Haemagglutination inhibition and virus microneutralisation serology assays: use of harmonised protocols and biological standards in seasonal influenza serology testing and their impact on inter-laboratory variation and assay correlation: A FLUCOP collaborative study.

Introduction: The haemagglutination inhibition assay (HAI) and the virus microneutralisation assay (MN) are long-established methods for quantifying antibodies against influenza viruses. Despite their widespread use, both assays require standardisation to improve inter-laboratory agreement in testing. The FLUCOP consortium aims to develop a toolbox of standardised serology assays for seasonal influenza. Building upon previous collaborative studies to harmonise the HAI, in this study the FLUCOP consortium carried out a head-to-head comparison of harmonised HAI and MN protocols to better understand the relationship between HAI and MN titres, and the impact of assay harmonisation and standardisation on inter-laboratory variability and agreement between these methods. Methods: In this paper, we present two large international collaborative studies testing harmonised HAI and MN protocols across 10 participating laboratories. In the first, we expanded on previously published work, carrying out HAI testing using egg and cell isolated and propagated wild-type (WT) viruses in addition to high-growth reassortants typically used influenza vaccines strains using HAI. In the second we tested two MN protocols: an overnight ELISA-based format and a 3-5 day format, using reassortant viruses and a WT H3N2 cell isolated virus. As serum panels tested in both studies included many overlapping samples, we were able to look at the correlation of HAI and MN titres across different methods and for different influenza subtypes. Results: We showed that the overnight ELISA and 3-5 day MN formats are not comparable, with titre ratios varying across the dynamic range of the assay. However, the ELISA MN and HAI are comparable, and a conversion factor could possibly be calculated. In both studies, the impact of normalising using a study standard was investigated, and we showed that for almost every strain and assay format tested, normalisation significantly reduced inter-laboratory variation, supporting the continued development of antibody standards for seasonal influenza viruses. Normalisation had no impact on the correlation between overnight ELISA and 3-5 day MN formats.

Long-Term Serological Investigations of Influenza A Virus in Free-Living Wild Boars (Sus scrofa) from Northern Italy (2007-2014).

Influenza A viruses (IAV) have been repeatedly demonstrated to circulate in wild suid populations. In this study, serum samples were collected from 2618 free-ranging wild boars in a protected area of Northern Italy between 2007 and 2014, and firstly screened by enzyme-linked immunosorbent assay (ELISA) for the presence of antibodies against IAV. The ELISA-positive samples were further tested by hemagglutination inhibition (HI) assays performed using antigen strains representative of the four major swine IAV (sIAV) lineages circulating in Italy: avian-like swine H1N1, pandemic-like swine H1N1, human-like swine H1N2 and human-like swine H3N2. An overall seroprevalence of 5.5% (145/2618) was detected by ELISA, with 56.7% (80/141) of screened sera tests positive by HI assay. Antibodies against H1N1 subtypes were the most prevalent beginning in 2009-with the highest detection in the first quarter of the year-until 2013, although at a low level. In addition, antibodies to H3N2 subtype were found during six years (2007, 2009, 2010, 2011, 2012 and 2014) whereas H1N2 antibodies were detected in 2012 only. Of the HI-positive samples, 30% showed reactivity to both H1N1 and H3N2 subtypes. These results provide additional insight into the circulation dynamics of IAV in wild suid populations, suggesting the occurrence of sIAV spillover events from pigs to wild boars.

Serologic Evidence of Occupational Exposure to Avian Influenza Viruses at the Wildfowl/Poultry/Human Interface.

