Immunomic analysis of the repertoire of T-cell specificities for influenza A virus in humans.

Continuing antigenic drift allows influenza viruses to escape antibody-mediated recognition, and as a consequence, the vaccine currently in use needs to be altered annually. Highly conserved epitopes recognized by effector T cells may represent an alternative approach for the generation of a more universal influenza virus vaccine. Relatively few highly conserved epitopes are currently known in humans, and relatively few epitopes have been identified from proteins other than hemagglutinin and nucleoprotein. This prompted us to perform a study aimed at identifying a set of human T-cell epitopes that would provide broad coverage against different virus strains and subtypes. To provide coverage across different ethnicities, seven different HLA supertypes were considered. More than 4,000 peptides were selected from a panel of 23 influenza A virus strains based on predicted high-affinity binding to HLA class I or class II and high conservancy levels. Peripheral blood mononuclear cells from 44 healthy human blood donors were tested for reactivity against HLA-matched peptides by using gamma interferon enzyme-linked immunospot assays. Interestingly, we found that PB1 was the major target for both CD4(+) and CD8(+) T-cell responses. The 54 nonredundant epitopes (38 class I and 16 class II) identified herein provided high coverage among different ethnicities, were conserved in the majority of the strains analyzed, and were consistently recognized in multiple individuals. These results enable further functional studies of T-cell responses during influenza virus infection and provide a potential base for the development of a universal influenza vaccine.

Control of mucosal virus infection by influenza nucleoprotein-specific CD8+ cytotoxic T lymphocytes.

BACKGROUND: MHC class I-restricted CD8+ cytotoxic T lymphocytes (CTL) are thought to play a major role in clearing virus and promoting recovery from influenza infection and disease. This has been demonstrated for clearance of influenza virus from the lungs of infected mice. However, human influenza infection is primarily a respiratory mucosal infection involving the nasopharynx and tracheobronchial tree. The role of CD8+ CTL directed toward the influenza nucleoprotein (NP) in defense against influenza virus infection at the respiratory mucosa was evaluated in two separate adoptive transfer experiments. METHODS: Influenza nucleoprotein (NP)-specific CD8+ CTL were generated from splenocytes obtained from Balb/c mice previously primed with influenza A/Taiwan/1/86 (H1N1) infection or with influenza A/PR/8/34 (H1N1)-derived NP plasmid DNA vaccine followed by infection with A/Hong Kong/68 (H3N2) virus. After in vitro expansion by exposure to an influenza NP-vaccinia recombinant, highly purified CD8+ T cells exhibited significant lysis in vitro of P815 target cells infected with A/Hong Kong/68 (H3N2) virus while the CD8- fraction (CD4+ T cells, B cells and macrophages) had no CTL activity. Purified CD8+ and CD8- T cells (1 x 107) were injected intravenously or interperitoneally into naive mice four hours prior to intranasal challenge with A/HK/68 (H3N2) virus. RESULTS: The adoptively transferred NP-vaccinia-induced CD8+ T cells caused significant reduction of virus titers in both the lungs and nasal passages when compared to CD8- cells. Neither CD8+ nor CD8- T cells from cultures stimulated with HIV gp120-vaccinia recombinant reduced virus titers. CONCLUSION: The present data demonstrate that influenza NP-specific CD8+ CTL can play a direct role in clearance of influenza virus from the upper respiratory mucosal surfaces.

Mice deficient for the wild-type p53-induced phosphatase gene (Wip1) exhibit defects in reproductive organs, immune function, and cell cycle control.

The Wip1 gene is a serine/threonine phosphatase that is induced in a p53-dependent manner by DNA-damaging agents. We show here that Wip1 message is expressed in moderate levels in all organs, but is present at very high levels in the testes, particularly in the postmeiotic round spermatid compartment of the seminiferous tubules. We have confirmed that Wip1 mRNA is induced by ionizing radiation in mouse tissues in a p53-dependent manner. To further determine the normal biological function of Wip1 in mammalian organisms, we have generated Wip1-deficient mice. Wip1 null mice are viable but show a variety of postnatal abnormalities, including variable male runting, male reproductive organ atrophy, reduced male fertility, and reduced male longevity. Mice lacking Wip1 show increased susceptibility to pathogens and diminished T- and B-cell function. Fibroblasts derived from Wip1 null embryos have decreased proliferation rates and appear to be compromised in entering mitosis. The data are consistent with an important role for Wip1 in spermatogenesis, lymphoid cell function, and cell cycle regulation.

Restoration of Retarded Influenza Virus-specific Immunoglobulin Class Switch in Aged Mice.

