Search

Age-related declines in immune function are associated with reduced humoral responses following vaccination or infection. Central to this defect is a decline in naïve CD4+ T cell function. In this review, we discuss factors intrinsic and extrinsic to the CD4+ T cell compartment that affect host immunity and propose means by which deficient CD4+ T cell function can be fully restored in the aged. [ABSTRACT FROM AUTHOR]

Campylobacter jejuni infection is a leading cause of foodborne disease, common to children, adult travelers, and military populations in low- to middle-income countries. In the absence of a licensed vaccine, efforts to evaluate prophylactic agents are underway. The prophylactic efficacy of a twice-daily, 550 mg dose of the antibiotic rifaximin demonstrated no efficacy against campylobacteriosis in a controlled human infection model (CHIM); however, samples from the CHIM study were utilized to assess how the human gut microbiome responds to C. jejuni infection, and if a 'protective' microbiota exists in study participants not developing campylobacteriosis. Statistically significant, but minor, differences in study participant beta diversity were identified during the challenge period (p = 0.002, R 2 = 0.042), but no significant differences were otherwise observed. Pre-challenge alpha diversity was elevated in study participants who did not develop campylobacteriosis compared to those who did (p < 0.001), but alpha diversity declined in all study participants from the pre-challenge period to post-discharge. Our work provides insight into gut microbiome shifts observed during a C. jejuni CHIM and following antibiotic treatment. This study utilized a high dose of 1.7 x 10 5 colony-forming units of C. jejuni ; future work could include CHIM studies performed with inocula more closely mimicking natural exposure as well as field studies involving naturally-occurring enteric infections., Competing Interests: BS is an employee of UES, Inc. JK, SI, AM, and RL are (or were at the time of the study) employees of the Henry M. Jackson Foundation for the Advancement of Military Medicine. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Stamps, Kuroiwa, Isidean, Schilling, Harro, Talaat, Sack, Tribble, Maue, Rimmer, Laird, Porter, Goodson and Poly.)

Dietary emulsifiers carboxymethylcellulose (CMC) and polysorbate-80 (P80) disturb gut microbiota, promoting chronic inflammation. Mice with minimal microbiota are protected against emulsifiers' effects, leading us to hypothesize that these compounds might provoke select pathobionts to promote inflammation. Gnotobiotic wild-type (WT) and interleukin-10 (IL-10) -/- mice were colonized with Crohn's-disease-associated adherent-invasive E. coli (AIEC) and subsequently administered CMC or P80. AIEC colonization of GF and altered Schaedler flora (ASF) mice results in chronic intestinal inflammation and metabolism dysregulations when consuming the emulsifier. In IL-10 -/- mice, AIEC mono-colonization results in severe intestinal inflammation in response to emulsifiers. Exposure of AIEC to emulsifiers in vitro increases its motility and ability to adhere to intestinal epithelial cells. Transcriptomic analysis reveals that emulsifiers directly induce expression of clusters of genes that mediate AIEC virulence and promotion of inflammation. To conclude, emulsifiers promote virulence and encroachment of pathobionts, providing a means by which these compounds may drive inflammation in hosts carrying such bacteria., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Enterotoxigenic Escherichia coli (ETEC), Campylobacter jejuni (CJ), and Shigella sp. are major causes of bacterial diarrhea worldwide, but there are no licensed vaccines against any of these pathogens. Most current approaches to ETEC vaccines are based on recombinant proteins that are involved in virulence, particularly adhesins. In contrast, approaches to Shigella and CJ vaccines have included conjugate vaccines in which Shigella lipopolysaccharides (LPS) or CJ capsule polysaccharides are chemically conjugated to proteins. We have explored the feasibility of developing a multi-pathogen vaccine by using ETEC proteins as conjugating partners for CJ and Shigella polysaccharides. We synthesized three vaccines in which two CJ polysaccharides were conjugated to two recombinant ETEC adhesins based on CFA/I (CfaEB) and CS6 (CssBA), and LPS from Shigella flexneri was also conjugated to CfaEB. The vaccines were immunogenic in mice as monovalent, bivalent and trivalent formulations. Importantly, functional antibodies capable of inducing hemaglutination inhibition (HAI) of a CFA/I expressing ETEC strain were induced in all vaccines containing CfaEB. These data suggest that conjugate vaccines could be a platform for a multi-pathogen, multi-serotype vaccine against the three major causes of diarrheal disease worldwide., (Published by Elsevier Ltd.)

