IL-15 adjuvanted multivalent vaccinia-based universal influenza vaccine requires CD4+ T cells for heterosubtypic protection.

Current influenza vaccines are ineffective against novel viruses and the source or the strain of the next outbreak of influenza is unpredictable; therefore, establishing universal immunity by vaccination to limit the impact of influenza remains a high priority. To meet this challenge, a novel vaccine has been developed using the immunogenic live vaccinia virus as a vaccine vector, expressing multiple H5N1 viral proteins (HA, NA, M1, M2, and NP) together with IL-15 as a molecular adjuvant. Previously, this vaccine demonstrated robust sterile cross-clade protection in mice against H5 influenza viruses, and herein its use has been extended to mediate heterosubtypic immunity toward viruses from both group 1 and 2 HA lineages. The vaccine protected mice against lethal challenge by increasing survival and significantly reducing lung viral loads against the most recent human H7N9, seasonal H3N2, pandemic-2009 H1N1, and highly pathogenic H7N7 influenza A viruses. Influenza-specific antibodies elicited by the vaccine failed to neutralize heterologous viruses and were unable to confer protection by passive transfer. Importantly, heterologous influenza-specific CD4(+) and CD8(+) T-cell responses that were elicited by the vaccine were effectively recalled and amplified following viral challenge in the lungs and periphery. Selective depletion of T-cell subsets in the immunized mice revealed an important role for CD4(+) T cells in heterosubtypic protection, despite low sequence conservation among known MHC-II restricted epitopes across different influenza viruses. This study illustrates the potential utility of our multivalent Wyeth/IL-15/5Flu as a universal influenza vaccine with a correlate of protective immunity that is independent of neutralizing antibodies.

Generation of Live Attenuated Influenza Virus by Using Codon Usage Bias.

UNLABELLED: Seasonal influenza epidemics and occasional pandemics threaten public health worldwide. New alternative strategies for generating recombinant viruses with vaccine potential are needed. Interestingly, influenza viruses circulating in different hosts have been found to have distinct codon usage patterns, which may reflect host adaptation. We therefore hypothesized that it is possible to make a human seasonal influenza virus that is specifically attenuated in human cells but not in eggs by converting its codon usage so that it is similar to that observed from avian influenza viruses. This approach might help to generate human live attenuated viruses without affecting their yield in eggs. To test this hypothesis, over 300 silent mutations were introduced into the genome of a seasonal H1N1 influenza virus. The resultant mutant was significantly attenuated in mammalian cells and mice, yet it grew well in embryonated eggs. A single dose of intranasal vaccination induced potent innate, humoral, and cellular immune responses, and the mutant could protect mice against homologous and heterologous viral challenges. The attenuated mutant could also be used as a vaccine master donor strain by introducing hemagglutinin and neuraminidase genes derived from other strains. Thus, our approach is a successful strategy to generate attenuated viruses for future application as vaccines. IMPORTANCE: Vaccination has been one of the best protective measures in combating influenza virus infection. Current licensed influenza vaccines and their production have various limitations. Our virus attenuation strategy makes use of the codon usage biases of human and avian influenza viruses to generate a human-derived influenza virus that is attenuated in mammalian hosts. This method, however, does not affect virus replication in eggs. This makes the resultant mutants highly compatible with existing egg-based vaccine production pipelines. The viral proteins generated from the codon bias mutants are identical to the wild-type viral proteins. In addition, our massive genome-wide mutational approach further minimizes the concern over reverse mutations. The potential use of this kind of codon bias mutant as a master donor strain to generate other live attenuated viruses is also demonstrated. These findings put forward a promising live attenuated influenza vaccine generation strategy to control influenza.

Viral reassortment as an information exchange between viral segments.