Ecological interactions between wild aquatic birds and outdoor-housed poultry can enhance spillover events of avian influenza viruses (AIVs) from wild reservoirs to domestic birds, thus increasing the related zoonotic risk to occupationally exposed workers. To assess serological evidence of AIV infection in workers operating in Northern Italy at the wildfowl/poultry interface or directly exposed to wildfowl, serum samples were collected between April 2005 and November 2006 from 57 bird-exposed workers (BEWs) and from 7 unexposed controls (Cs), planning three sample collections from each individual. Concurrently, AIV surveillance of 3587 reared birds identified 4 AIVs belonging to H10N7, H4N6 and H2N2 subtypes while serological analysis by hemagglutination inhibition (HI) assay showed recent infections caused by H1, H2, H4, H6, H10, H11, H12, and H13 subtypes. Human sera were analyzed for specific antibodies against AIVs belonging to antigenic subtypes from H1 to H14 by using HI and virus microneutralization (MN) assays as a screening and a confirmatory test, respectively. Overall, antibodies specific to AIV-H3, AIV-H6, AIV-H8, and AIV-H9 were found in three poultry workers (PWs) and seropositivity to AIV-11, AIV-H13-still detectable in October 2017-in one wildlife professional (WP). Furthermore, seropositivity to AIV-H2, accounting for previous exposure to the "extinct" H2N2 human influenza viruses, was found in both BEWs and Cs groups. These data further emphasize the occupational risk posed by zoonotic AIV strains and show the possible occurrence of long-lived antibody-based immunity following AIV infections in humans.

Epidemiological characteristics of COVID-19 cases and estimates of the reproductive numbers 1 month into the epidemic, Italy, 28 January to 31 March 2020.

BackgroundOn 20 February 2020, a locally acquired coronavirus disease (COVID-19) case was detected in Lombardy, Italy. This was the first signal of ongoing transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the country. The number of cases in Italy increased rapidly and the country became the first in Europe to experience a SARS-CoV-2 outbreak.AimOur aim was to describe the epidemiology and transmission dynamics of the first COVID-19 cases in Italy amid ongoing control measures.MethodsWe analysed all RT-PCR-confirmed COVID-19 cases reported to the national integrated surveillance system until 31 March 2020. We provide a descriptive epidemiological summary and estimate the basic and net reproductive numbers by region.ResultsOf the 98,716 cases of COVID-19 analysed, 9,512 were healthcare workers. Of the 10,943 reported COVID-19-associated deaths (crude case fatality ratio: 11.1%) 49.5% occurred in cases older than 80 years. Male sex and age were independent risk factors for COVID-19 death. Estimates of R0 varied between 2.50 (95% confidence interval (CI): 2.18-2.83) in Tuscany and 3.00 (95% CI: 2.68-3.33) in Lazio. The net reproduction number Rt in northern regions started decreasing immediately after the first detection.ConclusionThe COVID-19 outbreak in Italy showed a clustering onset similar to the one in Wuhan, China. R0 at 2.96 in Lombardy combined with delayed detection explains the high case load and rapid geographical spread. Overall, Rt in Italian regions showed early signs of decrease, with large diversity in incidence, supporting the importance of combined non-pharmacological control measures.

Whole genome and phylogenetic analysis of two SARS-CoV-2 strains isolated in Italy in January and February 2020: additional clues on multiple introductions and further circulation in Europe.

Whole genome sequences of SARS-CoV-2 obtained from two patients, a Chinese tourist visiting Rome and an Italian, were compared with sequences from Europe and elsewhere. In a phylogenetic tree, the Italian patient's sequence clustered with sequences from Germany while the tourist's sequence clustered with other European sequences. Some additional European sequences in the tree segregated outside the two clusters containing the patients' sequences. This suggests multiple SARS-CoV-2 introductions in Europe or virus evolution during circulation.

Eco-Virological Preliminary Study of Potentially Emerging Pathogens in Hedgehogs (Erinaceus europaeus) Recovered at a Wildlife Treatment and Rehabilitation Center in Northern Italy.

