Immunostimulant patch enhances immune responses to influenza virus vaccine in aged mice

Improvement in the immune response to influenza virus vaccination in the elderly represents the primary unmet need in influenza virus vaccination. We have shown that topical application of immunostimulating (IS) patches containing heat-labile enterotoxin of Escherichia coli (LT) enhances immune responses to injected vaccines. We extend these findings and show that LT-IS patch application enhances the antibody responses to influenza virus vaccination in both young and aged mice. LT-IS patches markedly increased influenza virus-specific immunoglobulin G (IgG), hemagglutination inhibition antibody, mucosal antibody, and T-cell responses. The magnitude of the immune responses in aged mice receiving an LT-IS patch was equivalent to or greater than that of the immune responses in young mice given vaccine alone. These results suggest that addition of an LT-IS patch may compensate for the deficient immune function seen in the aged in response to influenza virus vaccination. Therefore, use of an LT-IS patch could be a new, safe, and simple immunization strategy that may significantly improve the outcome of influenza virus vaccination in the elderly.     

Impaired immune responses in the lungs of aged mice following influenza infection

Background

Each year, influenza virus infection causes severe morbidity and mortality, particularly in the most susceptible groups including children, the elderly (>65 years-old) and people with chronic respiratory diseases. Among the several factors that contribute to the increased susceptibility in elderly populations are the higher prevalence of chronic diseases (e.g. diabetes) and the senescence of the immune system.

Methods

In this study, aged and adult mice were infected with sublethal doses of influenza virus (A/Puerto Rico/8/1934). Differences in weight loss, morbidity, virus titer and the kinetics of lung infiltration with cells of the innate and adaptive immune responses were analyzed. Additionally, the main cytokines and chemokines produced by these cells were also assayed.

Results

Compared to adult mice, aged mice had higher morbidity, lost weight more rapidly, and recovered more slowly from infection. There was a delay in the accumulation of granulocytic cells and conventional dendritic cells (cDCs), but not macrophages in the lungs of aged mice compared to adult animals. The delayed infiltration kinetics of APCs in aged animals correlated with alteration in their activation (CD40 expression), which also correlated with a delayed detection of cytokines and chemokines in lung homogenates. This was associated with retarded lung infiltration by natural killer (NK), CD4⁺ and CD8⁺ T-cells. Furthermore, the percentage of activated (CD69+) influenza-specific and IL-2 producer CD8+ T-cells was higher in adult mice compared to aged ones. Additionally, activation (CD69+) of adult B-cells was earlier and correlated with a quicker development of neutralizing antibodies in adult animals.

Conclusion

Overall, alterations in APC priming and activation lead to delayed production of cytokines and chemokines in the lungs that ultimately affected the infiltration of immune cells following influenza infection. This resulted in delayed activation of the adaptive immune response and subsequent delay in clearance of virus and prolonged illness in aged animals. Since the elderly are the fastest growing segment of the population in developed countries, a better understanding of the changes that occur in the immune system during the aging process is a priority for the development of new vaccines and adjuvants to improve the immune responses in this population.     

DHEAS improves learning and memory in aged SAMP8 mice but not in diabetic mice

Dehydroepiandrosterone sulfate (DHEAS) has been reported to improve memory in aged animals and suggested as a treatment for age-related dementias. The SAMP8 mouse, a model of Alzheimer's disease, has an age-related impairment in learning and memory and an increase in brain levels of amyloid precursor protein (APP) and amyloid beta protein (Abeta). Male SAMP8 mice also have a decrease in testosterone, to which DHEA is a precursor. Diabetes has been suggested as a model of aging and to be linked to Alzheimer's disease. Diabetics can have memory deficits and lower DHEAS levels. Here, we examined the effects of chronic oral DHEAS on acquisition and retention for T-maze footshock avoidance in 12 mo male SAMP8 mice and in CD-1 mice with streptozocin-induced diabetes. Learning and memory were improved in aged SAMP8 mice, but not in CD-1 mice with streptozocin-induced diabetes. These findings suggest that DHEAS is more effective in reversing the cognitive impairments associated with overexpression of Abeta than with diabetes.     

