An open-label phase I trial of a live attenuated H2N2 influenza virus vaccine in healthy adults.

BACKGROUND: Live attenuated influenza vaccines (LAIV) against a variety of strains of pandemic potential are being developed and tested. We describe the results of an open-label phase I trial of a live attenuated H2N2 virus vaccine. OBJECTIVES: To evaluate the safety, infectivity, and immunogenicity of a live attenuated H2N2 influenza virus vaccine. PARTICIPANTS/METHODS: The A/Ann Arbor/6/60 (H2N2) virus used in this study is the attenuated, cold-adapted, temperature-sensitive strain that provides the genetic backbone of seasonal LAIV (MedImmune). We evaluated the safety, infectivity, and immunogenicity of two doses of 10(7) TCID(50) of this vaccine administered by nasal spray 4 weeks apart to normal healthy seronegative adults. RESULTS: Twenty-one participants received a first dose of the vaccine; 18 participants received a second dose. No serious adverse events occurred during the trial. The most common adverse events after vaccination were headache and musculoskeletal pain. The vaccine was restricted in replication: 24% and 17% had virus detectable by culture or rRT-PCR after the first and second dose, respectively. Antibody responses to the vaccine were also restricted: 24% of participants developed an antibody response as measured by either hemagglutination-inhibition assay (10%), or ELISA for H2 HA-specific serum IgG (24%) or IgA (16%) after either one or two doses. None of the participants had a neutralizing antibody response. Vaccine-specific IgG-secreting cells as measured by enzyme-linked immunospot increased from a mean of 0·5 to 2·0/10(6) peripheral blood mononuclear cells (PBMCs); vaccine-specific IgA-secreting cells increased from 0·1 to 0·5/10(6) PBMCs. CONCLUSIONS: The live attenuated H2N2 1960 AA ca vaccine demonstrated a safety profile consistent with seasonal trivalent LAIV but was restricted in replication and minimally immunogenic in healthy seronegative adults.

Plasmablast-derived polyclonal antibody response after influenza vaccination.

Conventional measurement of antibody responses to vaccines largely relies on serum antibodies, which are primarily produced by bone marrow plasma cells and may not represent the entire vaccine-induced B cell repertoire, including important functional components such as those targeted to mucosal sites. After immunization or infection, activated B cells differentiate into plasmablasts in local lymphoid organs, then traffic through circulation to the target sites where they further develop into plasma cells. On day 7 after influenza vaccination, a burst of plasmablasts, highly enriched for vaccine-specific antibody secreting cells, appears in the peripheral blood. This provides a unique window to the overall B cell response to the vaccine, without interference of pre-existing cross-reactive serum antibody. In this study we isolated B cells from volunteers on day 7 after immunization with the inactivated influenza vaccine and cultured them ex vivo to collect plasmablast-derived polyclonal antibodies (PPAb). The PPAb contained secreted IgG and IgA, which was approximately 0.2ng per antibody secreting cell. Influenza-specific IgG and IgA binding activity was detected in PPAb at dilutions up to 10(5) by ELISA. The ratio of the titers of influenza-specific IgA to IgG by ELISA was 4-fold higher in PPAb than in day 28 post-vaccination sera, suggesting that vaccine-induced IgA is enriched in PPAb compared to sera. Functional activity was also detected in PPAb as determined by microneutralization and hemagglutination inhibition assays. In addition to bulk B cell cultures, we also cultured plasmablast subsets sorted by cell surface markers to generate PPAb. These results suggest that PPAb better reflects the mucosal IgA response than serum samples. Since PPAb are exclusively produced by recently activated B cells, it allows assessing vaccine-induced antibody response without interference from pre-existing cross-reactive serum antibodies and permits an assessment of antibody avidity based on antigen specific binding and antibody quantity. Therefore this assay is particularly useful for studying vaccine/infection-induced antibodies against antigens that might have previously circulated, such as antibody responses to rotavirus, dengue or influenza viruses in which cross-reactive antibodies against different virus serotypes/subtypes play a critical role in immunity and/or pathogenesis.

A whole genome transcriptional analysis of the early immune response induced by live attenuated and inactivated influenza vaccines in young children.