The Western European Hedgehog (Erinaceus europaeus) is one of the four hedgehog species belonging to the genus Erinaceus. Among them, E. amurensis is extant in East Asia's areas only, whereas E. europaeus, E. roumanicus and E. concolor are mainly found in Europe. E. europaeus is endemically distributed from western to central and southern Europe, including Italy. Western European hedgehogs' ecological and feeding habits, along with their high population densities, notable synanthropic attitudes, frequent contacts with sympatric wild and domestic species, including humans, implicate the possible involvement of E. europaeus in the ecology of potentially emerging viruses, such as coronaviruses, influenza A and influenza D viruses, canine distemper virus, pestiviruses and Aujeszky's disease virus. We examined 24 E. europaeus individuals found injured in urban and rural areas of Northern Italy. Of the 24 fecal samples collected and tested for the above-mentioned pathogens by both PCR-based and virus isolation methods, 14 were found PCR-positive for betacoronaviruses belonging to lineage C and related to the known Erinaceus coronaviruses (EriCoVs), as determined by partial sequencing of the virus genome. Our findings suggest that hedgehogs could be considered natural reservoirs of CoVs, and also act as chronic shedding carriers of these potentially emerging RNA viruses.

Moderate influenza vaccine effectiveness against A(H1N1)pdm09 virus, and low effectiveness against A(H3N2) subtype, 2018/19 season in Italy.

Background: Influenza vaccines are updated every year to match the vaccine strains with currently circulating viruses; consequently influenza vaccine effectiveness (IVE) has to be assessed annually.Research design and methods: A test-negative case-control study was conducted within the context of the Italian sentinel influenza surveillance network to estimate IVE by age group, virus subtype, and vaccine brand in medically attended laboratory-confirmed influenza.Results: In Italy, the 2018/19 influenza season was characterized by the co-circulation of influenza A(H1N1)pdm09 and A(H3N2) viruses. The adjusted IVE estimate in preventing influenza was moderate (44.8%, 95% CI: 18.8 to 62.5) against A(H1N1)pdm09, whereas there was no evidence of effectiveness (1.8%, 95% CI: -37.8 to 30.1) in persons affected by A(H3N2). IVE against A(H1N1)pdm09 decreased with age ranging from 65.7% to 13.1% among children/adolescents and elderly, respectively; moreover results suggest that Vaxigrip Tetra® was more effective against A(H1N1)pdm09 compared to Fluarix Tetra® [62.5% (95% CI: 34.3 to 78.6) vs 24.5% (95% CI: -40.6 to 59.6)]. Low effectiveness (35.2%, 95% CI: -50.8 to 72.1) against A(H3N2) was detected only in the elderly immunized with Fluad®.Conclusions: Findings suggest that influenza vaccines were low to moderately effective, probably due to a mismatch between circulating and vaccine strains.

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.

Virological Surveillance of Influenza in the eight epidemic seasons after the 2009 pandemic in Emilia-Romagna (Northern Italy).

BACKGROUND AND AIM OF THE WORK: Influenza virological surveillance is essential for monitoring the evolution of influenza viruses (IVs) as well as for annual updating of the vaccine composition. The aim of this study is to analyse IVs circulation in Emilia-Romagna during the eight epidemic seasons after the 2009 pandemic and to evaluate their match with seasonal vaccine strains. METHODS: A total of 7882 respiratory specimens from patients with influenza-like illness (ILI), were collected by regional sentinel practitioners and hospital physicians. Viral investigations were conducted by rRT-PCR assay. Genetic characterization was performed for a spatial-temporal representative number of influenza laboratory-confirmed specimens. RESULTS: Influenza-positive samples per season ranged between 28.9% (2013-2014) and 66.8% (2012-2013). Co-circulation of IVs type A and type B was observed in all seasons, although with a different intensity. In all seasons, the highest number of positive samples was recorded in younger patients aged 5-14 years with relative frequencies ranging from 40% in the 2013-2014 season and 78% in the 2012-2013 season. Since the 2009 pandemic, A/H1N1pdm09 IVs circulating were closely related to the vaccine strain A/California/7/2009. Antigenic mismatch between vaccine strain and A/H3N2 IVs was observed in the 2011-2012 and 2014-2015 seasons. During 2015-2016, 2016-2017 and 2017-2018 seasons a complete or nearly complete mismatch between the predominant influenza B lineage of IVs type B circulating and vaccine B lineage occurred. CONCLUSIONS: This analysis confirms the importance of the virological surveillance and highlights the need of a continuous monitoring of IVs circulation, to improve the most appropriate vaccination strategies. (www.actabiomedica.it).