Viruses have an extraordinary ability to diversify and evolve. For segmented viruses, reassortment can introduce drastic genomic and phenotypic changes by allowing a direct exchange of genetic material between coinfecting strains. For instance, multiple influenza pandemics were caused by reassortments of viruses typically found in separate hosts. What is unclear, however, are the underlying mechanisms driving these events and the level of intrinsic bias in the diversity of strains that emerge from coinfection. To address this problem, previous experiments looked for correlations between segments of strains that coinfect cells in vitro. Here, we present an information theory approach as the natural mathematical framework for this question. We study, for influenza and other segmented viruses, the extent to which a virus's segments can communicate strain information across an infection and among one another. Our approach goes beyond previous association studies and quantifies how much the diversity of emerging strains is altered by patterns in reassortment, whether biases are consistent across multiple strains and cell types, and if significant information is shared among more than two segments. We apply our approach to a new experiment that examines reassortment patterns between the 2009 H1N1 pandemic and seasonal H1N1 strains, contextualizing its segmental information sharing by comparison with previously reported strain reassortments. We find evolutionary patterns across classes of experiments and previously unobserved higher-level structures. Finally, we show how this approach can be combined with virulence potentials to assess pandemic threats.

A novel molecular test for influenza B virus detection and lineage differentiation.

Contemporary influenza B viruses are classified into two groups known as Yamagata and Victoria lineages. The co-circulation of two viral lineages in recent years urges for a robust and simple diagnostic test for detecting influenza B viruses and for lineage differentiation. In this study, a SYBR green-based asymmetric PCR assay has been developed for influenza B virus detection. Apart from identifying influenza B virus, the assay contains sequence-specific probes for lineage differentiation. This allows identifying influenza B virus and detecting influenza B viral lineage in a single reaction. The test has been evaluated by a panel of respiratory specimens. Of 108 influenza B virus-positive specimens, 105 (97%) were positive in this assay. None of the negative control respiratory specimens were positive in the test (N = 60). Viral lineages of all samples that are positive in the assay (N = 105) can also be classified correctly. These results suggest that this assay has a potential for routine influenza B virus surveillance.

Hemagglutinin-neuraminidase balance confers respiratory-droplet transmissibility of the pandemic H1N1 influenza virus in ferrets.

A novel reassortant derived from North American triple-reassortant (TRsw) and Eurasian swine (EAsw) influenza viruses acquired sustained human-to-human transmissibility and caused the 2009 influenza pandemic. To identify molecular determinants that allowed efficient transmission of the pandemic H1N1 virus among humans, we evaluated the direct-contact and respiratory-droplet transmissibility in ferrets of representative swine influenza viruses of different lineages obtained through a 13-y surveillance program in southern China. Whereas all viruses studied were transmitted by direct contact with varying efficiency, respiratory-droplet transmissibility (albeit inefficient) was observed only in the TRsw-like A/swine/Hong Kong/915/04 (sw915) (H1N2) virus. The sw915 virus had acquired the M gene derived from EAsw and differed from the gene constellation of the pandemic H1N1 virus by the neuraminidase (NA) gene alone. Glycan array analysis showed that pandemic H1N1 virus A/HK/415742/09 (HK415742) and sw915 possess similar receptor-binding specificity and affinity for α2,6-linked sialosides. Sw915 titers in differentiated normal human bronchial epithelial cells and in ferret nasal washes were lower than those of HK415742. Introducing the NA from pandemic HK415742 into sw915 did not increase viral replication efficiency but increased respiratory-droplet transmissibility, despite a substantial amino acid difference between the two viruses. The NA of the pandemic HK415742 virus possessed significantly higher enzyme activity than that of sw915 or other swine influenza viruses. Our results suggest that a unique gene constellation and hemagglutinin-neuraminidase balance play a critical role in acquisition of efficient and sustained human-to-human transmissibility.

Entry of influenza A Virus with a α2,6-linked sialic acid binding preference requires host fibronectin.

The receptor binding specificity of influenza A virus is one of the major determinants of viral tropism and host specificity. In general, avian viral hemagglutinin prefers to bind to α2,3-linked sialic acid, whereas the human viral hemagglutinin prefers to bind to α2,6-linked sialic acid. Here, we demonstrate that host fibronectin protein plays an important role in the life cycle of some influenza A viruses. Treating cells with anti-fibronectin antibodies or fibronectin-specific small interfering RNA can inhibit the virus replication of human H1N1 influenza A viruses. Strikingly, these inhibitory effects cannot be observed in cells infected with H5N1 viruses. By using reverse genetics techniques, we observed that the receptor binding specificity, but not the origin of the hemagglutinin subtype, is responsible for this differential inhibitory effect. Changing the binding preference of hemagglutinin from α2,6-linked sialic acid to α2,3-linked sialic acid can make the virus resistant to the anti-fibronectin antibody treatment and vice versa. Our further characterizations indicate that anti-fibronectin antibody acts on the early phase of viral replication cycle, but it has no effect on the initial binding of influenza A virus to cell surface. Our subsequent investigations further show that anti-fibronectin antibody can block the postattachment entry of influenza virus. Overall, these results indicate that the sialic acid binding preference of influenza viral hemagglutinin can modulate the preferences of viral entry pathways, suggesting that there are subtle differences between the virus entries of human and avian influenza viruses.