Cellular immune responses of mice to influenza virus infection

Mice infected with influenza virus develop cytotoxic T lymphocytes (CTL) specific for viral antigens prior to the appearance of virus-specific antibody-forming cells (AFCs). Effector T cells were detected at a time coincident with a precipitous decline in pulmonary virus titer. CTLs of draining lymph nodes and spleen were found to be cross-reactive among H-2 compatible cells infected with influenza type A virus subtypes. AFCs were observed to be primarily hemagglutinin specific. Virus-specific IgA-secreting AFCs were detected in mediastinal lymph nodes of infected mice.     

Developmental exposure to bisphenol a modulates innate but not adaptive immune responses to influenza A virus infection

Bisphenol A (BPA) is used in numerous products, such as plastic bottles and food containers, from which it frequently leaches out and is consumed by humans. There is a growing public concern that BPA exposure may pose a significant threat to human health. Moreover, due to the widespread and constant nature of BPA exposure, not only adults but fetuses and neonates are also exposed to BPA. There is mounting evidence that developmental exposures to chemicals from our environment, including BPA, contribute to diseases late in life; yet, studies of how early life exposures specifically alter the immune system are limited. Herein we report an examination of how maternal exposure to a low, environmentally relevant dose of BPA affects the immune response to infection with influenza A virus. We exposed female mice during pregnancy and through lactation to the oral reference dose for BPA listed by the US Environmental Protection Agency, and comprehensively examined immune parameters directly linked to disease outcomes in adult offspring following infection with influenza A virus. We found that developmental exposure to BPA did not compromise disease-specific adaptive immunity against virus infection, or reduce the host's ability to clear the virus from the infected lung. However, maternal exposure to BPA transiently reduced the extent of infection-associated pulmonary inflammation and anti-viral gene expression in lung tissue. From these observations, we conclude that maternal exposure to BPA slightly modulates innate immunity in adult offspring, but does not impair the anti-viral adaptive immune response, which is critical for virus clearance and survival following influenza virus infection.     

Humoral immune responses in Cr2-/- mice: enhanced affinity maturation but impaired antibody persistence

Deficiency in CD21/CD35 by disruption of the Cr2 loci leads to impaired humoral immune responses. In this study, we detail the role of CD21/CD35 on Ab responses to the hapten (4-hydroxy-3-nitrophenyl)acetyl conjugated to chicken gamma-globulin. Surprisingly, Cr2-/- mice generate significant Ab responses and germinal center (GC) reactions to low doses of this Ag in alum, although the magnitude of their responses is much reduced in comparison with those of Cr2+/- and C57BL/6 controls. Increasing Ag dose partially corrected this deficit. In situ study of the somatic genetics of GC B cells demonstrated that VDJ hypermutation does not require CD21/CD35, and Cr2-/- mice exhibited enhanced affinity maturation of serum Ab in the post-GC phase of the primary response. On the other hand, Cr2-/- mice displayed accelerated loss of serum Ab and long-lived Ab-forming cells. These observations suggest that B cell activation/survival signals mediated by CD21 and/or the retention of Ag by CD21/CD35 play important roles in the generation, quality, and maintenance of serum Ab.     

The immune responses to bacterial antigens encountered in vivo at mucosal surfaces

Mammals have evolved a sophisticated immune system for handling antigens encountered at their mucosal surfaces. The way in which mucosally delivered antigens are handled influences our ability to design effective mucosal vaccines. Live attenuated derivatives of pathogens are one route towards the development of mucosal vaccines. However, some molecules, described as mucosal immunogens, are inherently immunogenic at mucosal surfaces. Studies on mucosal immunogens may facilitate the identification of common characteristics that contribute to mucosal immunogenicity and aid the development of novel, non-living mucosal vaccines and immunostimulators.     

Blockage of regulatory T cells augments induction of protective immune responses by influenza virus-like particles in aged mice

Elderly humans over 65 years old are at great risk to pathogenesis by influenza virus infection. However, although influenza vaccines provide effective protection in healthy young adults, protection of elderly adults is substantially lower even with a good match between the vaccine and the circulating influenza virus. To gain insight of the underlying mechanism for the reduced immunogenicity of influenza vaccines in the aged population, we investigated immunogenicity of influenza virus-like particle vaccines in aged mice, which represent a useful model for studying aging associated impairment in immune responses. Specifically, we investigated the effect of inhibiting regulatory T cells in aged mice on induction of protective immune responses by influenza vaccines. Our results showed that injecting anti-CD25 antibodies could down-regulate CD25 on the surface of regulatory T cells and significantly increase the levels of antibody responses induced by VLP immunization in aged mice. Further, the profiles of antibody responses were also changed towards Th1 type by regulatory T cell blockage in aged mice. Moreover, aged mice that were treated by anti-CD25 antibodies prior to vaccination were more effectively protected against lethal influenza virus challenge.     