The protective mechanisms of influenza vaccines in young children are not completely understood. A phase 2 clinical study was conducted in 85 children 12-35 months of age to describe and compare the immune responses to live attenuated influenza vaccine (LAIV) with trivalent inactivated influenza vaccine (TIV). To better understand the biology of vaccine effects, oligonucleotide microarrays were employed to measure the genome-wide changes in transcript profiles in whole blood at approximately 7 days after 1 dose of LAIV or TIV. Of the total 265 differentially expressed genes identified in this study, 6 clusters of genes were identified to be tightly coexpressed, many of which are likely modulated by cytokines including type 1 interferons (IFNs) and granulocyte-macrophage colony-stimulating factor. Additional functional analyses revealed that the type 1 IFN pathway and cell cycle regulation-related genes are enriched in the 6 coexpressed gene sets. Promoter characterization of these coexpressed genes also supported this conclusion. Moreover, it is suggested that the IFN-stimulated response element is likely to be a potential bidirectional promoter, and the CCAAT/enhancer-binding protein might cooperate with the E2F transcription factor family in the regulation of the cell cycle in the early immune response induced by the influenza vaccine. Overall, our study clearly indicates that the expression profile changes induced by LAIV are significantly different from those induced by TIV. These results suggest that the pattern of overexpression of type 1 IFN-stimulated genes can potentially be used as a biomarker to identify the early vaccination response of LAIV and may also explain, to a certain extent, previous clinical study observations of LAIV-induced protection against influenza-like illness in the first 2 weeks after administration.

Influence of prior influenza vaccination on antibody and B-cell responses.

Currently two vaccines, trivalent inactivated influenza vaccine (TIV) and live attenuated influenza vaccine (LAIV), are licensed in the USA. Despite previous studies on immune responses induced by these two vaccines, a comparative study of the influence of prior influenza vaccination on serum antibody and B-cell responses to new LAIV or TIV vaccination has not been reported. During the 2005/6 influenza season, we quantified the serum antibody and B-cell responses to LAIV or TIV in adults with differing influenza vaccination histories in the prior year: LAIV, TIV, or neither. Blood samples were collected on days 0, 7-9 and 21-35 after immunization and used for serum HAI assay and B-cell assays. Total and influenza-specific circulating IgG and IgA antibody secreting cells (ASC) in PBMC were detected by direct ELISPOT assay. Memory B cells were also tested by ELISPOT after polyclonal stimulation of PBMC in vitro. Serum antibody, effector, and memory B-cell responses were greater in TIV recipients than LAIV recipients. Prior year TIV recipients had significantly higher baseline HAI titers, but lower HAI response after vaccination with either TIV or LAIV, and lower IgA ASC response after vaccination with TIV than prior year LAIV or no vaccination recipients. Lower levels of baseline HAI titer were associated with a greater fold-increase of HAI titer and ASC number after vaccination, which also differed by type of vaccine. Our findings suggest that the type of vaccine received in the prior year affects the serum antibody and the B-cell responses to subsequent vaccination. In particular, prior year TIV vaccination is associated with sustained higher HAI titer one year later but lower antibody response to new LAIV or TIV vaccination, and a lower effector B-cell response to new TIV but not LAIV vaccination.

Baseline levels of influenza-specific CD4 memory T-cells affect T-cell responses to influenza vaccines.

BACKGROUND: Factors affecting immune responses to influenza vaccines have not been studied systematically. We hypothesized that T-cell and antibody responses to the vaccines are functions of pre-existing host immunity against influenza antigens. METHODOLOGY/PRINCIPAL FINDINGS: During the 2004 and 2005 influenza seasons, we have collected data on cellular and humoral immune reactivity to influenza virus in blood samples collected before and after immunization with inactivated or live attenuated influenza vaccines in healthy children and adults. We first used cross-validated lasso regression on the 2004 dataset to identify a group of candidate baseline correlates with T-cell and antibody responses to vaccines, defined as fold-increase in influenza-specific T-cells and serum HAI titer after vaccination. The following baseline parameters were examined: percentages of influenza-reactive IFN-gamma(+) cells in T and NK cell subsets, percentages of influenza-specific memory B-cells, HAI titer, age, and type of vaccine. The candidate baseline correlates were then tested with the independent 2005 dataset. Baseline percentage of influenza-specific IFN-gamma(+) CD4 T-cells was identified as a significant correlate of CD4 and CD8 T-cell responses, with lower baseline levels associated with larger T-cell responses. Baseline HAI titer and vaccine type were identified as significant correlates for HAI response, with lower baseline levels and the inactivated vaccine associated with larger HAI responses. Previously we reported that baseline levels of CD56(dim) NK reactivity against influenza virus inversely correlated with the immediate T-cell response to vaccination, and that NK reactivity induced by influenza virus depended on IL-2 produced by influenza-specific memory T-cells. Taken together these results suggest a novel mechanism for the homeostasis of virus-specific T-cells, which involves interaction between memory helper T-cells, CD56(dim) NK and DC. SIGNIFICANCE: These results demonstrate that assessment of baseline biomarkers may predict immunologic outcome of influenza vaccination and may reveal some of the mechanisms responsible for variable immune responses following vaccination and natural infection.

