Enhanced humoral response to influenza vaccine in aged mice with a novel adjuvant, rOv-ASP-1.

Immunization is the best way to prevent seasonal epidemics and pandemics of influenza. There are two kinds of influenza vaccines available in the United States: an inactivated vaccine (TIV) and an attenuated vaccine; however, only TIV is approved for immunization of the elderly population. While the aged population has the highest rate of influenza vaccination, the protective efficacy is low as evidenced by elderly individuals having the highest mortality associated with influenza. Recently, we reported that an adjuvant derived from the helminth parasite Onchocerca volvulus, named O. volvulus activation-associated secreted protein-1 (Ov-ASP-1), can significantly enhance the protective efficacy of an inactivated vaccine (TIV) in young adult mice. In the current study, we examined whether this recombinant Ov-ASP-1 (rOv-ASP-1) can enhance the efficacy of TIV in aged mice as well. While primary immunization with TIV alone produced only a low level of influenza-specific antibodies (total IgG, IgG1, and IgG2c) in aged mice, the antibody levels were significantly increased after immunization with TIV+rOv-ASP-1. More importantly, the level of the total IgG in aged mice administered TIV+rOv-ASP-1 was comparable to that of young adult mice immunized with TIV alone. Co-administration of rOv-ASP-1 induced a low level of cross-reactive antibody and enhanced the protective efficacy of TIV in aged mice, reflected by significantly increased survival after challenge with a heterologous influenza virus. rOv-ASP-1 was also superior to the conventional adjuvant alum in inducing specific IgG after TIV immunization in aged mice, and in conferring protection after challenge. These results demonstrate that rOv-ASP-1 may serve as a potential adjuvant for influenza vaccine to improve the efficacy of protection in the elderly.

Antigen sparing and enhanced protection using a novel rOv-ASP-1 adjuvant in aqueous formulation with influenza vaccines.

Influenza is one of the most common infectious diseases endangering the health of humans, especially young children and the elderly. Although vaccination is the most effective means of protection against influenza, frequent mutations in viral surface antigens, low protective efficacy of the influenza vaccine in the elderly, slow production process and the potential of vaccine supply shortage during a pandemic are significant limitations of current vaccines. Adjuvants have been used to enhance the efficacy of a variety of vaccines; however, no adjuvant is included in current influenza vaccines approved in the United States. In this study, we found that a novel adjuvant, rOv-ASP-1, co-administrated with inactivated influenza vaccine using an aqueous formulation, substantially improved the influenza-specific antibody response and protection against lethal infection in a mouse model. rOv-ASP-1 enhanced the magnitude of the specific antibody response after immunization with low doses of influenza vaccine, allowing antigen-sparring by 10-fold. The rOv-ASP-1 formulated vaccine induced a more rapid response and a stronger Th1-associated antibody response compared to vaccine alone and to the vaccine formulated with the adjuvant alum. Importantly, rOv-ASP-1 significantly enhanced cross-reactive antibody responses and protection against challenge with an antigenically distinct strain. These results demonstrate that rOv-ASP-1 is an effective adjuvant that: (1) accelerates and enhances the specific antibody response induced by influenza vaccine; (2) allows for antigen sparing; and (3) augments a Th1-biased and cross-reactive antibody response that confers protection against an antigenically distinct strain.

Impaired specific CD8 T cell response with aging is not due to decreased expression of CD90 on TCR transgenic T cells.

BACKGROUND: CD90 (Thy-1) is a small glycoprotein that is particularly abundant on the surface of mouse thymocytes and peripheral T cells, and is often used as a marker in adoptive transfer experiments to distinguish donor and recipient T cells with different CD90 subtypes. We have performed adoptive transfer experiments with T cell receptor transgenic (TCR Tg) mice to study the impaired CD8 T cell response with aging. FINDINGS: After stimulation with a CD8 T cell epitope, HA518-524, the response of TCR Tg CD8 T cells from aged mice was decreased compared to the response of TCR Tg T cells from young mice. CD90 expression was also substantially decreased on the TCR Tg CD8 T cells of aged mice. However, the responses of CD90hi and CD90low CD8 T cells of the aged mice were similar in both early activation and proliferation, demonstrating that the impaired Tg T cell response with aging is not associated with the altered CD90 expression on CD8 T cells. CONCLUSIONS: The impaired Tg CD8 T cell response in aged mice is not due to age-associated changes in CD90 expression on Tg CD8 T cells.