Immune responses to T-dependent and T-independent antigens during visceral leishmaniasis in mice: evidence for altered T-cell regulation of immune responses to non-parasite antigens

Antibody responses to T-dependent and T-"independent" antigens were studied in disease-susceptible (BALB/c and C57BL/10) and disease-resistant (A/J) mice infected with Leishmania donovani chagasi. Disease-susceptible mice but not disease-resistant mice showed a transient decrease in PFC responses to TNP on a T-dependent carrier (BGG) during the period of 4-8 weeks after infection. Infected disease-susceptible animals also showed increased responses to TNP on a type II T-independent carrier (Ficoll), which persisted until at least 14 weeks after infection. The increased responses were associated with a significant increase in anti-TNP antibody of the IgG2b subclass. When T-enriched spleen cells from infected mice and B-enriched spleen cells from uninfected mice were transferred to irradiated recipients immunized with TNP-Ficoll, increased anti-TNP PFC were observed over numbers seen in irradiated recipients which received both B and T cells from uninfected mice. Increased responses to TNP-Ficoll were also induced by prior administration of soluble leishmania extract in CFA. Infected mice immunized with TNP-LPS, a T-independent type I antigen, also had increased anti-TNP antibody responses, but had normal anti-LPS antibody responses. The elevated antibody production which occurred in response to the T-"independent" antigens could not be attributed to the relatively low polyclonal response which occurred in both disease-resistant and disease-susceptible mice infected with L. donovani chagasi. The observations are consistent with leishmania induced, transient alterations in some T-cell functions including response to haptens on T-dependent carriers, and a lack of down regulation of T-"independent" responses. Subtle lesions in immunoregulation may be important correlates of successful protozoal infection and may be responsible for some of the immunologic manifestations of the disease.     

Recombinant murine IL-3 fails to stimulate T or B lymphopoiesis in vivo, but enhances immune responses to T cell-dependent antigens

We have explored the in vivo effect of IL-3 on the lymphopoiesis and humoral responses of mice bearing osmotic minipumps loaded with murine rIL-3 for 1 to 4 wk. A marked splenomegaly due to the accumulation of hemopoietic precursors was seen, but no increase was found in the lymphoid organs in the total number of cells belonging to the T or B lymphocyte lineage, i.e., of L3T4+ or Lyt-2+, or of allospecific cytotoxic T lymphocyte precursor for the T lineage, or of sIg+ or B220+ cells, or of B colony-forming cells for the B lineage; total activity of natural killer and lymphokine-activated killer cells was decreased. In contrast to the splenomegaly, a marked diminution in the number of thymocytes was observed, suggesting that rIL-3 in large amounts does suppress the T lymphopoiesis, perhaps as the result of the selective stimulation of early progenitor cells toward the hemopoietic pathway. rIL-3 perfusion during immunization increased the IgM and IgG responses to a T cell-dependent antigen, human IgG, and prevented tolerance induction by the deaggregated human IgG, although in the same conditions it did not modify the response to a T cell-independent antigen. Our results suggest that in vivo IL-3 does not act directly on lymphocytes or their precursors, but may potentiate the humoral immune response to T cell-dependent antigens, presumably by acting on accessory cells.     

Partial reactivation of impaired immune competence in aged mice by synthetic thymus factors

The stability of mouse uterine cytosol receptor-[³H]estradiol complex was evaluated in the presence of neutral salts of the Hoffmeister series. Marked increases in the rate of dissociation of the complex were observed with the more chaotropic anions (SCN^?, $\text{ClO}{}_4{}^{\mathrm{?}}$, $\text{NO}{}_3{}^{\mathrm{?}}$, Br^?), and the effects of these ions were greater at lower temperatures, where water assumes a more rigid structure. At higher temperatures F^? and CH₃COO^?, which tend to stabilize water structure, led to retardation of the rate of dissociation of the hormone-receptor complex. There was essentially no change in steroid specificity in the presence of the markedly chaotropic salts. The perturbation of water structure adjacent to the steroid binding site is a factor to be considered in the isolation of steroid receptor complexes.     