The epidemiological signature of influenza B virus and its B/Victoria and B/Yamagata lineages in the 21st century.

We describe the epidemiological characteristics, pattern of circulation, and geographical distribution of influenza B viruses and its lineages using data from the Global Influenza B Study. We included over 1.8 million influenza cases occurred in thirty-one countries during 2000-2018. We calculated the proportion of cases caused by influenza B and its lineages; determined the timing of influenza A and B epidemics; compared the age distribution of B/Victoria and B/Yamagata cases; and evaluated the frequency of lineage-level mismatch for the trivalent vaccine. The median proportion of influenza cases caused by influenza B virus was 23.4%, with a tendency (borderline statistical significance, p = 0.060) to be higher in tropical vs. temperate countries. Influenza B was the dominant virus type in about one every seven seasons. In temperate countries, influenza B epidemics occurred on average three weeks later than influenza A epidemics; no consistent pattern emerged in the tropics. The two B lineages caused a comparable proportion of influenza B cases globally, however the B/Yamagata was more frequent in temperate countries, and the B/Victoria in the tropics (p = 0.048). B/Yamagata patients were significantly older than B/Victoria patients in almost all countries. A lineage-level vaccine mismatch was observed in over 40% of seasons in temperate countries and in 30% of seasons in the tropics. The type B virus caused a substantial proportion of influenza infections globally in the 21st century, and its two virus lineages differed in terms of age and geographical distribution of patients. These findings will help inform health policy decisions aiming to reduce disease burden associated with seasonal influenza.

Effectiveness of the trivalent MF59 adjuvated influenza vaccine in preventing hospitalization due to influenza B and A(H1N1)pdm09 viruses in the elderly in Italy, 2017 - 2018 season.

Background: Evidence on influenza vaccine effectiveness (VE) in preventing mortality and morbidity in the elderly is weak. Our aim was to measure the VE against severe outcomes in the elderly. Methods: We conducted a multicentre hospital-based test-negative design (TND) case-control study, during the 2017/18 season, in four Italian hospitals. The study population included individuals aged ≥65 years hospitalized with Severe Acute Respiratory Infections (SARI). Patients were classified as cases and controls based on the results of the PCR influenza testing. We estimated VE by virus subtypes and specific VE for the trivalent adjuvanted vaccine (TIVadj). Results: 502 patients with SARI were enrolled: 118 (23.5%) tested positive (cases) and 384 (76.5%) tested negative (controls) for influenza. The adjusted VE of 48.5% for all vaccines was comparable to the adjusted VE for the TIVadj vaccine (48.3%). Adjusted VE for the TIVadj vaccine was 67.5% for A(H1N1)pdm09 and 44.5% for B viruses. Conclusion: We show a moderate adjusted VE of the TIVadj against all viruses, a good adjusted VE against A(H1N1)pdm09 strains and a moderate adjusted VE against B strains, despite a mismatch between the B circulating lineage and the lineage included in the vaccine. This is likely due to the cross-protection among B strains induced by the TIVadj in elderly patients.

Integrase Defective Lentiviral Vector as a Vaccine Platform for Delivering Influenza Antigens.