Intrinsic defects in CD8 T cells with aging contribute to impaired primary antiviral responses.

Aging is associated with altered immune responses, particularly with a diminished CD8 T cell response. Although both intrinsic and extrinsic factors are hypothesized to impact this decreased T cell response, the direct evidence of an intrinsic deficiency in virus-specific CD8 T cells is limited. In this study, a TCR transgenic (Tg) P14 mouse model was utilized to compare the activation and proliferation of the Tg CD8 T cells of young and aged P14 mice upon stimulation with antigen or infection with virus. The proliferation of purified Tg CD8 T cells of aged mice was significantly lower than that of young mice when cultured in vitro with both the LCMV specific peptide and antigen presenting cells from young wild type mice. In addition, expression of the activation markers, CD69, CD25, and CD44, was delayed on Tg T cells of aged mice after stimulation. Importantly, while adoptive transfer of purified Tg CD8 T cells of young or aged mice into young wild type mice resulted in expansion of the Tg CD8 T cells of both ages after LCMV infection, the expansion of the Tg T cells from aged mice was significantly decreased compared with that of the Tg T cells from young mice. However, while the number of IFN-γ secreting Tg CD8 T cells from aged mice was significantly decreased compared to that of young mice, the percentages of Tg CD8 T cells producing IFN-γ were similar in young and aged mice, demonstrating that proliferation, but not function, of the Tg CD8 T cells of aged mice was impaired. Importantly, chronological age alone was not sufficient to predict an altered proliferative response; rather, expression of high levels of CD44 on CD8 T cells of aged mice reflected a decreased proliferative response. These results reveal that alterations intrinsic to CD8 T cells can contribute to the age-associated defects in the primary CD8 T cell response during viral infection.

Differential effects of stimulatory factors on natural killer cell activities of young and aged mice.

Age-associated influences on natural killer (NK) cell functions following cytokine stimulation were examined in splenocytes from C57BL/6 mice. NK cells of both young and aged mice exhibited significantly increased: interferon-γ production after interleukin (IL)-12 or IL-15 alone or any combination of IL-12, IL-18, and IL-2; cytotoxicity after IL-2 or IL-15; and granzyme B expression after IL-15. The only significant age-associated differences were observed in interferon-γ production after IL-15 or IL-12 + 18 + 2 and in granzyme B expression following IL-2 or IL-15. Perforin expression did not increase following stimulation; however, NK cells from aged mice expressed significantly higher levels than young mice. These results underscore the complexity of the cytokine-induced functional activities of NK cells and illustrate the differential response of NK cells from young and aged mice to cytokine stimulation.

Characterization of the metabolic phenotype of rapamycin-treated CD8+ T cells with augmented ability to generate long-lasting memory cells.

BACKGROUND: Cellular metabolism plays a critical role in regulating T cell responses and the development of memory T cells with long-term protections. However, the metabolic phenotype of antigen-activated T cells that are responsible for the generation of long-lived memory cells has not been characterized. DESIGN AND METHODS: Using lymphocytic choriomeningitis virus (LCMV) peptide gp33-specific CD8(+) T cells derived from T cell receptor transgenic mice, we characterized the metabolic phenotype of proliferating T cells that were activated and expanded in vitro in the presence or absence of rapamycin, and determined the capability of these rapamycin-treated T cells to generate long-lived memory cells in vivo. RESULTS: Antigen-activated CD8(+) T cells treated with rapamycin gave rise to 5-fold more long-lived memory T cells in vivo than untreated control T cells. In contrast to that control T cells only increased glycolysis, rapamycin-treated T cells upregulated both glycolysis and oxidative phosphorylation (OXPHOS). These rapamycin-treated T cells had greater ability than control T cells to survive withdrawal of either glucose or growth factors. Inhibition of OXPHOS by oligomycin significantly reduced the ability of rapamycin-treated T cells to survive growth factor withdrawal. This effect of OXPHOS inhibition was accompanied with mitochondrial hyperpolarization and elevation of reactive oxygen species that are known to be toxic to cells. CONCLUSIONS: Our findings indicate that these rapamycin-treated T cells may represent a unique cell model for identifying nutrients and signals critical to regulating metabolism in both effector and memory T cells, and for the development of new methods to improve the efficacy of adoptive T cell cancer therapy.

