Innocent N. Mbawuike

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

ABSTRACT 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.

Enhancement of the protective efficacy of inactivated influenza A virus vaccine in aged mice by IL-2 liposomes.

A dose-dependent, vaccine-induced protection of aged and young Balb/c mice against lethal influenza A virus challenge has been demonstrated. Low dose formalin-inactivated influenza A virus vaccine was protective in young mice, but not in aged mice, while a higher dose was protective in both. Administration of low dose vaccine with IL-2 liposomes conferred protection comparable to the high dose in aged mice. Serum neutralizing antibody responses were stimulated by vaccine in a dose-dependent manner while IL-2 liposomes significantly enhanced responses in the low dose paralleled protection in young but not in aged mice. Lung interferon levels paralleled lung virus titres in young but not in aged mice. CTL responses in infected mice were generally higher in young than aged mice. These results demonstrate efficacy of IL-2 liposomes as an adjuvant for influenza virus vaccines in the aged.

Cytokines and impaired CD8+ CTL activity among elderly persons and the enhancing effect of IL-12.

We have previously demonstrated that about 70% of elderly persons exhibit deficient cytotoxic T lymphocyte (CD8+ CTL) responses against influenza viruses when compared to young persons. Since IFN-gamma, a Th1 cytokine and IL-4, a Th2 cytokine, stimulate and inhibit CD8+ CTL responses respectively, their role(s) in the age-related CTL deficiency was investigated. Lymphocytes from young adults (34 +/- 5 years old) and elderly subjects (71 +/- 1 years old) were stimulated in vitro with influenza A/H3N2, A/H1N1 or influenza B virus for 6-7 days. The CD8+ CTL activity against virus-infected autologous target cells was significantly lower among the elderly than the young subjects (P < 0.01). Following stimulation with influenza virus, IL-4 production in both age groups was similar on day 3 but significantly higher among elderly persons on day 6 (P < 0.05). In contrast, T cells from the elderly produced significantly lower IFN-gamma than did those from young persons on both days (P < 0.05). Treatment of T cells from young and elderly adults with recombinant human IL-12, a pivotal cytokine that stimulates Th1 cytokines, resulted in enhancement of CD8+ CTL activity and IFN-gamma production in a dose dependent manner (P < 0.01). IL-12-dependent enhancement of CTL activity was not always abrogated by anti-IFN-gamma antibody treatment. These results suggest that deficient influenza virus-specific CTL activity among the elderly is attributable to a Th1 to Th2 cytokine production switch. Immunotherapy with IL-12 could represent a useful approach to correct the CD8+ CTL deficiency and cytokine imbalance among elderly humans.

HLA-Restricted CD8+ Cytotoxic T Lymphocyte, Interferon-γ, and Interleukin-4 Responses to Respiratory Syncytial Virus Infection in Infants and Children

CD8+ cytotoxic T lymphocyte (CTL) activity, interferon (IFN)-gamma, and interleukin (IL)-4 production were evaluated in a blinded manner among respiratory syncytial virus (RSV)-infected newborns and their mothers for 3 epidemic seasons. Most mothers (80%) exhibited RSV-specific CD8+ CTL activity. Twenty (80%) of the 26 infants exhibited significant RSV-specific CTL activity during or after their first RSV season. CTL frequency increased with RSV infection rate, reaching 75% by the end of the third season. Most infants who shed virus (75%) had a medically attended lower respiratory tract disease (LRD). In the first year, RSV-infected infants (virus culture and antibody increase) were more likely to develop CTL activity (10 of 13) than were uninfected infants (1 of 5; P=.02). Infants with CTL activity in the first year were less likely to have an LRD in the second year. CD8+ CTL levels correlated positively with IFN-gamma (P<.001) and inversely with IL-4 (P=.03). Contribution of CD8+ CTL and IFN-gamma in the control of RSV disease in infants and children is implicated.

Studies on reactogenicity and immunogenicity of attenuated bivalent cold recombinant influenza type A (CRA) and inactivated trivalent influenza virus (TI) vaccines in infants and young children

Fifty-two infants seronegative to or without prior infection with influenza type A viruses were enrolled in a study to evaluate reactogenicity and immunogenicity of three bivalent cold recombinant type A (CRA) and two trivalent inactivated influenza (TI) vaccines. Controls consisted of infants receiving normal saline by nose drops (Pli.n.) or intramuscularly (Pli.m.). CRA and TI vaccines were monitored for local and systemic reactions after vaccination. Serum specimens obtained prior to and 6 weeks postvaccination were analysed for neutralizing antibody to influenza H1N1 and H3N2 viruses. CRA vaccines and Pli.n. recipients had similar numbers of acute respiratory infections and comparable rates of illnesses during the trial. Significantly fewer CRA vaccinees without an intercurrent viral infection had fever (0/16 versus 4/10, p = 0.04) and cough (4/16 versus 9/10, p = 0.002) than CRA vaccinees with a confirmed intercurrent viral infection. Recipients of TI vaccine and Pli.m. did not develop reactions at the injection site. For each of the CRA vaccines tested, a dominant CRA virus was identified. The dominant CRA viruses were isolated from a greater number of infants or for a longer duration than the non-dominant CRA viruses. All 14 non-dominant CRA viruses were recovered from infants within the first week after vaccination; 24 of 77 dominant CRA viruses were recovered more than 7 days after vaccination. The immunogenicity of CRA vaccines was not affected by a confirmed intercurrent viral infection or low titres of influenza-specific antibody.(ABSTRACT TRUNCATED AT 250 WORDS)

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 104 TCID50 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-specif ic 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-γ 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 hlstopathological, 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.

