after ID than IM-MF59, yet both vaccines stimulated lower responses against the B strain than against the two A strains. The two vaccines induced favorable results also against four epidemic drifted A(H3N2) viruses circulating in Winter 2011-2012. Six months after vaccination, the HI titers decreased in both groups. CONCLUSION: The responses induced by IM-MF59 and ID vaccines in institutionalized elderly people were similar against the A(H3N2) and A(H1N1) strains but frequently higher, for the ID, against the B strain. The two vaccines induced positive responses against drifted A(H3N2) circulating viruses.

Is there a relation between genetic or social groups of mallard ducks and the circulation of low pathogenic avian influenza viruses?

We investigated the circulation dynamics of low pathogenic avian influenza viruses (LPAIVs) in the mallard (Anas platyrhynchos) reservoir in Italy. In particular, we evaluated the temporal distribution of virologic findings by combining virus isolation data with a new population genetic-based study approach. Thus, during 11 consecutive sampling periods (wintering periods between 1993/94 and 2003/04), categorised into 40 sampling sub-periods, cloacal swab samples were collected from 996 wild and 16 captive-reared mallards, to be screened by RT-PCR before attempting influenza A virus isolation in embryonated eggs. Forty-eight LPAIVs were isolated from wild mallards and antigenically characterised by haemagglutination-inhibition and neuraminidase-inhibition assays. When considering LPAIV antigenic subtypes in which more than one mallard tested virus isolation positive (H1N1, n. 22; H2N3, n. 2; H5N3, n. 2; H6N5, n. 3; H6N8, n. 2; H7N3, n. 3; H11N6, n. 5), at least two birds infected with a specific HN subtype clustered within one same sampling sub-period. In the context of the novel population genetic approach, total DNA was extracted from a subset of 16 captive-reared and 65 wild ducks (2000/01 and 2001/02 sampling periods) to assess genetic diversity by amplified fragment length polymorphisms (AFLP) markers. Analyses of AFLP results showed that captive-reared mallards clustered together, whereas two main independent clusters characterised the distribution pattern of most wild mallards. Within this subset of samples, nearly identical H7N3 LPAIV strains were isolated from two wild mallards belonging to the same genetic cluster. Blood sera were also collected from the above subset of mallards and examined for antibodies to the homologous H7N3 virus strain. Four out of six wild mallards testing H7N3-seropositive by haemagglutination-inhibition assay (2001/02 period) belonged to the genetic cluster including H7N3 virus shedding ducks. Overall, our data raise the possibility of an enhanced transmission and circulation of LPAIVs in genetic or social groups of wild mallards, gathered in flocks possibly related by parentage and/or geographic origin.

Chlamydophila pneumoniae, Mycoplasma pneumoniae, and influenza in children with respiratory infections in Alexandria, Egypt.

INTRODUCTION: Acute respiratory infections (ARI) are the leading cause of pediatric morbidity and mortality worldwide. Information about etiological agents of ARI in developing countries is still limited. METHODOLOGY: Throat swabs collected from children hospitalized with ARI between December 2009 and May 2010 were investigated for Chlamydophila pneumoniae, Mycoplasma pneumoniae, and influenza viruses by molecular analyses. RESULTS: This study conducted in Alexandria, Egypt, was designed to determine the prevalence of several microorganisms in 156 children hospitalized with ARI. Overall, samples from 76 individuals (49%) were found to be positive for at least one pathogen, and 10 of them were positive for two agents. C. pneumoniae was the most commonly detected agent, followed by M. pneumonia and H1N1 pandemic influenza virus. Positivity for C. pneumoniae was associated with colder months and mild disease of the upper respiratory tract such as laryngitis. CONCLUSIONS: Further studies are needed to identify other possible agents of ARI (e.g., RSV, adenoviruses, other bacterial infections) in this population and to better understand the causal role of atypical bacteria detected in respiratory samples.

Modified vaccinia virus Ankara expressing the hemagglutinin of pandemic (H1N1) 2009 virus induces cross-protective immunity against Eurasian 'avian-like' H1N1 swine viruses in mice.

OBJECTIVES: To examine cross-reactivity between hemagglutinin (HA) derived from A/California/7/09 (CA/09) virus and that derived from representative Eurasian "avian-like" (EA) H1N1 swine viruses isolated in Italy between 1999 and 2008 during virological surveillance in pigs. DESIGN: Modified vaccinia virus Ankara (MVA) expressing the HA gene of CA/09 virus (MVA-HA-CA/09) was used as a vaccine to investigate cross-protective immunity against H1N1 swine viruses in mice. SAMPLE: Two classical swine H1N1 (CS) viruses and four representative EA-like H1N1 swine viruses previously isolated during outbreaks of respiratory disease in pigs on farms in Northern Italy were used in this study. SETTING: Female C57BL/6 mice were vaccinated with MVA/HA/CA/09 and then challenged intranasally with H1N1 swine viruses. MAIN OUTCOME MEASURES: Cross-reactive antibody responses were determined by hemagglutination- inhibition (HI) and virus microneutralizing (MN) assays of sera from MVA-vaccinated mice. The extent of protective immunity against infection with H1N1 swine viruses was determined by measuring lung viral load on days 2 and 4 post-challenge. RESULTS AND CONCLUSIONS: Systemic immunization of mice with CA/09-derived HA, vectored by MVA, elicited cross-protective immunity against recent EA-like swine viruses. This immune protection was related to the levels of cross-reactive HI antibodies in the sera of the immunized mice and was dependent on the similarity of the antigenic site Sa of H1 HAs. Our findings suggest that the herd immunity elicited in humans by the pandemic (H1N1) 2009 virus could limit the transmission of recent EA-like swine HA genes into the influenza A virus gene pool in humans.