Expansion of regulatory T cells in aged mice following influenza infection.

While it has been established that Treg cells can down-modulate an immune response, no study has addressed if the observed increase in Treg cells in aged mice is related to the decreased and delayed specific CD8 T cell responses seen following primary influenza infection. In this study, phenotypic characteristics and function of Treg cells were analyzed in young (4-6 months) and aged (18-22 months) mice prior to and during the course of primary influenza infection. Upon infection, aged, but not young, mice have a significant expansion of Treg cells. In addition, Treg cells of aged mice demonstrate both a higher percentage and higher expression per cell of CD69 both at baseline and during infection compared to young mice. However, Treg cells isolated from young and aged mice comparably suppress CD8 T cells and suppression is dose dependent. These results suggest that the increase in the percentage of Treg cells in aged mice may contribute to the diminished CD8 T cell response to primary influenza infection.

CD8 T cell responses to influenza virus infection in aged mice.

Influenza is one of the most common infectious diseases afflicting humans, particularly the elderly. The murine model has been widely employed for investigation of immunity to influenza virus infection. In this paper, we review the recent advances in understanding the diminished CD8 T cell immune response to influenza virus infection in aged mice. Possible mechanisms of impaired CD8 T cell responses with aging are addressed, including: (1) the role of dendritic cells (DCs); (2) the effect of age-associated changes in the T cell repertoire; and (3) the interactions with CD4 T cells, including T regulatory (Treg) cells and CD4 T helper cells. The aged murine model of the CD8 T cell response to influenza virus is helping to elucidate the mechanisms of immunosenescence which can lead to therapeutic improvements in the primary CD8 T cell response to new infections, as well as the development of new strategies for immunization to prevent influenza in the elderly.

Enhancement of virus-specific expansion of transgenic CD8 T cells in aged mice by dendritic cells.

Aging is associated with a decreased CD8 T cell response to multiple antigens and to virus infection. Although both intrinsic and extrinsic factors have been shown to contribute to the decrease, the mechanisms are still largely unknown. In this study, the role of dendritic cells (DCs) in the age-associated decrease was examined. Influenza-specific TCR transgenic CD8 T cells of young mice demonstrated limited expansion in response to influenza infection when adoptively transferred to aged compared to young mice. This decreased response in aged mice could be significantly enhanced when DCs of young mice were co-transferred. Co-transfer of DCs had no impact in young recipient mice. Adoptive transfer of the DCs also increased the endogenous CD8 T cell response of intact aged mice, although to a lesser degree. These results suggest that the diminished CD8 T cell response to virus infection in aged mice is partially attributable to age-associated changes in DCs.

Limited expansion of virus-specific CD8 T cells in the aged environment.

The mechanisms responsible for the diminished immune response seen with aging are unclear. In this study, we investigate the contributions of alterations in the lymphoid microenvironment to this decrease. Using adoptive transfer of virus-specific transgenic CD8 T cells, we demonstrate that the aged environment inhibits the clonal expansion of specific CD8 T cells from young mice during virus infection. Transferred specific CD8 T cells from young mice demonstrated a response reflecting the CD8 T cell response of the intact aged host: the CD8 T cells expand more slowly and have a decreased maximal expansion in an aged compared to a young environment. While isolated DCs (MHC II(+) CD11c(+)) of aged mice maintain their ability to support CD8 T cell Ag-specific expansion in vitro, splenocytes demonstrated an age-associated decrease in this ability. Since the percentages of various populations of DCs in splenocytes demonstrate no significant alteration with age, this diminished APC activity of splenocytes of aged mice may reflect inhibitory activity of other cell populations. The results of this study demonstrate that elements of the aged environment play an important role in the alteration of T cell response to virus infection in the aged.