Background: Campylobacter species are a leading cause of diarrheal disease globally with significant morbidity. Primary prevention efforts have yielded limited results. Rifaximin chemoprophylaxis decreases rates of travelers' diarrhea and may be suitable for high-risk persons. We assessed the efficacy of rifaximin in the controlled human infection model for Campylobacter jejuni., Methods: Twenty-eight subjects were admitted to an inpatient facility and randomized to a twice-daily dose of 550 mg rifaximin or placebo. The following day, subjects ingested 1.7 × 105 colony-forming units of C. jejuni strain CG8421. Subjects continued prophylaxis for 3 additional days, were followed for campylobacteriosis for 144 hours, and were subsequently treated with azithromycin and ciprofloxacin. Samples were collected to assess immunologic responses to CG8421., Results: There was no difference (P = 1.0) in the frequency of campylobacteriosis in those receiving rifaximin (86.7%) or placebo (84.6%). Additionally, there were no differences in the clinical signs and symptoms of C. jejuni infection to include abdominal pain/cramps (P = 1.0), nausea (P = 1.0), vomiting (P = .2), or fever (P = 1.0) across study groups. Immune responses to the CG8421 strain were comparable across treatment groups., Conclusions: Rifaximin did not prevent campylobacteriosis in this controlled human infection model. Given the morbidity associated with Campylobacter infection, primary prevention efforts remain a significant need., Clinical Trials Registration: NCT02280044.

Campylobacter jejuni infections are a leading cause of bacterial food-borne diarrhoeal illness worldwide, and Campylobacter infections in children are associated with stunted growth and therefore long-term deficits into adulthood. Despite this global impact on health and human capital, how zoonotic C. jejuni responds to the human host remains unclear. Unlike other intestinal pathogens, C. jejuni does not harbour pathogen-defining toxins that explicitly contribute to disease in humans. This makes understanding Campylobacter pathogenesis challenging and supports a broad examination of bacterial factors that contribute to C. jejuni infection. Here, we use a controlled human infection model to characterize C. jejuni transcriptional and genetic adaptations in vivo, along with a non-human primate infection model to validate our approach. We found that variation in 11 genes is associated with either acute or persistent human infections and includes products involved in host cell invasion, bile sensing and flagella modification, plus additional potential therapeutic targets. In particular, a functional version of the cell invasion protein A (cipA) gene product is strongly associated with persistently infecting bacteria and we identified its biochemical role in flagella modification. These data characterize the adaptive C. jejuni response to primate infections and suggest therapy design should consider the intrinsic differences between acute and persistently infecting bacteria. In addition, RNA sequencing revealed conserved responses during natural host commensalism and human infections. Thirty-nine genes were differentially regulated in vivo across hosts, lifestyles and C. jejuni strains. This conserved in vivo response highlights important C. jejuni survival mechanisms such as iron acquisition and evasion of the host mucosal immune response. These advances highlight pathogen adaptability across host species and demonstrate the utility of multidisciplinary collaborations in future clinical trials to study pathogens in vivo.

Introduction: Endemic Burkitt's lymphoma (eBL) is associated with Epstein-Barr virus and repeated malaria infections. A defining feature of eBL is the translocation of the c-myc oncogene to the control of the immunoglobulin promoter. Activation-induced cytidine deaminase (AID) has been shown to be critical for this translocation. Malaria infection induces AID in germinal center B cells, but whether malaria infection more broadly affects AID activation in extrafollicular B cells is unknown., Methods: We either stimulated purified B cells from AID-green fluorescence protein (GFP) reporter mice or infected AID-GFP mice with Plasmodium chabaudi , AID fluorescence was monitored in B cell subsets by flow cytometry., Results: In vitro analysis of B cells from these mice revealed that CpG (a Toll-like receptor 9 ligand) was a potent inducer of AID in both mature and immature B cell subsets. Infection of AID-GFP mice with Plasmodium chabaudi demonstrated that AID expression occurs in transitional and marginal zone B cells during acute malaria infection. Transitional B cells were also capable of differentiating into antibody secreting cells when stimulated in vitro with CpG when isolated from a P. chabaudi -infected mouse., Conclusions: These data suggest that P. chabaudi is capable of inducing AID expression in B cell subsets that do not participate in the germinal center reaction, suggesting an alternative role for malaria in the etiology of eBL.