Molecular detection of human H7N9 influenza A virus causing outbreaks in China.

BACKGROUND: A novel subtype of influenza A virus (H7N9) was recently identified in humans. The virus is a reassortant of avian viruses, but these human isolates contain mutations [hemagglutinin (HA) Q226L and PB2 E627K] that might make it easier for the virus to adapt to mammalian hosts. Molecular tests for rapid detection of this virus are urgently needed. METHODS: We developed a 1-step quantitative real-time reverse-transcription PCR assay to detect the novel human H7N9 virus. The primer set was specific to the hemagglutinin (HA) gene of the H7N9 viruses currently causing the outbreak in China and had mismatches to all previously known avian or mammalian H7 HA sequences. In addition, the assay was evaluated using influenza A viruses of various genetic backgrounds and other negative controls. RESULTS: The detection limit of the assay was approximately 0.04 TCID50 (median tissue culture infective dose) per reaction. The assay specificity was high and all negative control samples, including 8 H7 viruses not closely related to the human H7N9 virus, tested negative. CONCLUSIONS: The established assay allows rapid detection of the novel human H7N9 virus, thereby allowing better pandemic preparedness.

Rapid genotyping of swine influenza viruses.

The emergence of pandemic (H1N1) 2009 virus highlighted the need for enhanced surveillance of swine influenza viruses. We used real-time reverse-transcription PCR-based genotyping and found that this rapid and simple genotyping method may identify reassortants derived from viruses of Eurasian avian-like, triple reassortant-like, and pandemic (H1N1) 2009 virus lineages.

DNA intercalator stimulates influenza transcription and virus replication.

Influenza A virus uses its host transcription machinery to facilitate viral RNA synthesis, an event that is associated with cellular RNA polymerase II (RNAPII). In this study, various RNAPII transcription inhibitors were used to investigate the effect of RNAPII phosphorylation status on viral RNA transcription. A low concentration of DNA intercalators, such as actinomycin D (ActD), was found to stimulate viral polymerase activity and virus replication. This effect was not observed in cells treated with RNAPII kinase inhibitors. In addition, the loss of RNAPII(a) in infected cells was due to the shift of nonphosphorylated RNAPII (RNAPII(a)) to hyperphosphorylated RNAPII (RNAPII(o)).

Rapid detection of reassortment of pandemic H1N1/2009 influenza virus.

BACKGROUND: Influenza viruses can generate novel reassortants in coinfected cells. The global circulation and occasional introductions of pandemic H1N1/2009 virus in humans and in pigs, respectively, may allow this virus to reassort with other influenza viruses. These possible reassortment events might alter virulence and/or transmissibility of the new reassortants. Investigations to detect such possible reassortants should be included as a part of pandemic influenza surveillance plans. METHODS: We established a real-time reverse-transcription (RT)-PCR-based strategy for the detection of reassortment of pandemic H1N1/2009 virus. Singleplex SYBR green-based RT-PCR assays specific for each gene segment of pandemic H1N1/2009 were developed. These assays were evaluated with influenza viruses of various genetic backgrounds. RESULTS: All human pandemic H1N1 (n = 27) and all seasonal human (n = 58) isolates were positive and negative, respectively, for all 8 segments. Of 48 swine influenza viruses isolated from our ongoing surveillance program of influenza viruses in swine, 10 were positive in all reactions. All 8 viral segments of these 10 samples were confirmed to be of pandemic H1N1 origin, indicating that these were caused by zoonotic transmissions from human to pigs. The 38 swine viruses that were nonpandemic H1N1/2009 had 1-6 gene segments positive in the tests. Further characterization of these nonpandemic H1N1/2009 swine viruses indicated that these PCR-positive genes were the precursor genes of the pandemic H1N1/2009 virus. CONCLUSIONS: Our results demonstrated that these assays can detect reintroductions of pandemic H1N1/2009 virus in pigs. These assays might be useful screening tools for identifying viral reassortants derived from pandemic H1N1/2009 or its precursors.