Variable requirement of dendritic cells for recruitment of NK and T cells to different TLR agonists.

TLRs initiate the host immune response to microbial pathogens by activating cells of the innate immune system. Dendritic cells (DCs) can be categorized into two major groups, conventional DCs (including CD8(+) and CD8(-) DCs) and plasmacytoid DCs. In mice, these subsets of DCs express a variety of TLRs, with conventional DCs responding in vitro to predominantly TLR3, TLR4, TLR5, and TLR9 ligands, and plasmacytoid DCs responding mainly to TLR7 and TLR9 ligands. However, the in vivo requirement of DCs to initiate immune responses to specific TLR agonists is not fully known. Using mice depleted of >90% of CD11c(+) MHC class II(+) DCs, we demonstrate that cellular recruitment, including CD4(+) T cell and CX5(+)DX5(+) NK cell recruitment to draining lymph nodes following the footpad administration of TLR4 and TLR5 agonists, is dramatically decreased upon reduction of DC numbers, but type I IFN production can partially substitute for DCs in response to TLR3 and TLR7 agonists. Interestingly, TLR ligands can activate T cells and NK cells in the draining lymph nodes, even with reduced DC numbers. The findings reveal considerable plasticity in the response to TLR agonists, with TLR4 and TLR5 agonists sharing the requirement of DCs for subsequent lymph node recruitment of NK and T cells.

Aging affects initiation and continuation of T cell proliferation.

Aging is associated with a decline in immune responses, particularly within the T cell compartment. While the expansion of specific T cells in response to virus infections is consistently decreased in aged mice, the differences in T cell activation between young and aged mice as demonstrated in each round of proliferation remain poorly defined. In the present study, we utilized the T cell mitogen, ConA, to explore if fewer T cells of aged mice initiate proliferation upon mitogen stimulation or if similar numbers of T cells of aged mice begin proliferation but undergo fewer rounds of division. We also examined whether these age-associated changes in proliferation are reflected by differences in T cell activation by comparing activation markers (CD25, CD69, CD44, and CD62L) on T cells of young and aged mice at each round of proliferation. Not only was the kinetics of the expression of these markers greatly different between young and aged mice on the entire CD8 T cell population, but also at each round of proliferation. Our results demonstrate that a larger percentage of CD8 T cells of aged mice do not proliferate at all upon stimulation. Of the CD8 T cells of aged mice that do proliferate, a larger percentage start later and stop sooner. These results suggest that multiple levels of alteration may need to be considered when trying to maximize the immune response of aged individuals.

T-kininogen: a biomarker of aging in Fisher 344 rats with possible implications for the immune response.

T-kininogen (T-KG) is a reliable biomarker of aging in male Sprague-Dawley rats. Here we confirm, in a longitudinal study, a similar behavior in Fisher 344 rats of both sexes. In males, the increase in serum levels of T-KG follows an exponential curve, whereas in females the increase is best fitted by a linear curve. In both genders, dietary restriction delays the increase in T-KG. We have previously shown that T-KG inhibits T lymphocyte proliferation. Here we show that serum T-KG levels correlate negatively with the ability of splenocytes (most likely B cells) to proliferate in response to lipopolysaccharide. A similar correlation was not observed with other markers of inflammation, including alpha1-acid glycoprotein (AGP), haptoglobin, or interleukin-10. We conclude that the increase in serum T-KG represents a useful biomarker of aging in Fisher 344, and it correlates with decreased lymphocyte proliferation with age, although a cause-effect relationship has not been established.

Age-related changes in the immune response to influenza vaccination in a racially diverse, healthy elderly population.