Phenotypic changes in influenza-specific CD8+ T cells after immunization of children and adults with influenza vaccines.

The effect of trivalent inactivated influenza vaccine (TIV) or live attenuated influenza vaccine (LAIV) on the phenotypes of circulating influenza-specific CD8+ T cells was analyzed by interferon (IFN)-gamma flow cytometry and tetramer staining. In adults, the expression of the T cell differentiation marker CD27 on virus-specific CD8+ T cells decreased after LAIV but increased after TIV. In children, expression of the cytotoxicity molecule perforin in influenza-specific CD8+ T cells increased after TIV but not after LAIV. Among children aged 6 months to 4 years who had not been vaccinated previously and who received 2 doses of TIV, CD27 expression decreased after each dose, whereas perforin expression increased after the second dose. These findings indicate that the phenotypic changes of influenza-specific CD8+ T cells differ depending on the type of vaccine and the age of the vaccinee. These differences are potentially affected by the different routes of vaccination and pathways of antigen presentation for TIV and LAIV.

Comparison of the influenza virus-specific effector and memory B-cell responses to immunization of children and adults with live attenuated or inactivated influenza virus vaccines.

Cellular immune responses to influenza virus infection and influenza virus vaccination have not been rigorously characterized. We quantified the effector and memory B-cell responses in children and adults after administration of either live attenuated (LAIV) or inactivated (TIV) influenza virus vaccines and compared these to antibody responses. Peripheral blood mononuclear cells were collected at days 0, 7 to 12, and 27 to 42 after immunization of younger children (6 months to 4 years old), older children (5 to 9 years old), and adults. Influenza virus-specific effector immunoglobulin A (IgA) and IgG circulating antibody-secreting cells (ASC) and stimulated memory B cells were detected using an enzyme-linked immunospot assay. Circulating influenza virus-specific IgG and IgA ASC were detected 7 to 12 days after TIV and after LAIV immunization. Seventy-nine percent or more of adults and older children had demonstrable IgG ASC responses, while IgA ASC responses were detected in 29 to 53% of the subjects. The IgG ASC response rate to LAIV immunization in adults was significantly higher than the response rate measured by standard serum antibody assays (26.3% and 15.8% by neutralization and hemagglutination inhibition assays, respectively). IgG ASC and serum antibody responses were relatively low in the younger children compared to older children and adults. TIV, but not LAIV, significantly increased the percentage of circulating influenza virus-specific memory B cells detected at 27 to 42 days after immunization in children and adults. In conclusion, although both influenza vaccines are effective, we found significant differences in the B-cell and antibody responses elicited after LAIV or TIV immunization in adults and older children and between young children and older age groups.

Cellular immune responses in children and adults receiving inactivated or live attenuated influenza vaccines.

The patterns of cellular immune responses induced by live attenuated influenza vaccine (LAIV) versus those of the trivalent inactivated influenza vaccine (TIV) have not been studied extensively, especially in children. The goals of this study were to evaluate the effects of TIV and LAIV immunization on cellular immunity to live influenza A virus in children and adults and to explore factors associated with variations in responses to influenza vaccines among individuals. A gamma interferon (IFN-gamma) flow cytometry assay was used to measure IFN-gamma-producing (IFN-gamma+) NK and T cells in peripheral blood mononuclear cell cultures stimulated with a live influenza A virus strain before and after LAIV or TIV immunization of children and adults. The mean percentages of influenza A virus-specific IFN-gamma+ CD4 and CD8 T cells increased significantly after LAIV, but not TIV, immunization in children aged 5 to 9 years. No increases in the mean levels of influenza A virus-reactive IFN-gamma+ T cells and NK cells were observed in adults given LAIV or TIV. TIV induced a significant increase in influenza A virus-reactive T cells in 6-month- to 4-year-old children; LAIV was not evaluated in this age group. The postvaccination changes (n-fold) in the percentages of influenza A virus-reactive IFN-gamma+ T and NK cells in adults were highly variable and correlated inversely with the prevaccination percentages, in particular with that of the CD56(dim) NK cell subset. In conclusion, our findings identify age, type of vaccine, and prevaccination levels of immune reactivity to influenza A virus as factors significantly associated with the magnitude of cellular immune responses to influenza vaccines.