OBJECTIVE: The declined immune response to infection causes significant higher morbidity and mortality in aging in spite of the coexisted hyperimmunoglobulinemia (HIG). This study is to reveal the cellular basis of HIG and mechanism of weakened HA-specific IgG response in aged mice and to test cell therapy in the treatment of age-related IgG antibody production deficiency with immunocyte adoptive transfer. METHODS: BALB/c mice was immunized with Influenza A/Taiwan vaccine and challenged with the same strain of virus. ELISA was used to assess the levels of total immunoglobulins and antigen specific antibody response. The flow cytometry and ELISPOT were used to evaluate the frequencies of total immunoglobulin- and specific antibody-producing and secreting B lymphocytes. In vitro expanded mononuclear cells, CD4+ T lymphocytes and CD20+ B lymphocytes from old and young mice were adoptively transferred into influenza virus-challenged aged mice, and HA-specific IgG responses were observed. RESULTS: It is found that old mice exhibited higher levels of total serum IgG, IgM and IgA, higher frequencies of IgG+, IgM+ and IgA+ cells, and greater antigen-specific IgM and IgA responses to influenza infection, in comparison to young mice. However, influenza antigen- specific IgG and its subclass responses in old mice were significantly lower. CONCLUSION: The retarded specific IgG response could be attributed to an insufficiency of immunoglobulin class switch in aging. Correlation analysis indicated that HIG and deficient specific IgG production in aged mice could be independent to each other in their pathogenesis. Correction of deficient specific IgG production by adoptive transfer of in vitro expanded and unexpanded CD4+ cells from immunized young mice suggests the CD4+ cell dysfunction contributes to the insufficiency of immunoglobulin class switch in aged mice. The transfusion of in vitro expanded lymphocytes could be a potential effective therapy for the age-related immunodeficiency and could play a role in the infection prevention in aging.

Cell mediated immune responses following revaccination with an influenza A/H5N1 vaccine.

PURPOSE: The study aims were to determine whether inactivated influenza A/H5N1 vaccine administration elicited cell mediated immune (CMI) responses and the impact of adjuvant, vaccine dose and subject age on these responses. METHODS: Adults who were previously primed with either adjuvanted or unadjuvanted, inactivated, A/H5N1/Vietnam/1203/2004 (Clade 1) vaccine or unprimed (received placebo) in previous vaccine studies were randomized to receive one (primed) or two (unprimed) 15- or 90-mcg doses of inactivated, A/H5N1/Indonesia/05/05 (Clade 2) vaccine. Peripheral blood mononuclear cells (PBMCs) were collected and analyzed from a subset of vaccinees to assess CMI responses using IFN-γ and granzyme B ELISPOT assays. Cytokine measurements were performed on PBMC supernatants after stimulation with H5N1 virus. RESULTS: PBMCs were available from 177 participants; 88 and 89 received 15-mcg and 90-mcg of unadjuvanted clade 2 vaccine, respectively. Following H5N1 clade 1 stimulation, IFN-γ but not granzyme B normalized spot-forming cell numbers had statistically significant increased numbers at each of the post-vaccination timepoints compared to baseline in pooled analyses of all vaccine doses and age groups. Clade 2 stimulation resulted in statistically significant increased numbers of IFN-γ cells only 180 days following the last vaccination. Responses were similar among younger and older study participants, as were responses among those primed with alum-adjuvanted or non-adjuvanted clade 1 H5N1 vaccines. The dosage of clade 2 vaccine did not impact CMI responses among primed subjects, but responses were statistically significantly greater in unprimed recipients of the 90-mcg dosage compared to unprimed recipients of the 15-mcg dosage. IFN-γ levels in the supernatants of stimulated PBMC were strongly correlated with IFN-γ ELISPOT results. CONCLUSION: CMI responses occur in adults administered influenza A/H5N1 inactivated influenza vaccine.

An aged mouse model for RSV infection and diminished CD8(+) CTL responses.

Recent studies indicate that respiratory syncytial virus (RSV), like influenza, causes significant morbidity and mortality among elderly persons. There are currently no animal models to study the effects of aging on RSV disease and immunity. This manuscript provides an initial description of such a model. Aged and young BALB/c mice (22-24 and 2-4 months, respectively) were infected with 10(4) TCID(50) of RSV A2. RSV was detected by culture in lung and nose wash specimens obtained 4-6 days following infection at a slightly higher titer in old mice in comparison with young mice. RT-PCR assay detected RSV in the lungs and nose washes of all mice on 4, 8, and 21 days postinoculation, with only a slightly less frequency in young mice. Splenic lymphocytes from old mice exhibited significantly lower RSV-specific MHC class I-restricted CD8(+) CTL responses (P < 0.01-0001), and reduced IFN-production (P < 0.03) than young mice. Conversely, IL-4 production was somewhat elevated in old mice. These results demonstrate diminished RSV virus-specific CD8(+)CTL responses and IFN-gamma production in old mice in comparison with young. It is speculated that the deficient RSV-specific CTL responses may account for the increased morbidity and mortality from RSV infections in elderly persons. Although detailed histopathological, virological, and immunological analyses are incomplete at present, the old BALB/c RSV infection model described provides an opportunity to evaluate the role of CD8(+)CTL and cytokines in RSV disease in aging.