Exercise training-induced adaptations of immune response are mediated by beta-adrenergic receptors in aged but not young mice.

Beta-adrenergic blockade was used to determine whether the exercise training-induced adaptations of immune response to viral infection were mediated by catecholamines in young and old mice. Young (2 mo) and older (16 mo) male BALB/c mice were randomly assigned to an exercise or control group, and half of the mice in each group received the beta-adrenergic receptor antagonist nadolol. After 8 wk of moderate exercise training, mice were challenged with herpes simplex virus (HSV) 24 h postexercise. The results showed that exercise treatment increased anti-HSV IgM antibody, enhanced IL-10, and altered the kinetics of IFN-gamma and IL-2 production in young and old mice. Unique to older mice, exercise decreased mitogen-induced proliferation, increased splenocytes, and tended to decrease memory cells (CD44(hi+)). In contrast, exercise increased mitogen-induced proliferation but decreased the number of splenic lymphocyte and CD4+ cells in young mice. beta-Adrenergic blockade blunted the exercise-induced changes in anti-HSV IgM, IL-2, IFNgamma, and mitogen-induced proliferation in old but not young mice. The findings suggest that some of the immunomodulatory effects of chronic exercise are mediated via beta-adrenergic receptors and that the role of beta-adrenergic receptors is age dependent.     

CpG-ODN but not other TLR-ligands restore the antitumor responses in old mice: the implications for vaccinations in the aged

Aim There is accumulative evidence indicating that targeting antigen presenting cells (APCs) with different types of adjuvants could result in the induction of antitumor immune responses. It has been hypothesized that APCs function may be altered in the elderly contributing to a decline in the immune function. We evaluated whether targeting APCs following injection with Poly I:C, LPS, flagellin, imiquimod and CpG-ODN would induce an antitumor response in the old. Materials and methods The immune and antitumor responses induce Poly I:C, LPS, flagellin, imiquimod and CpG-ODN were compared in young (2 month old) and old (18 months) mice. Results Our results indicated that only intratumoral (i.t.) injections of CpG-ODN completely rejected the tumor in both young and old mice. Injections of Poly I:C also induced the rejection of tumors in the young but not in the old. Furthermore, i.t. injections of CpG-ODN promoted the development of protective memory responses in the young and the old. Analysis of the immune responses in the old indicated that CpG-ODN but not Poly-I:C induces: a pro-inflammatory Th1 type response; accumulation and activation of CD4⁺, CD8⁺ T and, NK cell responses; activation of APCs; and reduction in the number of T_regs. The activation of these immune-parameters positively correlates with the induction of an antitumor response. Conclusions These studies indicate that there are differences in the level of stimulation with TLR-ligands between young and old APCs and that the aged immune responses can be rescued and exploited for the induction of tumor immunity by targeting APCs with specific TLR-ligands. These results have important clinical implications for developing immunization strategies containing TLR-ligands that will be effective in both the young and old.     

Defective primary and secondary IgG responses to thymic-dependent antigens in autoimmune PN mice

Palmerston North (PN) mice spontaneously develop autoimmune disease resembling SLE. Because immune responsiveness has not been defined in this strain, a study was designed to assay primary splenic plaque-forming cell (PFC) responses to thymus-dependent (TD) and thymus-independent (TI) Ag. Initial surveys of PN mice inoculated with the TD Ag SRBC showed adequate production of IgM PFC, but small numbers of IgG PFC were developed with polyspecific antiserum. In contrast, H-2-compatible DBA/1 control mice gave the expected responses to SRBC (IgG plaques elevated twofold compared with IgM plaques). PN mice had the usual responses to Ag that are largely TI; both PN and DBA/1 mice had active IgM and modest IgG responses to TNP-LPS and TNP-Ficoll. Additional experiments determined that PN mice had similar patterns of defective IgG responses to several different TD Ag (SRBC, horse RBC, and DNP-keyhole limpet hemocyanin). In each instance, the usual predominance of IgG1 plaques was absent, and total numbers of plaques developed with antisera specific for IgG isotypes were suppressed. Defective PN IgG production was evident as early as 3 wk of age, was not influenced by aging to 43 wk, and was not corrected by increasing the antigenic challenge 10-fold. PN spleen cells treated with monoclonal anti-Thy-1.2 and C were injected with pools of DBA/1 T cells into 850-rad irradiated (DBA/1 x PN)F1 hybrids. These recipients expressed low IgG1 responses to SRBC, suggesting that the B cell-containing fraction that was not lysed by anti-Thy-1.2 transferred the PN defect. PN mice, which do not respond to TD Ag with active IgG production, contradict the proposal that autoimmunity is associated with hyper-responsiveness to TD and TI Ag.     