Proliferation to phytohemagglutinin (PHA) and cell-mediated responses to influenza vaccine (FLU) were assessed in healthy elderly Caucasians, African Americans, Latinos and young Caucasians before and after immunization. Both PHA- and FLU-induced proliferation were reduced in elderly subgroups. FLU-induced proliferation increased post-vaccination in all elderly, except African Americans, but responses were lower than young. IL-2 and IFN-gamma levels did not change after vaccination of elderly, regardless of race. The altered response to FLU in elderly African Americans, compared to elderly Caucasians and Latinos, indicates that racial background is important when drawing conclusions about immune responses to FLU in mixed elderly populations.

Nutritional formula improved immune profiles of seniors living in nursing homes.

OBJECTIVES: To assess whether an experimental nutritional formula (EXP) supports immune function in seniors living in long-term care facilities. DESIGN: Prospective, randomized, double-blind, controlled trial conducted September 2002 through January 2003. SETTING: North central Florida nursing homes. PARTICIPANTS: Subjects aged 65 and older (n = 157). INTERVENTION: Subjects received 240 mL/d of EXP or standard liquid nutrition (CON) for 4 weeks before and 6 weeks after an influenza vaccination. MEASUREMENTS: Influenza vaccine antibody responses, immunophenotyping, lymphocyte activation, cytokines, and clinical measures (fever, number of prescribed antibiotics). RESULTS: Ninety-two subjects (n = 40, CON; n = 52, EXP) completed the study. Geometric mean antibody titers were similar between groups, yet the percentage of subjects with H1N1 antibody titers greater than 100 postvaccination was higher in the EXP group than in the CON group (43% vs 23%, P=.047). Similar trends were found for the percentage of subjects (intent to treat) with fourfold increases against the B/Hong Kong component (64% vs 46%, P = .09) or with H3N2 antibody titers of 40 or more (97% vs 89%, P=.06). EXP subjects had higher levels of influenza-activated lymphocytes (CD69+ and CD25+). Cytokine production after mitogen activation was lower in EXP than CON subjects (interleukin (IL)-6: 20+/-3 vs 29+/-3 ng/mL, P = .045; IL-10: 310+/-60 vs 603+/-140 pg/mL, P = .06). Fewer EXP subjects were treated for fever (5% vs 16%, P = .02) or prescribed antibiotics (7 vs 11 new antibiotics/100 days of study, P = .06). CONCLUSION: Seniors consuming the EXP formula demonstrated enhanced immune function, indicated by increased influenza vaccine response and lymphocyte activation, less fever, and fewer newly prescribed antibiotics than those consuming a standard ready-to-drink nutritional supplement.

Depletion of T cells by type I interferon: differences between young and aged mice.

Type I IFN (IFN-I or IFN-alphabeta) plays an important role in the innate immune response against viral infection. Here we report that a potent inducer of IFN-alphabeta, polyinosinic-polycytidylic acid [poly(I:C)], led to the depletion of T cells in young, but not aged mice, and that this depletion was limited to central memory, but not effector memory, T cells. Although early activation of T cells in vivo by poly(I:C), as demonstrated by CD69, was not impaired with aging, the expression of active caspase-3 was higher in young compared with aged mice. This depletion of T cells and induction of active caspase-3 in young mice and of CD69 in both young and aged mice by poly(I:C) were blocked by anti-IFN-alphabeta Ab. Although poly(I:C) stimulated lower circulating levels of IFN-alphabeta in aged mice, administration of IFN-alphabeta after poly(I:C) did not induce depletion of T cells in aged mice. These results indicate that IFN-alphabeta plays a critical role in the depletion of T cells of young mice, and further suggest that the lower level of functional IFN-alphabeta and decreased induction of active caspase-3 in T cells of aged mice after poly(I:C) may be responsible for the increased resistance of T cells of aged mice to depletion.

Response of aged mice to primary virus infections.