Human cytomegalovirus plasmid-based amplicon vector system for gene therapy.

We have constructed and evaluated the utility of a helper-dependent virus vector system that is derived from Human Cytomegalovirus (HCMV). This vector is based on the herpes simplex virus (HSV) amplicon system and contains the HCMV orthologs of the two cis-acting functions required for replication and packaging of HSV genomes, the complex HCMV viral DNA replication origin (oriLyt), and the cleavage packaging signal (the a sequence). The HCMV amplicon vector replicated independently and was packaged into infectious virions in the presence of helper virus. This vector is capable of delivering and expressing foreign genes in infected cells including progenitor cells such as human CD34+ cells. Packaged defective viral genomes were passaged serially in fibroblasts and could be detected at passage 3; however, the copy number appeared to diminish upon serial passage. The HCMV amplicon offers an alternative vector strategy useful for gene(s) delivery to cells of the hematopoietic lineage.

T cell-dependent production of IFN-gamma by NK cells in response to influenza A virus.

The role of human NK cells in viral infections is poorly understood. We used a cytokine flow-cytometry assay to simultaneously investigate the IFN-gamma response of NK and T lymphocytes to influenza A virus (fluA). When PBMCs from fluA-immune adult donors were incubated with fluA, IFN-gamma was produced by both CD56(dim) and CD56(bright) subsets of NK cells, as well as by fluA-specific T cells. Purified NK cells did not produce IFN-gamma in response to fluA, while depletion of T lymphocytes reduced to background levels the fluA-induced IFN-gamma production by NK cells, which indicates that T cells are required for the IFN-gamma response of NK cells. The fluA-induced IFN-gamma production of NK cells was suppressed by anti-IL-2 Ab, while recombinant IL-2 replaced the helper function of T cells for IFN-gamma production by NK cells. This indicates that IL-2 produced by fluA-specific T cells is involved in the T cell-dependent IFN-gamma response of NK cells to fluA. Taken together, these results suggest that at an early stage of recurrent viral infection, NK-mediated innate immunity to the virus is enhanced by preexisting virus-specific T cells.

Analysis of the frequencies and of the memory T cell phenotypes of human CD8+ T cells specific for influenza A viruses.

We characterized the human CD8+ T cell response against influenza A viruses by a flow cytometry-based assay. Peripheral blood mononuclear cells (PBMCs) were incubated with inactivated influenza virus preparation, for 17 h, and were stained for intracellular interferon-gamma. Major histocompatibility complex class I-restricted memory CD8+ T cells specific for influenza antigens were detected in PBMCs from all 19 adult donors, at an average frequency of 0.39%. On average, 83% of influenza virus-specific CD8+ T cells expressed the differentiation-associated marker CD27, a percentage that is significantly higher than that of CD8+ T cells specific for pp65 of human cytomegalovirus (53%). These observations indicate that class I-restricted immunity against influenza A viruses is characterized by the persistence, after clearance of infection, of circulating antigen-specific CD8+ T cells. The different patterns of CD27 expression in influenza virus- and cytomegalovirus-specific CD8+ T cells suggest that influenza virus-specific memory and effector CD8+ T cells can be differentiated by phenotypic analysis.

Cytomegalovirus UL97 phosphotransferase mutations that affect susceptibility to ganciclovir.

Most ganciclovir (GCV)-resistant cytomegalovirus (CMV) isolates contain UL97 gene mutations at codon 460 or 520 or between codons 590 and 607, where an increasing variety of mutations have been detected, including deletions. To determine their phenotypic effect, 9 UL97 mutations not previously studied were transferred to drug-sensitive laboratory CMV strains that contained unique restriction sites developed for this purpose. Deletion of the entire codon range 591-607 conferred a 6-fold increase in GCV resistance, with little effect on viral replication. Some mutations found in clinical isolates, including C592G and A594T, conferred only 2-3-fold decreases in GCV susceptibility. For C592G, this phenotype was confirmed by transfer to different CMV strains and by restoration of full drug susceptibility after removal of the mutation. Low drug levels resulting from oral GCV therapy may predispose the virus to the initial selection of these low-grade UL97 resistance mutations and to later accumulation of other mutations and greater resistance.