Cytotoxic T lymphocyte responses of infants after natural infection or immunization with live cold‐recombinant or inactivated influenza A virus vaccine

The cytotoxic T lymphocyte (CTL) response of infants after immunization with either inactivated trivalent subvirion vaccine (TIV) or bivalent attenuated cold‐recombinant (CR) vaccine or occurrence of natural influenza virus infection were compared in a blinded, placebo‐controlled study during the 1987–1988 and 1988–1989 influenza epidemic seasons. Healthy infants between 6 and 13 months of age were randomly assigned and administered a single dose of intranasal bivalent (A/H3N2/A/H1N1) CR vaccine, a two‐dose regimen of TIV (A/H3N2/A/H1N1/B) influenza vaccine, or placebo. Peripheral blood lymphocytes were obtained prior to and 2–8 weeks after vaccination and at the end of the epidemic season and stimulated with virus in vitro for 6 or 7 days. Lysis of autologous virus‐infected target cells was assessed in a 4 hr 51Cr release assay. MHC class l‐restricted influenza A‐specific CTL was stimulated following natural influenza A virus infection but not after immunization with CR influenza A virus vaccine or TIV. These results demonstrate for the first time induction of influenza virus‐specific CTL activity in infants under 1 year of age.

Influenza A subtype cross-protection after immunization of outbred mice with a purified chimeric NS1/HA2 influenza virus protein

Influenza A/PR/8/34-derived chimeric (D) protein (SK&F 106160) composed of the first 81 amino acids (aa) of NS1 fused to the conserved 157 C-terminal aa of HA2 (NS1 1-81-HA2 65-222) was previously shown to induce H-2d-restricted protective cytotoxic T-lymphocyte (CTL) immunity in inbred mice. However, D protein, like other small peptides, exhibited haplotype dependence and was not immunogenic in H-2b and H-2K mice. A potential use of this antigen in humans and the role of T cells in any protection were evaluated in outbred Swiss and inbred CBF6F1 (H-2d/b) mice. Mice immunized with D protein and challenged by small-particle aerosol with a lethal dose of influenza virus were significantly protected against mortality from influenza A/H1N1 and A/H2N2 (p < 0.05-< 0.0000001), but not from A/H3N2 and influenza B viruses when compared with control mice. D protein did not induce serum virus-neutralizing antibody but caused virus to be cleared faster in immunized mice. Protection was long-lasting. In vivo depletion of either Lyt2 (CD8+) or L3T4 (CD4+) T cells with monoclonal antibodies led to abrogation of in vitro-generated CTL activity in CF6F1 mice and significant reduction in the protective efficacy of D protein against virus challenge in both Swiss and CF6F1 mice. These results suggest that protection was mediated by CD8+ and/or CD4+ cells and not antibody. Thus D protein, via a conserved sequence on the HA2 polypeptide, has the potential to induce partially cross-reactive CTL that may protect against influenza virus disease in humans.

Rectification of Age-Associated Deficiency in Cytotoxic T Cell Response to Influenza A Virus by Immunization with Immune Complexes

Decline in cellular immunity in aging compromises protection against infectious diseases and leads to the increased susceptibility of the elderly to infection. In particular, Ag-specific cytotoxic T lymphocyte (CTL) response against virus is markedly reduced in an aged immune system. It is of great importance to explore novel strategy in eliciting effective antiviral CTL activity in the elderly. In this study, the efficacy and mechanisms of immunization with immune complexes in overcoming age-associated deficiency in cellular immunity were investigated. In this study, we show that the severely depressed CTL response to influenza A in aged mice can be significantly restored by immunization with immune complexes consisting of influenza A virus and mAb to influenza A nucleoprotein. The main mechanisms underlying this recovery of CTL response induced by immune complex immunization in aged mice are enhanced dendritic cell function and elevated production of IFN-γ in both CD4+ Th1 and CD8+ CTLs. Thus, these results demonstrate that immune complex immunization may represent a novel strategy to elicit effective virus-specific cytotoxic response in an aged immune system, and possibly, to overcome age-related immune deficiency in general.

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

BackgroundMHC 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.MethodsInfluenza 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 × 107) were injected intravenously or interperitoneally into naive mice four hours prior to intranasal challenge with A/HK/68 (H3N2) virus.ResultsThe 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.ConclusionThe 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.