Role of viral antigens in destructive cellular immune responses to adenovirus vector-transduced cells in mouse lungs.

Adenoviruses missing E1 have been used as gene delivery vectors to the lungs for the treatment of cystic fibrosis. Transient expression of the recombinant gene and the development of inflammation have been two major limitations to the application of first-generation recombinant adenoviruses for gene therapy. Studies with mouse models of liver- and lung-directed gene therapy suggested that CD8+ cytotoxic T lymphocytes (CTLs) are effectors that contribute to extinction of transgene expression. The precise antigens responsible for activation of CTLs have not been identified. In this study, we examine the relative contributions of viral proteins versus the transgene product to the activation of CTLs which eliminate transgene-containing cells in mouse lungs. Instillation of a lacZ-expressing virus into the lungs of C57BL/6 mice elicited CTL responses to both viral proteins and the transgene product, beta-galactosidase, which collectively contribute to loss of trans-gene expression in mouse airways. Similar results were obtained in two experimental models in which the animals should be tolerant to the transgene, i.e., lacZ virus delivered to an animal transgenic for lacZ and a virus expressing the liver-specific enzyme ornithine transcarbamylase administered to the lungs of various strains of immune-competent mice. These data confirm the hypothesis that CTLs specific for viral antigens contribute to the problem of transgene instability in mouse lungs and indicate that CTLs specific for transgene product alone cannot account for the observed problem.     

Studies on immune responses to parasite antigens in mice: VI. Delayed-type hypersensitivity to blood cells from Plasmodium berghei-infected mice

A delayed-type hypersensitivity response to washed Plasmodium berghei-infected blood cells can be elicited in sensitized BALB/c, CBA/H, and SJL/J mice. The response is T-cell dependent and the genotype of the cells used to elicit the response does not play any obvious role. The eliciting antigens, therefore, are unlikely to be modified polymorphic host cell surface components and appear to be parasite antigens.     

Herpes simplex virus type 2 virion host shutoff protein regulates alpha/beta interferon but not adaptive immune responses during primary infection in vivo

The herpes simplex virus (HSV) virion host shutoff (vhs) protein, the product of the UL41 (vhs) gene, is an important determinant of HSV virulence. vhs has been implicated in HSV interference with host antiviral immune responses, down-regulating expression of major histocompatibility complex molecules to help HSV evade host adaptive immunity. The severe attenuation of vhs-deficient viruses in vivo could reflect their inability to escape immune detection. To test this hypothesis, BALB/c or congenic SCID mice were infected intravaginally (i.vag.) with the HSV type 2 (HSV-2) vhs null mutant 333d41 or the vhs rescue virus 333d41(R). vhs-deficient virus remained severely attenuated in SCID mice compared with rescue virus, indicating that vhs regulation of adaptive immune responses does not influence HSV pathogenesis during acute infection. Innate antiviral effectors remain intact in SCID mice; prominent among these is alpha/beta interferon (IFN-alpha/beta). The attenuation of HSV-2 vhs mutants could reflect their failure to suppress IFN-alpha/beta-mediated antiviral activity. To test this hypothesis, 129 and congenic IFN-alpha/beta receptor-deficient (IFN-alpha/betaR(-/-)) mice were infected i.vag. with wild-type virus, vhs null mutants 333-vhsB or 333d41, or the vhs rescue virus 333d41(R). Whereas vhs-deficient viruses showed greatly reduced replication in the genital mucosa of 129 mice compared with wild-type or vhs rescue viruses, they were restored to nearly wild-type levels of replication in IFN-alpha/betaR(-/-) mice over the first 2 days postinfection. Only wild-type and vhs rescue viruses caused severe genital disease and hind limb paralysis in 129 mice, but infection of IFN-alpha/betaR(-/-) mice restored the virulence of vhs-deficient viruses. vhs-deficient viruses replicated as vigorously as wild-type and rescue viruses in the nervous systems of IFN-alpha/betaR(-/-) mice. Restoration was specific for the vhs mutation, because thymidine kinase-deficient HSV-2 did not regain virulence or the capacity to replicate in the nervous systems of IFN-alpha/betaR(-/-) mice. Furthermore, the defect in the IFN-alpha/beta response was required for restoration of vhs-deficient virus replication and virulence, but the IFN-alpha/beta-stimulated protein kinase R pathway was not involved. Finally, vhs of HSV-2 has a unique capacity to interfere with the IFN-alpha/beta response in vivo, because an HSV-1 vhs null mutant did not recover replication and virulence after i.vag. inoculation into IFN-alpha/betaR(-/-) mice. These results indicate that vhs plays an important role early in HSV-2 pathogenesis in vivo by interfering with the IFN-alpha/beta-mediated antiviral response.     