Viral vectors represent an attractive technology for vaccine delivery. We exploited the integrase defective lentiviral vector (IDLV) as a platform for delivering relevant antigens within the context of the ADITEC collaborative research program. In particular, Influenza virus hemagglutinin (HA) and nucleoprotein (NP) were delivered by IDLVs while H1N1 A/California/7/2009 subunit vaccine (HAp) with or without adjuvant was used to compare the immune response in a murine model of immunization. In order to maximize the antibody response against HA, both IDLVs were also pseudotyped with HA (IDLV-HA/HA and IDLV-NP/HA, respectively). Groups of CB6F1 mice were immunized intramuscularly with a single dose of IDLV-NP/HA, IDLV-HA/HA, HAp alone, or with HAp together with the systemic adjuvant MF59. Six months after the vaccine prime all groups were boosted with HAp alone. Cellular and antibody responses to influenza antigens were measured at different time points after the immunizations. Mice immunized with HA-pseudotyped IDLVs showed similar levels of anti-H1N1 IgG over time, evaluated by ELISA, which were comparable to those induced by HAp + MF59 vaccination, but significantly higher than those induced by HAp alone. The boost with HAp alone induced an increase of antibodies in all groups, and the responses were maintained at higher levels up to 18 weeks post-boost. The antibody response was functional and persistent overtime, capable of neutralizing virus infectivity, as evaluated by hemagglutination inhibition and microneutralization assays. Moreover, since neuraminidase (NA)-expressing plasmid was included during IDLV preparation, immunization with IDLV-NP/HA and IDLV-HA/HA also induced functional anti-NA antibodies, evaluated by enzyme-linked lectin assay. IFNγ-ELISPOT showed evidence of HA-specific response in IDLV-HA/HA immunized animals and persistent NP-specific CD8+ T cell response in IDLV-NP/HA immunized mice. Taken together our results indicate that IDLV can be harnessed for producing a vaccine able to induce a comprehensive immune response, including functional antibodies directed toward HA and NA proteins present on the vector particles in addition to a functional T cell response directed to the protein transcribed from the vector.

Correction to: Detection and full genome characterization of two beta CoV viruses related to Middle East respiratory syndrome from bats in Italy.

CORRECTION: After Publication of the article [1], it has been brought to our attention that an author's name has been spelt incorrectly. The correct spelling should be "Massimo Ciccozzi", but it was previously included as "Massimo Cicozzi". The original version has now been revised to reflect this.

Quantitative Multiplexed Imaging Analysis Reveals a Strong Association between Immunogen-Specific B Cell Responses and Tonsillar Germinal Center Immune Dynamics in Children after Influenza Vaccination.

Generation of Ag-specific humoral responses requires the orchestrated development and function of highly specialized immune cells in secondary lymphoid organs. We used a multiparametric approach combining flow cytometry, confocal microscopy, and histocytometry to analyze, for the first time to our knowledge in children, tonsils from seasonal influenza-vaccinated children. We used these novel imaging assays to address the mucosal immune dynamics in tonsils investigating the spatial positioning, frequency, and phenotype of immune cells after vaccination. Vaccination was associated with a significantly higher frequency of follicular helper CD4 T cells compared with the unvaccinated control group. The imaging analysis revealed that potential suppressor (FOXP3hi) CD4 T cells are mainly located in extrafollicular areas. Furthermore, a significantly reduced frequency of both follicular and extrafollicular FOXP3hi CD4 T cells was found in the vaccine group compared with the control group. Levels of circulating CXCL13 were higher in those vaccinated compared with controls, mirroring an increased germinal center reactivity in the tonsils. Notably, a strong correlation was found between the frequency of tonsillar T follicular helper cells and tonsillar Ag-specific Ab-secreting cells. These data demonstrate that influenza vaccination promotes the prevalence of relevant immune cells in tonsillar follicles and support the use of tonsils as lymphoid sites for the study of germinal center reactions after vaccination in children.

Detection and full genome characterization of two beta CoV viruses related to Middle East respiratory syndrome from bats in Italy.