Protection against influenza infection by cytokine-enhanced aerosol genetic immunization.

BACKGROUND: Conventional vaccine development for newly emerging pandemic influenza virus strains would likely take too long to prevent devastating global morbidity and mortality. If DNA vaccines can be distributed and delivered efficiently, genetic immunization could be an attractive solution to this problem, since plasmid DNA is stable, easily engineered to encode new protein antigens, and able to be quickly produced in large quantities. METHODS: We compared two novel genetic immunization methods in a mouse model of influenza to evaluate protective effects: aerosol delivery of polyethylenimine (PEI)-complexed hemagglutinin (HA)-expressing plasmid and intravenous (IV) delivery of the plasmid complexed with macroaggregated albumin/PEI. Serial serum samples were obtained for assay of neutralizing antibodies against HA. Mice were then challenged in the airway with influenza virus, and production of infectious virus in the lungs was titered. RESULTS: Most mice immunized with HA plasmid alone by aerosol and all mice immunized IV developed protective immune responses, whereas none administered control plasmid were protected. Aerosol co-administration of HA plasmid with plasmids encoding the cytokines interleukin 12 (IL12) and granulocyte-macrophage colony stimulating factor (GM-CSF) markedly increased neutralizing antibody responses, so that all aerosol immunized mice were protected from high level virus proliferation. CONCLUSIONS: Cytokine-enhanced aerosol delivery of plasmid vaccines can elicit robust protective immune responses against influenza. Thus, aerosol delivery has the potential to address the need for rapid widespread immunization against new influenza virus strains, and may have applications for other infectious and toxic disease processes.

Cationic liposome-mediated enhanced generation of human HLA-restricted RSV-specific CD8+ CTL+.

Generation of human CD8+ cytotoxic T-lymphocyte (CTL) activity against respiratory syncytial virus (RSV) using peripheral blood leukocytes (PBL) in vitro is inefficient. Lipofectamine, a polycationic liposome, previously shown to enhance the transfection efficiency of DNA in cells, was evaluated for enhancing RSV CTL activity. Stimulator cells were prepared by infecting human PBL with RSV with or without Lipofectamine for 3 hr and then transferred to responder cells. After 8 days of incubation, CTL lysis of autologous target cells infected with RSV (also treated with Lipofectamine) was determined in a 4-hr 5'chromium release assay. Lipofectamine treatment significantly enhanced HLA-restricted RSV-specific CD8+ CTL activity (up to sevenfold, P < 0.05-0.001). Lipofectamine treatment also enhanced cell surface RSV antigen expression and increased the frequencies of HLA-A,B,C+/RSV+ and HLA-DR+/RSV+ leukocytes as demonstrated by flow cytometry. These results demonstrate the usefulness of cationic liposomes in augmenting cell surface antigen expression and increasing the efficiency of generation of human RSV-specific CD8+ CTL activity.

Immunoglobulin A-deficient mice exhibit altered T helper 1-type immune responses but retain mucosal immunity to influenza virus.

We have previously demonstrated that immunoglobulin A (IgA)(-/-) knockout (KO) mice exhibit levels of susceptibility to influenza virus infection that are similar to those of their normal IgA(+/+) littermates. To understand the mechanism of this apparent mucosal immunity without IgA, immunoglobulin isotype and T helper 1 (Th1)-type [interferon-gamma (IFN-gamma)] and Th2-type [interleukin (IL)-4, IL-5)] cytokine responses to influenza vaccine were evaluated. Intranasal immunization with influenza virus subunit vaccine plus cholera toxin/cholera toxin B subunit (CT/CTB) induced significant influenza virus-specific immunoglobulin G (IgG) antibody in the serum and nasal passages of both IgA(-/-) and IgA(+/+) mice, while IgA antibodies were induced only in IgA(+/+) mice. IgA KO mice exhibited an IgG1 subclass haemagglutinin (HA)-specific response but no detectable IgG2a and IgG2b responses. In contrast, IgA(+/+) mice exhibited significant IgG1 as well as IgG2a responses. This indicates a predominant Th2-type response in IgA KO mice compared to normal mice. Following stimulation with influenza virus in vitro, splenic lymphocytes from immunized IgA(-/-) mice produced significantly lower levels of IFN-gamma than IgA(+/+) mice (P < 0.001), but elaborated similar levels of IL-4 and IL-5. This was true at both protein and mRNA levels. Immunized mice were challenged intranasally with a small inoculum of influenza virus to allow deposition of virus in the nasal mucosal passages. Compared to non-immunized mice, immunized IgA(-/-) and IgA(+/+) mice exhibited significant, but similar levels of reduction in virus titres in the nose and lung. These results demonstrate that in addition to IgA deficiency, IgA gene deletion also resulted in down-regulated Th1-type immune responses and confirm our previous data that IgA antibody is not indispensable for the prevention of influenza virus infection.