Background: Patients with Crohn's disease (CD) have serologic responses to various microbial antigens. Serologic markers are associated with aggressive forms of disease and can be detected before onset of symptoms. Their utility in pre-clinical disease or prediction of complicated disease course before diagnosis is unclear., Aim: To evaluate the pattern of serologic anti-microbial antibodies long prior to diagnosis and the subsequent risk of complicated Crohn's disease at diagnosis., Methods: Sera from 100 US military personnel with Crohn's disease were obtained from the Department of Defense Serum Repository. For each patient, four samples were obtained at different time points before and around diagnosis, and were tested for 6 microbiota-directed antibodies (ASCA-IgA, ASCA-IgG, anti-OmpC, anti-CBir1, anti-A4-Fla2 and anti-FlaX). Associations between the presence and accumulation of Crohn's disease anti-microbial antibodies before diagnosis and with the later development of complications were evaluated., Results: Overall, 65 patients were positive for at least one Crohn's disease associated anti-microbial antibody in the earliest available sample, at a median of 6 years before Crohn's disease diagnosis (interquartile range, 5.6-8.2). The number of positive anti-microbial antibodies increased up to the time of Crohn's disease diagnosis. Complicated disease developed around the time of diagnosis in 24 patients. The proportion of positive antimicrobial antibodies before diagnosis was higher in patients with complicated vs. noncomplicated Crohn's disease. There was an inverse relationship between the time to first complication and the magnitude of serologic response before diagnosis., Conclusion: The presence and accumulation of circulating anti-microbial antibodies years before Crohn's disease diagnosis was associated with complicated Crohn's disease at or shortly after diagnosis., (© 2016 John Wiley & Sons Ltd.)

Background: There is a recognized need for biological markers to facilitate diagnoses of irritable bowel syndrome (IBS) and to distinguish it from other functional and organic disorders. As postinfectious IBS (PI-IBS) is believed to account for as many as one third of all IBS cases, here we sought to identify differences in specific cytokines and serologic responses across patients with idiopathic IBS and PI-IBS and healthy controls., Methods: At total of 120 US military personnel were identified from the Defense Medical Surveillance System-based International Classification of Diseases, 9th Revision, Clinical Modification (ICD9-CM) codes recorded during medical encounters and were grouped based on infectious gastroenteritis (IGE) episode (Shigella, Campylobacter, Salmonella, or an unspecified pathogen) followed by IBS, IBS without antecedent IGE, or IGE without subsequent IBS within 2 years of the IGE exposure. Sera from subjects were assayed for cytokine levels and antibodies against a panel of microbiome antigens., Results: In total, 10 of 118 markers considered were shown to differ between IBS patients and healthy controls, including cytokines interleukin-6 (IL-6), IL-8, IL-1β, and macrophage inflammatory protein-1β (MIP-1β), as well as antibody responses to microbial antigens. Antimicrobial antibody response profiles also differed between PI-IBS cases compared with IBS cases without an antecedent episode of acute IGE. Comparisons also suggest that immunoglobulin A (IgA) and IgG profiles may point to pathogen-specific origins among PI-IBS cases., Conclusion: Taken together, these results provide further evidence as to the molecular distinctness of classes of IBS cases and that serum biomarkers may prove useful in elucidating their pathobiological pathways., (Published 2015. This article has been contributed to by US Government employees and their work is in the public domain in the USA.)

Campylobacter jejuni is a leading cause of human gastroenteritis worldwide; however, our understanding of the human immune response to C. jejuni infection is limited. A previous human challenge model has shown that C. jejuni elicits IFNγ production by peripheral blood mononuclear cells, a response associated with protection from clinical disease following re-infection. In this study, we investigate T lymphocyte profiles associated with campylobacteriosis using specimens from a new human challenge model in which C. jejuni-naïve subjects were challenged and re-challenged with C. jejuni CG8421. Multiparameter flow cytometry was used to investigate T lymphocytes as a source of cytokines, including IFNγ, and to identify cytokine patterns associated with either campylobacteriosis or protection from disease. Unexpectedly, all but one subject evaluated re-experienced campylobacteriosis after re-challenge. We show that CD4+ T cells make IFNγ and other pro-inflammatory cytokines in response to infection; however, multifunctional cytokine response patterns were not found. Cytokine production from peripheral CD4+ T cells was not enhanced following re-challenge, which may suggest deletion or tolerance. Evaluation of alternative paradigms or models is needed to better understand the immune components of protection from campylobacteriosis.