BACKGROUND: Middle East respiratory syndrome coronavirus (MERS-CoV), which belongs to beta group of coronavirus, can infect multiple host species and causes severe diseases in humans. Multiple surveillance and phylogenetic studies suggest a bat origin. In this study, we describe the detection and full genome characterization of two CoVs closely related to MERS-CoV from two Italian bats, Pipistrellus kuhlii and Hypsugo savii. METHODS: Pool of viscera were tested by a pan-coronavirus RT-PCR. Virus isolation was attempted by inoculation in different cell lines. Full genome sequencing was performed using the Ion Torrent platform and phylogenetic trees were performed using IQtree software. Similarity plots of CoV clade c genomes were generated by using SSE v1.2. The three dimensional macromolecular structure (3DMMS) of the receptor binding domain (RBD) in the S protein was predicted by sequence-homology method using the protein data bank (PDB). RESULTS: Both samples resulted positive to the pan-coronavirus RT-PCR (IT-batCoVs) and their genome organization showed identical pattern of MERS CoV. Phylogenetic analysis showed a monophyletic group placed in the Beta2c clade formed by MERS-CoV sequences originating from humans and camels and bat-related sequences from Africa, Italy and China. The comparison of the secondary and 3DMMS of the RBD of IT-batCoVs with MERS, HKU4 and HKU5 bat sequences showed two aa deletions located in a region corresponding to the external subdomain of MERS-RBD in IT-batCoV and HKU5 RBDs. CONCLUSIONS: This study reported two beta CoVs closely related to MERS that were obtained from two bats belonging to two commonly recorded species in Italy (P. kuhlii and H. savii). The analysis of the RBD showed similar structure in IT-batCoVs and HKU5 respect to HKU4 sequences. Since the RBD domain of HKU4 but not HKU5 can bind to the human DPP4 receptor for MERS-CoV, it is possible to suggest also for IT-batCoVs the absence of DPP4-binding potential. More surveillance studies are needed to better investigate the potential intermediate hosts that may play a role in the interspecies transmission of known and currently unknown coronaviruses with particular attention to the S protein and the receptor specificity and binding affinity.

Influenza vaccine effectiveness in Italy: Age, subtype-specific and vaccine type estimates 2014/15 season.

The 2014/15 influenza season in Europe was characterised by the circulation of influenza A(H3N2) viruses with an antigenic and genetic mismatch from the vaccine strain A/Texas/50/2012(H3N2) recommended for the Northern hemisphere for the 2014/15 season. Italy, differently from other EU countries where most of the subtyped influenza A viruses were H3N2, experienced a 2014/15 season characterized by an extended circulation of two influenza viruses: A(H1N1)pdm09 and A(H3N2), that both contributed substantially to morbidity. Within the context of the existing National sentinel influenza surveillance system (InfluNet) a test-negative case-control study was established in order to produce vaccine effectiveness (VE) estimates. The point estimates VE were adjusted by age group (<5; 5-15; 15-64; 65+ years), the presence of at least one chronic condition, target group for vaccination and need help for walking or bathing. In Italy, adjusted estimates of the 2014/15 seasonal influenza VE against medically attended influenza-like illness (ILI) laboratory-confirmed as influenza for all age groups were 6.0% (95%CI: -36.5 to 35.2%), 43.6% (95%CI: -3.7 to 69.3%), -84.5% (95%CI: (-190.4 to -17.2%) and 50.7% (95% CI: -2.5 to 76.3%) against any influenza virus, A(H1N1)pdm09, A(H3N2) and B, respectively. These results suggest evidence of good VE against A(H1N1)pdm09 and B viruses in Italy and evidence of lack of VE against A(H3N2) virus due to antigenic and genetic mismatch between circulating A(H3N2) and the respective 2014/15 vaccine strain.

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.

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.

Exploring mucosal immunization with a recombinant influenza virus carrying an HIV-polyepitope in mice with pre-existing immunity to influenza.

HIV-1 vaccines based on recombinant vectors have been developed to elicit immune responses; however, the failure of the STEP HIV-1 vaccine trial has caused concern regarding the impact on vaccine efficacy of pre-existing vector seropositivity in humans. By using a mouse model of infection, we evaluated the immune responses elicited by intranasal and vaginal immunization with the recombinant influenza virus WSN/CKG carrying the PCLUS3-P18 peptide and a Gag epitope in its hemagglutinin, and the impact of pre-existing vector immunity on protection against recombinant vaccinia virus challenge. We found that despite the protective immunity induced in naïve mice by the WSN/CKG virus via either route, the vaginal immunization of mice with pre-existing influenza immunity restricted vPE16 replication more significantly in the ovaries than intranasal immunization. Thus, successful vaccination strategies under limiting conditions, such as pre-existing vector immunity, require the local induction of mucosal immunity at the site of virus infection.