Unrelated cord blood transplantation for severe combined immunodeficiency and other primary immunodeficiencies.

HCT is currently the treatment of choice for children with severe primary immunodeficiencies (PIDs). Frequently, these patients lack an HLA-identical sibling donor, and umbilical cord blood (UCB) transplantation may be an option; however, experience in this field remains scant. Fifteen children with PID (SCID 11, X-linked lymphoproliferative syndrome 2, Omenn's syndrome 1, Wiskott-Aldrich syndrome 1) received a UCB transplant. The donor was unrelated in 14 cases and related in 1. Median age at transplant was 11.6 months (range, 2.9-68.0) and median weight 7 kg (range, 4-21). Thirteen patients were conditioned with busulphan and cyclophosphamide and 2 with fludarabine and melphalan. Nine patients received antithymocyte globulin. Median NC x 10(7)/kg infused was 7.9 (range, 2.9-25.0) and median CD34 x 10(5)/kg 2.9 (range, 1.0-7.9). All patients engrafted. Median days to >0.5 x 10(9)/l neutrophils was 31. Eight patients developed acute graft-versus-host disease (GvHD) grades II-IV and one chronic GvHD. Viral and fungal infections were frequent. Four patients died: three from GvHD grade IV complicated by infection and one from progressive interstitial lung disease. Five-year survival was 0.73+/-0.12. All surviving patients presented complete immunologic reconstitution. No patient is intravenous immunoglobulin (IVIg) replacement therapy-dependent. UCB transplantation is a valid option for children with PID who lack an HLA-identical sibling donor.

Heterologous influenza vRNA segments with identical non-coding sequences stimulate viral RNA replication in trans.

The initiation of transcription and replication of influenza A virus requires the 5' and 3' ends of vRNA. Here, the role of segment-specific non-coding sequences of influenza A virus on viral RNA synthesis was studied. Recombinant viruses, with the nonstructural protein (NS) segment-specific non-coding sequences replaced by the corresponding sequences of the neuraminidase (NA) segment, were characterized. The NS and NA vRNA levels in cells infected with these mutants were much higher than those of the wild type, whereas the NS and NA mRNA levels of the mutants were comparable to the wild-type levels. By contrast, the PB2 vRNA and mRNA levels of all the tested viruses were similar, indicating that vRNA with heterologous segment-specific non-coding sequences was not affected by the mutations. The observations suggested that, with the cooperation between the homologous 5' and 3'segment-specific sequences, the introduced mutations could specifically enhance the replication of NA and NS vRNA.

[Conservative treatment of pneumatosis intestinalis and pneumoperitoneum after bone marrow transplantation]. / Tratamiento conservador de la neumatosis intestinal y el neumoperitoneo postrasplante de médula osea.

OBJECTIVE: Pneumatosis intestinalis (PI) is a radiological sign that can be accompanied by pneumoperitoneum. It is not exclusive of neonatal necrotizing enterocolitis. It can also appear after bone marrow transplantation. We describe our experience with 6 patients diagnosed of PI after bone marrow transplantation (BMT) who were treated conservatively without surgery in any case and good outcome. PATIENTS AND METHOD: We have reviewed the patients diagnosed of PI from 2000 to 2007 after BMT in our center. RESULTS: Six patients have had 7 episodes of PI with pneumoperitoneum in 3. All cases previously developed intestinal graft-versus-host disease. PI was diagnosed from 1 to 4 months after transplantation. At diagnosis, any patient presented peritoneal signs. Computed tomography was used for PI diagnosis with colonic predominance (5), pneumomediastinum (1) and retropneumoperitoneum (2). The treatment was conservative with intestinal rest, antibiotics and total parenteral nutrition. Enteral feeding was initiated progressively between 1 and 2 months after diagnosis but in one case PI reappeared and it required to start again the conservative treatment. In the other cases, outcome was very satisfactory, improving the pneumatosis and with a correct oral feeding without needing of surgery in any case. COMMENTS: PI with or without pneumoperitoneum is an condition to have in mind in bone marrow transplantation patients. Pneumoperitoneum with good general condition and no sign of peritonitis is not indicative of surgery in these patients. Conservative treatment with antibiotics and parenteral nutrition allows resolution spontaneously

Combined use of live-attenuated and inactivated influenza vaccines to enhance heterosubtypic protection.