Campylobacter jejuni is a major cause of diarrheal disease and results in high levels of morbidity and economic loss in both industrialized and developing regions of the world. To date, prior vaccine approaches have failed to confer protection against this enteric pathogen. Key challenges to the development of a practical Campylobacter vaccine for human use include a lack of understanding of Campylobacter pathogenesis and well-defined immune correlates of protection. With the discovery that C. jejuni expresses a capsule polysaccharide associated with virulence, a conjugate vaccine approach is currently being evaluated. Conjugate vaccines have been successfully developed and implemented against other invasive mucosal pathogens including Streptococcus pneumoniae, Neisseria meningitidis, and Hemophilus influenzae. Furthermore, Shigella-based conjugate vaccines based on lipopolysaccharide have shown promising results in field trials. A prototype C. jejuni conjugate vaccine is currently entering human testing.

High rates of coinfection occur in malaria endemic regions, leading to more severe disease outcomes. Understanding how coinfecting pathogens influence the immune system is important in the development of treatment strategies that reduce morbidity and mortality. Using the Plasmodium chabaudi mouse model of malaria and immunization with model Ags that are either T-dependent (4-hydroxy-3-nitrophenyl [NP]-OVA) or T-independent (NP-Ficoll), we analyzed the effects of acute malaria on the development of humoral immunity to secondary Ags. Total Ig and IgG1 NP-specific Ab responses to NP-OVA were significantly decreased in the P. chabaudi-infected group compared with the uninfected group, whereas NP-specific IgG2c Ab was significantly increased in the P. chabaudi-infected group. In contrast, following injection with T-independent NP-Ficoll, the P. chabaudi-infected group had significantly increased NP-specific total Ig, IgM, and IgG2c Ab titers compared with controls. Treatment with anti-IFN-γ led to an abrogation of the NP-specific IgG2c Ab induced by P. chabaudi infection but did not affect other NP-specific Ab isotypes or titers. IFN-γ depletion also increased the percentage of plasma cells in both P. chabaudi-infected and uninfected groups but decreased the percentage of B cells with a germinal center (GC) phenotype. Using immunofluorescent microscopy, we were able to detect NP(+) GCs in the spleens of noninfected mice, but there were no detectible NP(+) GCs in mice infected with P. chabaudi. These data suggest that during P. chabaudi infection, there is a shift toward an extrafollicular Ab response that could be responsible for decreased Ab responses to secondary T-dependent Ags.

Background: Campylobacter jejuni is a common cause of diarrhea and is associated with serious postinfectious sequelae. Although symptomatic and asymptomatic infections are recognized, protective immunity is not well understood. Previous data suggests that interferon γ (IFN-γ) may be associated with protection. To better define the clinical and immunologic development of protective immunity to C. jejuni, we assessed the ability of an initial infection to prevent clinical illness after a second experimental infection., Methods: Subjects with no clinical or immunologic evidence of prior infection with C. jejuni received an initial challenge with C. jejuni CG8421 with rechallenge 3 months later. The primary endpoint was campylobacteriosis, as defined by diarrhea and/or systemic signs. Close inpatient monitoring was performed. Serum immunoglobulin A (IgA) and immunoglobulin G (IgG), fecal IgA, IgA antibody-secreting cells (ASCs), and IFN-γ production were evaluated. All subjects were treated with antibiotics and were clinically well at discharge., Results: Fifteen subjects underwent a primary infection with C. jejuni CG8421; 14 (93.3%) experienced campylobacteriosis. Eight subjects received the second challenge, and all experienced campylobacteriosis with similar severity. Immune responses after primary infection included serum IgA, IgG, ASC, and IFN-γ production. Responses were less robust after secondary infection., Conclusions: In naive healthy adults, a single infection with CG8421 did not protect against campylobacteriosis. Although protection has been demonstrated with other strains and after continuous environmental exposure, our work highlights the importance of prior immunity, repeated exposures, and strain differences in protective immunity to C. jejuni., Clinical Trials Registration: NCT01048112.