Antibody but not memory B-cell responses are tuned-down in vertically HIV-1 infected children and young individuals being vaccinated yearly against influenza.

Yearly immunization against seasonal influenza is highly recommended for HIV-1 infected individuals but evaluating the success of vaccination by serological markers may not be fully informative in this population. Recently, it has been hypothesized that the generation of long-lasting immune responses may depend on whether similar antigens challenge the immune system frequently and intermittently. In the present study, in order to search for additional correlates of vaccine-induced protective immunity and to further dissect this theory, both humoral and memory B-cell responses to the trivalent 2012-2013 seasonal influenza vaccination has been evaluated by strain-specific (separately for H1N1, H3N2 and B strain) standard hemagglutination inhibition (HI) assay and B-cell enzyme-linked immunosorbent spot (ELISpot) in a cohort of vertically HIV-1 infected children and young individuals as compared to age-matched healthy controls. A high number of HIV-1 infected individuals had protective antibody levels prior to vaccination and showed low seroconversion rates after vaccination as compared to healthy controls. On the contrary, similar frequencies of influenza-specific memory B-cells were detected by B-cell ELISpot in both groups suggesting that an adequate B-cell response has been elicited. Data from the H1N1 strain, which is recurrent in seasonal influenza vaccines since 2009, pointed out decreasing antibody but not memory B-cell responses for HIV-1 infected patients being vaccinated for a greater number of years. Further investigations are required to standardize the influenza-specific B-cell ELISpot and to understand whether it could be used routinely as an additional tool to evaluate response to influenza vaccination in immune-compromised individuals being vaccinated yearly.

Pandemic influenza A/H1N1pdm in Italy: age, risk and population susceptibility.

BACKGROUND: A common pattern emerging from several studies evaluating the impact of the 2009 A/H1N1 pandemic influenza (A/H1N1pdm) conducted in countries worldwide is the low attack rate observed in elderly compared to that observed in children and young adults. The biological or social mechanisms responsible for the observed age-specific risk of infection are still to be deeply investigated. METHODS: The level of immunity against the A/H1N1pdm in pre and post pandemic sera was determined using left over sera taken for diagnostic purposes or routine ascertainment obtained from clinical laboratories. The antibody titres were measured by the haemagglutination inhibition (HI) assay. To investigate whether certain age groups had higher risk of infection the presence of protective antibody (≥1∶40), was calculated using exact binomial 95% CI on both pre- and post- pandemic serological data in the age groups considered. To estimate age-specific susceptibility to infection we used an age-structured SEIR model. RESULTS: By comparing pre- and post-pandemic serological data in Italy we found age- specific attack rates similar to those observed in other countries. Cumulative attack rate at the end of the first A/H1N1pdm season in Italy was estimated to be 16.3% (95% CI 9.4%-23.1%). Modeling results allow ruling out the hypothesis that only age-specific characteristics of the contact network and levels of pre-pandemic immunity are responsible for the observed age-specific risk of infection. This means that age-specific susceptibility to infection, suspected to play an important role in the pandemic, was not only determined by pre-pandemic levels of H1N1pdm antibody measured by HI. CONCLUSIONS: Our results claim for new studies to better identify the biological mechanisms, which might have determined the observed pattern of susceptibility with age. Moreover, our results highlight the need to obtain early estimates of differential susceptibility with age in any future pandemics to obtain more reliable real time estimates of critical epidemiological parameters.

Reassortment ability of the 2009 pandemic H1N1 influenza virus with circulating human and avian influenza viruses: public health risk implications.

Exploring the reassortment ability of the 2009 pandemic H1N1 (A/H1N1pdm09) influenza virus with other circulating human or avian influenza viruses is the main concern related to the generation of more virulent or new variants having implications for public health. After different coinfection experiments in human A549 cells, by using the A/H1N1pdm09 virus plus one of human seasonal influenza viruses of H1N1 and H3N2 subtype or one of H11, H10, H9, H7 and H1 avian influenza viruses, several reassortant viruses were obtained. Among these, the HA of H1N1 was the main segment of human seasonal influenza virus reassorted in the A/H1N1pdm09 virus backbone. Conversely, HA and each of the three polymerase segments, alone or in combination, of the avian influenza viruses mainly reassorted in the A/H1N1pdm09 virus backbone. Of note, A/H1N1pdm09 viruses that reassorted with HA of H1N1 seasonal human or H11N6 avian viruses or carried different combination of avian origin polymerase segments, exerted a higher replication effectiveness than that of the parental viruses. These results confirm that reassortment of the A/H1N1pdm09 with circulating low pathogenic avian influenza viruses should not be misjudged in the prediction of the next pandemic.