The limited protection of current commerical vaccines necessitates the investigation of novel vaccine strategies for unpredictable outbreaks. To investigate the feasibility of using vaccines derived from Group 1 influenza A virus to induce broadly cross-reactive immune responses against multiple influenza subtypes, we tested a panel of sequential 4-dose immunization regimens in mice. Mice were treated with inactivated (seasonal H1N1, pandemic H1N1 and H5N1) and vaccinia virus-based H5N1 live-attenuated vaccines in different combinations. Mice were then challenged by viruses of either Group 1 (H1N1) or Group 2 (H3N2, H7N7) influenza virus. All studied sequential 4-dose vaccinations could induce some degrees of heterosubtypic protection in mice. Amongst all these regimens, the combined use of inactivated and live-attenuated vaccines could achieve the best heterologous protection. These results highlight the synergistic effect of combining different vaccine platforms to enhance heterosubtypic protection against influenza viruses.

Protection by universal influenza vaccine is mediated by memory CD4 T cells.

There is a diverse array of influenza viruses which circulate between different species, reassort and drift over time. Current seasonal influenza vaccines are ineffective in controlling these viruses. We have developed a novel universal vaccine which elicits robust T cell responses and protection against diverse influenza viruses in mouse and human models. Vaccine mediated protection was dependent on influenza-specific CD4+ T cells, whereby depletion of CD4+ T cells at either vaccination or challenge time points significantly reduced survival in mice. Vaccine memory CD4+ T cells were needed for early antibody production and CD8+ T cell recall responses. Furthermore, influenza-specific CD4+ T cells from vaccination manifested primarily Tfh and Th1 profiles with anti-viral cytokine production. The vaccine boosted H5-specific T cells from human PBMCs, specifically CD4+ and CD8+ T effector memory type, ensuring the vaccine was truly universal for its future application. These findings have implications for the development and optimization of T cell activating vaccines for universal immunity against influenza.

Reliable universal RT-PCR assays for studying influenza polymerase subunit gene sequences from all 16 haemagglutinin subtypes.

Realizable one-step RT-PCR assays specific for influenza PB2, PB1 and PA segments are described in this report. The designs of the consensus primers were based on more than five thousands polymerase genes derived from avian or mammalian viral strains. All the viral RNA tested in this study could be consistently amplified by the assays. The reaction products were specific and could be used for direct DNA sequencing. These assays might be useful tools to study the sequences of these genes.

Allogeneic hemopoietic stem cell transplantation (HSCT) for Wiskott-Aldrich syndrome: a report of the Spanish Working Party for Blood and Marrow Transplantation in Children (GETMON).

Allogeneic stem cell transplantation is the only curative treatment for Wiskott-Aldrich syndrome. The authors retrospectively analyzed the outcome with this procedure in 13 patients with severe Wiskott-Aldrich syndrome transplanted in 5 Spanish centers from 1989 to 2006. A patient was transplanted twice from the same donor due to a late engraftment failure. Age at transplant ranged from 7 to 192 months (median 30 months). There were 10 matched donors (3 related and 7 unrelated), 2 mismatched unrelated, and 1 haploidentical. Conditioning regimen consisted of busulfan and cyclophosphamide (BuCy) in 11 cases and fludarabine and melfalan (1) or BuCy (1). ATG was added in transplants from non-genetically matched donors. GvHD prophylaxis consisted of cyclosporine and methotrexate in most patients plus T-cell depletion in the haploidentical HSCT. Nine of the 13 transplanted patients are alive with complete clinical, immunologic, and hematologic recovery 8-204 months (median 101 months) after HSCT. Eight surviving patients had been transplanted from matched donors (3 related and 5 unrelated) and 1 from a haploidentical donor. Four patients died, 2 transplanted from matched donors (1 from acute GvHD and organ failure, 1 from a lymphoproliferative disorder after a second transplant), and 2 transplanted from mismatched unrelated donors (1 from acute GvHD and organ failure, 1 from graft failure and infection). Allogeneic hemopoietic stem cell transplantation must be utilized in all patients with severe Wisckott-Aldrich syndrome, using the most suitable graft variant for each patient.