Clinical immunity to malaria declines in the absence of repeated parasite exposure. However, little is known about how B cell populations and antigen-specific memory B cells change in the absence of P. falciparum infection. A successful indoor residual insecticide spraying campaign in a highland area of western Kenya, led to an absence of blood-stage P. falciparum infection between March 2007 and April 2008. We assessed memory B cell responses in 45 adults at the beginning (April 2008) and end (April 2009) of a subsequent 12-month period during which none of the adults had evidence of asymptomatic parasitemia or clinical disease. Antibodies and memory B cells to the 42-kDa portion of the merozoite surface protein-1 (MSP-142) were measured using ELISA and ELISPOT assays, respectively. B cell populations were characterized by flow cytometry. From 2008 to 2009, the prevalence of MSP-142-specific memory B cells (45% vs. 55%, respectively, P = 0.32) or antibodies (91% vs. 82%, respectively, P = 0.32) did not differ significantly, although specific individuals did change from positive to negative and vice versa, particularly for memory B cells, suggesting possible low-level undetected parasitemia may have occurred in some individuals. The magnitude of MSP-142-specific memory B cells and levels of antibodies to MSP-142 also did not differ from 2008 to 2009 (P>0.10 for both). However, from 2008 to 2009 the proportions of both class-switched atypical (CD19+IgD-CD27-CD21-IgM-) and class-switched activated (CD19+IgD-CD27+CD21-IgM-) memory B cells decreased (both P<0.001). In contrast, class-switched resting classical memory B cells (CD19+IgD-CD27+CD21+IgM-) increased (P<0.001). In this area of seasonal malaria transmission, a one- year absence of detectable P. falciparum infection was not associated with changes in the prevalence or level of MSP-142 specific memory B cells, but was associated with major changes in overall memory B cell subsets.

Campylobacter jejuni is a major cause of bacterial diarrheal disease worldwide. The organism is characterized by a diversity of polysaccharide structures, including a polysaccharide capsule. Most C. jejuni capsules are known to be decorated nonstoichiometrically with methyl phosphoramidate (MeOPN). The capsule of C. jejuni 81-176 has been shown to be required for serum resistance, but here we show that an encapsulated mutant lacking the MeOPN modification, an mpnC mutant, was equally as sensitive to serum killing as the nonencapsulated mutant. A nonencapsulated mutant, a kpsM mutant, exhibited significantly reduced colonization compared to that of wild-type 81-176 in a mouse intestinal colonization model, and the mpnC mutant showed an intermediate level of colonization. Both mutants were associated with higher levels of interleukin 17 (IL-17) expression from lamina propria CD4(+) cells than from cells from animals infected with 81-176. In addition, reduced levels of Toll-like receptor 4 (TLR4) and TLR2 activation were observed following in vitro stimulation of human reporter cell lines with the kpsM and mpnC mutants compared to those with wild-type 81-176. The data suggest that the capsule polysaccharide of C. jejuni and the MeOPN modification modulate the host immune response.

CD4 T cells, and especially T follicular helper cells, are critical for the generation of a robust humoral response to an infection or vaccination. Importantly, immunosenescence affects CD4 T-cell function, and the accumulation of intrinsic defects decreases the cognate helper functions of these cells. However, much less is known about the contribution of the aged microenvironment to this impaired CD4 T-cell response. In this study, we have employed a preclinical model to determine whether the aged environment contributes to the defects in CD4 T-cell functions with aging. Using an adoptive transfer model in mice, we demonstrate for the first time that the aged microenvironment negatively impacts at least three steps of the CD4 T-cell response to antigenic stimulation. First, the recruitment of CD4 T cells to the spleen is reduced in aged compared to young hosts, which correlates with dysregulated chemokine expression in the aged organ. Second, the priming of CD4 T cells by DCs is reduced in aged compared to young mice. Finally, naïve CD4 T cells show a reduced transition to a T follicular helper cell phenotype in the aged environment, which impairs the subsequent generation of germinal centers. These studies have provided new insights into how aging impacts the immune system and how these changes influence the development of immunity to infections or vaccinations., (© 2012 The Authors. Aging Cell © 2012 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.)