Aging is associated with an increased morbidity to virus infections as well as a delay in clearance of symptoms after infection. Studies of sublethal virus infections of aged mice closely mirror the human situation: there is a delay in clearance of virus. The delay in virus clearance is accompanied by a delay and a decrease in T-cell response, particularly of CD8(+) T cells. Intrinsic alterations of T cells of aged mice contribute to this decrease in virus-specific T-cell response; however, evidence suggests that environmental or innate components of the aged host also influence this age-associated decline in clearance of virus. While the changes in the adaptive immune response have been carefully described, the early events in the generation of the T-cell response after virus infection have received limited attention. Importantly, age-associated changes in the innate response to virus infection, particularly production of and response to interferon (IFN)-alpha/beta, cytotoxicity and IFN-gamma production by natural killer cells, interleukin-12 induction, and depletion of non-specific T cells early during virus infection need further evaluation.

Nutritional formula enhanced immune function and reduced days of symptoms of upper respiratory tract infection in seniors.

OBJECTIVES: To assess whether an experimental nutritional formula, given as a supplement, would reduce days of symptoms of upper respiratory tract infection (URTI) and affect antibody and lymphocyte proliferative responses to influenza vaccine. DESIGN: A prospective, randomized, double-blind, controlled trial was conducted between October 1999 and April 2000. SETTING: Assisted- and independent-living facilities in North Central Florida. PARTICIPANTS: Sixty-six individuals, aged 65 and older. INTERVENTION: Subjects received 8 oz/d of an experimental formula containing antioxidants, zinc, selenium, fermentable oligosaccharides, and structured triacylglycerol or an isoenergetic, isonitrogenous control formula for 183 days. MEASUREMENTS: Subjects recorded daily symptoms of URTI. Antibody titers and lymphocyte proliferation to three influenza vaccine components were measured on Days 57 and 183. RESULTS: Eighteen subjects in the control group and 16 subjects in the experimental group consumed an average of 7 ounces of formula daily and completed the 183-day study. Median days of symptoms of URTI were 3 (range 0-69, total days=156) and 0 (range 0-49, total days=78) for the control and experimental groups, respectively (P=.049). On Day 57, seven of 17 (41%) subjects in the control group and 13 of 15 (87%) subjects in the experimental group achieved a fourfold or greater increase in serum antibody titer to A/Beijing (P=.012). Lymphocyte proliferation to influenza vaccine components was greater in the experimental (median=1,365 cpm, range=0-14,955 cpm) than the control group (median=136 cpm, range=0-4,270 cpm) (P=.013). CONCLUSION: Subjects consuming an experimental nutritional formula experienced enhanced immune function and fewer days of URTI symptoms.

Cuttine edge: T cells from aged mice are resistant to depletion early during virus infection.

Aging is associated with decreased expansion of T cells upon stimulation. In young mice, infection induces a transient T cell depletion followed by the development of an Ag-specific T cell response that controls the infection. We found that T cells were depleted early after infection with E55 + murine leukemia retrovirus in young, but not aged, mice. Adoptive transfer experiments showed donor T cells of young, but not aged, mice were depleted due to apoptosis in various tissues of young recipients. However, T cells of neither young nor aged donors were depleted in aged recipients. These results indicate that both environmental and intrinsic cellular properties limit depletion of T cells of aged mice and suggest a novel explanation for the decreased T cell response associated with aging.

Age-related changes in Type 1 and Type 2 cytokine production in humans.

Although aging is accompanied by several changes in immune function, altered T cell function represents the most consistent and dramatic change. Since cytokines modulate the immune response, it has been postulated that these age-associated changes in T cell function may be due to alterations in cytokine production. Data from murine studies generally support an age-related shift from a Th1-like (IL-2, IFN-gamma) to a Th2-like (IL-4, IL-6, IL-10) cytokine response; however, whether or not such an age-related shift to a Type 2 cytokine response occurs in humans is not certain. This review of over 60 studies in humans, in which Type 1 and Type 2 cytokines were evaluated either alone or together, suggests that age-associated changes in cytokine production are inconsistent. Further, these age-associated changes in cytokine production do not always induce a shift to a Type 2 cytokine response. Importantly, these studies highlight that the stimulus used to induce cytokines influences both the level and pattern of immune response. Additional comprehensive evaluations in human studies are both critical and necessary to clearly identify the impact of altered cytokine production on age-related changes in immune function.