A simplified approach to improve the efficiency and safety of ex vivo hematopoietic gene therapy in fanconi anemia patients.

Fanconi anemia (FA) is an inherited DNA repair disorder characterized by genetic instability of cells lacking a functional FA/BRCA pathway. Previous studies have shown that in vitro stimulation of bone marrow cells (BMCs) from FA mice promotes apoptosis, reduces the reconstitution ability of the stem cells, and induces myelodysplasia and myeloid leukemia upon reinfusion of the cells. This suggests the convenience of adapting standard protocols of gene therapy to FA. Here we show that the reserve of BM progenitors in FA patients is generally below 20% of normal values. Because this reduced reserve could activate the cycling of BM progenitors, we developed a simplified protocol to transduce BMCs from FA patients with gammaretroviral vectors. We demonstrate that a short in vitro manipulation (12-24 hr) of fresh mononuclear BMCs is sufficient to transduce 42% of hematopoietic progenitors from FA-A patients, in the absence of in vitro prestimulation. When FANCA-expressing vectors were used, this simple procedure reversed the phenotype of the BM progenitors from these patients. We propose that our approach will be more efficient and safer compared with standard gene therapy protocols for FA.

Stalking influenza by vaccination with pre-fusion headless HA mini-stem.

Inaccuracies in prediction of circulating viral strain genotypes and the possibility of novel reassortants causing a pandemic outbreak necessitate the development of an anti-influenza vaccine with increased breadth of protection and potential for rapid production and deployment. The hemagglutinin (HA) stem is a promising target for universal influenza vaccine as stem-specific antibodies have the potential to be broadly cross-reactive towards different HA subtypes. Here, we report the design of a bacterially expressed polypeptide that mimics a H5 HA stem by protein minimization to focus the antibody response towards the HA stem. The HA mini-stem folds as a trimer mimicking the HA prefusion conformation. It is resistant to thermal/chemical stress, and it binds to conformation-specific, HA stem-directed broadly neutralizing antibodies with high affinity. Mice vaccinated with the group 1 HA mini-stems are protected from morbidity and mortality against lethal challenge by both group 1 (H5 and H1) and group 2 (H3) influenza viruses, the first report of cross-group protection. Passive transfer of immune serum demonstrates the protection is mediated by stem-specific antibodies. Furthermore, antibodies induced by these HA stems have broad HA reactivity, yet they do not have antibody-dependent enhancement activity.

High survival rate in infant acute leukemia treated with early high-dose chemotherapy and stem-cell support. Groupo Español de Trasplante de Médula Osea en Niños.

PURPOSE: Infants with acute leukemia have a poor prognosis when treated with conventional chemotherapy. It is still unknown if stem-cell transplantation (SCT) can improve the outcome of these patients. In the present study, we review our experience with SCT in infant acute leukemia to clarify this issue. PATIENTS AND METHODS: We report the results of 26 infants who were submitted to a SCT for acute leukemia. There were 15 cases of acute myeloid leukemia and 10 cases of acute lymphoid leukemia. One patient had a bilineal leukemia. Twenty-two patients were in their first complete response (CR1), three were in their second CR, and one was in relapse. Eight patients were submitted to allogeneic SCT, and 18 underwent autologous SCT. RESULTS: With a median follow-up of 67 months, the 5-year overall survival and disease-free survival (DFS) are 64% (SE = 9%) and 63% (SE = 10%), respectively. Autologous and allogeneic SCT offered similar outcome. There was not any transplant-related mortality, and all deaths were caused by relapse in the first 6 months after SCT. In multivariate analysis, the single factor associated with better DFS was an interval between CR1 and SCT of less than 4 months (P: <.025). CONCLUSION: SCT is a valid option in the treatment of infant acute leukemia, and it may overcome the high risk of relapse with conventional chemotherapy showing very reduced toxicity. This study suggests that SCT should be performed in CR1 in the early phase of the disease.