Blk was identified two decades ago as a B-cell-specific member of the Src family of tyrosine kinases. Recent studies, however, have discovered that Blk is expressed in many cell types outside of the B lineage, including early thymic precursors, interleukin-17-producing γδ T cells and pancreatic β-cells. In light of these recent discoveries, we performed a more comprehensive analysis of Blk expression patterns in hematopoietic cells and found that Blk is differentially expressed in mature B-cell subsets, with marginal zone (MZ) B cells expressing high levels, B1 B cells expressing intermediate-to-high levels and follicular (FO) B cells expressing low levels of Blk. To determine whether these differences in Blk expression levels reflected differential requirements for Blk in MZ, B1 and FO B-cell development, we analyzed the effects of reducing and eliminating Blk expression on B-cell development. We report that both Blk haploinsufficiency and Blk deficiency impaired the generation of MZ B cells. Moreover, although there were fewer MZ B cells in Blk(+/-) and Blk(-/-) mice as compared with Blk(+/+) mice, Blk-mutant MZ B cells were hyper-responsive to B-cell receptor stimulation, both in vitro and in vivo. Thus, this study has revealed a previously unappreciated role for Blk in the development and activation of MZ B cells.

Campylobacter jejuni remains a major cause of bacterial diarrhea worldwide and is associated with numerous sequelae, including Guillain Barré Syndrome, inflammatory bowel disease, reactive arthritis, and irritable bowel syndrome. C. jejuni is unusual for an intestinal pathogen in its ability to coat its surface with a polysaccharide capsule (CPS). These capsular polysaccharides vary in sugar composition and linkage, especially those involving heptoses of unusual configuration and O-methyl phosphoramidate linkages. This structural diversity is consistent with CPS being the major serodeterminant of the Penner scheme, of which there are 47 C. jejuni serotypes. Both CPS expression and expression of modifications are subject to phase variation by slip strand mismatch repair. Although capsules are virulence factors for other pathogens, the role of CPS in C. jejuni disease has not been well defined beyond descriptive studies demonstrating a role in serum resistance and for diarrhea in a ferret model of disease. However, perhaps the most compelling evidence for a role in pathogenesis are data that CPS conjugate vaccines protect against diarrheal disease in non-human primates. A CPS conjugate vaccine approach against this pathogen is intriguing, but several questions need to be addressed, including the valency of CPS types required for an effective vaccine. There have been numerous studies of prevalence of CPS serotypes in the developed world, but few studies from developing countries where the disease incidence is higher. The complexity and cost of Penner serotyping has limited its usefulness, and a recently developed multiplex PCR method for determination of capsule type offers the potential of a more rapid and affordable method. Comparative studies have shown a strong correlation of the two methods and studies are beginning to ascertain CPS-type distribution worldwide, as well as examination of correlation of severity of illness with specific CPS types.

Immunosenescence influences many components of the immune system. Most importantly, profound changes in T cell function are evident in older individuals. The impact of aging on specific T cell subsets has been difficult to examine, but recent advances in murine model systems and new insights into T cell function have allowed for the more precise examination of how T cell responses change with aging. Importantly, recent studies have shown that age-related enhancement of both Th17 generation and regulatory T cell function may contribute to significant changes in immune function. In this review, we summarize the current views on how aging influences the factors that impact T cell function and how this can affect the immune response to infections, vaccinations, and tumors.

It is well established that increasing age is associated with a decreased capacity of the immune system to mediate effective immune responses to vaccination and invading pathogens. Because of the inherent limitations of conducting experiments in humans, much of what we have learned is owed to the utility of experimental mouse models of aging. Recent studies performed in the mouse have demonstrated mechanisms responsible for age-related declines in the function of CD4(+) and CD8(+) cells. This review describes key findings regarding age-related defects in T-cell function and discusses the impact these defects have on vaccine efficacy and immunity.

Age-related declines in humoral responses contribute to the reduced efficacy of vaccines in older populations. Using an adoptive transfer model, we have shown that age-related intrinsic declines in CD4 T cell function contribute significantly to the reduced humoral responses observed with aging, resulting in reduced B cell expansion and differentiation as well as reduced IgG production. In this current study, we show that the helper function of aged CD4 T cells can be enhanced using a TLR-binding adjuvant or an adjuvant containing proinflammatory (PI) cytokines. The helper function of aged CD4 T cells was also enhanced when PI cytokines were added during in vitro CD4 effector generation. Enhanced helper activity resulted in improved expansion and differentiation of B cells and affinity maturation of IgG. PI cytokines also induced significant production of effector cytokines, including IL-4, IFN-gamma, IL-17, and IL-21, by both young and aged CD4 T cells. Importantly, we also show that proinflammatory adjuvants can significantly enhance the humoral response in intact aged animals. We propose that one of the mechanisms involved in the ability of adjuvants to enhance both young and aged T cell responses includes driving multifaceted T cell differentiation and production of multiple cytokines by responding CD4 T cells.