Suppression of viral specific primary T-cell response following intense physical exercise in young but not old mice.

Intense exercise to exhaustion leads to increased susceptibility and severity of infections. T cells play an essential role in control of viral infections. Whereas immune suppression is considered as a likely mechanism for exhaustive exercise-induced susceptibility to infection, we know little about viral-specific T-cell response following exhaustive exercise in young or old mice. In this study, one group of female young (10-12 wk) and old (22-24 mo) C57BL/6 mice was exposed to a single bout of intense exercise to exhaustion and immediately infected with lymphocytic choriomeningitis virus (LCMV). Eight days later, at the peak of expansion phase of T-cell response, we used tetramers of MHC class I molecules containing viral peptides to directly visualize antigen-specific CD8 T cells and a sensitive functional assay measuring interferon-gamma production at the single-cell level to quantitate the CD8 and CD4 T-cell response. To evaluate the impact of intense exercise during both the initiation and evolution of the expansion phase of the T-cell response, a second group of young and old mice continued their daily bouts of intense exercise to exhaustion over the next 8 days. Our data show that, in young mice, LCMV infection following exhaustive exercise leads to suppression of LCMV-specific CD8 and CD4 T-cell responses, and this suppression effect occurs at the initiation of the expansion phase of viral-specific T cells. However, in old mice, unlike young mice, exhaustive exercise does not cause suppression of LCMV-specific T-cell responses.     

Receptor activator of NF-kappa B ligand inhibition suppresses bone resorption and hypercalcemia but does not affect host immune responses to influenza infection

Receptor activator of NF-kappaB (RANK) and its ligand (RANKL) are essential for osteoclast formation, function, and survival. Osteoprotegerin (OPG) inhibits RANK signaling by sequestering RANKL. This study evaluated the antiosteoclast and immunoregulatory effects of mouse rRANK-Fc, which, similar to OPG, can bind RANKL. The effect of RANKL inhibition by RANK-Fc on osteoclast function was determined by inhibition of vitamin D(3) (1,25(OH)(2)D(3))-induced hypercalcemia. Mice were injected with a single dose of 0, 10, 100, 500, or 1000 microg of RANK-Fc; 100 microg of OPG-Fc; or 5 microg of zoledronate 2 h before 1,25(OH)(2)D(3) challenge on day 0, and sacrificed on days 1, 2, 4, 6, 8, 12, 16, and 20. RANK-Fc doses of 100 or 500 microg were tested in a mouse respiratory influenza virus host-resistance model. A single dose of RANK-Fc > or =100 microg suppressed elevation of serum calcium levels and suppressed the bone turnover marker serum pyridinoline at day 4 and later time points, similar to those observed with OPG-Fc and zoledronate (p < or = 0.01 vs controls). By day 6, both immature and mature osteoclasts were depleted by high doses of RANK-Fc (500 and 1000 microg) or 100 microg of OPG-Fc. RANK-Fc doses of 100 or 500 microg had no detectable effect on immune responses to influenza infection, as measured by activation of cytotoxic T cell activity, influenza-specific IgG response, and virus clearance. RANK-Fc inhibition of RANKL has antiosteoclast activity at doses that have no detectable immunoregulatory activity, suggesting that RANKL inhibitors be further studied for their potential to treat excess bone loss.