Understanding how aging impacts the function of memory CD4 T cells is critical for designing effective vaccines. Our studies show that immunological memory generated during youth functions well into old age, whereas that generated later in life functions poorly. This is the result of declines in the function of naive CD4 T cells from aged individuals and contributes to reduced efficacy of vaccines in the elderly. To begin to identify the cause of this defect, we examined the function of memory T cells generated from bone marrow precursor cells (BMPC) from young or aged mice in young hosts. In two different models, memory cells derived from young and aged BMPC exhibit good ex vivo and in vivo function. Importantly, memory CD4 T cells generated from aged BMPC exhibit potent cognate helper function for humoral responses, which are critical for effective immunization. These results indicate that there are no apparent age-related intrinsic defects in BMPC with regards to generation of functional memory T cells.

The potency of genetic immunization observed in the mouse has demonstrated the utility of DNA vaccines to induce cell-mediated and humoral immune responses. However, it has been relatively difficult to generate comparable responses in non-rodent species. The use of molecular adjuvants may increase the magnitude of these suboptimal responses. In this study, we demonstrate that the co-administration of plasmid-encoded GM-CSF and CD80/CD86 with a novel ESAT-6:CFP10 DNA vaccine against bovine tuberculosis enhances antigen-specific cell-mediated immune responses. ESAT-6:CFP10+GM-CSF+CD80/CD86 DNA vaccinated animals exhibited significant (p<0.01) antigen-specific proliferative responses compared to other DNA vaccinates. Increased expression (p< or =0.05) of CD25 on PBMC from ESAT-6:CFP10+GM-CSF+CD80/CD86 DNA vaccinates was associated with increased proliferation, as compared to control DNA vaccinates. Significant (p<0.05) numbers of ESAT-6:CFP10-specific IFN-gamma producing cells were evident from all ESAT-6:CFP10 DNA vaccinated animals compared to control DNA vaccinates. However, the greatest increase in IFN-gamma producing cells was from animals vaccinated with ESAT-6:CFP10+GM-CSF+CD80/CD86 DNA. In a low-dose aerosol challenge trial, calves vaccinated as neonates with Mycobacterium bovis BCG and ESAT-6:CFP10+GM-CSF+CD80/CD86 DNA exhibited decreased lesion severity in the lung and lung-associated lymph nodes following viruluent M. bovis challenge compared to other vaccinated animals or non-vaccinated controls. These data suggest that a combined vaccine regimen of M. bovis BCG and a candidate ESAT-6:CFP10 DNA vaccine may offer greater protection against tuberculosis in cattle than vaccination with BCG alone.

Cell-mediated immune responses are critical for protective immunity to mycobacterial infections. Recent progress in defining mycobacterial antigens has determined that region of difference 1 (RD1) gene products induce strong T-cell responses, particularly the early secretory antigenic target 6-kDa (ESAT-6) protein and culture filtrate protein 10 (CFP10). However, comprehensive analysis of the immune response towards these antigens is incompletely characterized. To evaluate recall responses to ESAT-6 and CFP10, peripheral blood mononuclear cells from M. bovis-infected cattle were stimulated in vitro with a recombinant ESAT-6 (rESAT-6)-CFP10 fusion protein and compared to responses induced by M. bovis-derived purified protein derivative. Following antigenic stimulation, activation marker expression was evaluated. Significant proliferative responses (P < 0.05) were evident in CD4(+), CD8(+), immunoglobulin M-positive, and CD172a(+) cell fractions after 6 days of culture. Expression of CD25 and CD26 was increased (P < 0.05) on CD4(+), CD8(+), and gammadelta T-cell-receptor-positive cells. CD4(+) and CD8(+) cells also exhibited significant changes (P < 0.05) in expression of CD45 isoforms. Using a flow cytometry-based proliferation assay, it was determined that CD45R expression is downregulated (P < 0.05) and that CD45RO expression is upregulated (P < 0.05) on proliferating (i.e., activated) CD4(+) cells. Collectively, data indicate that recall immune responses directed toward the rESAT-6-CFP10 fusion protein or purified protein derivative are comparable and that recall to mycobacterial antigens correlates with a CD45RO(+) phenotype.

DNA vaccination is known to elicit robust cellular and humoral responses to encoded antigen. The co-administration of costimulatory molecules CD80 (B7-1), CD86 (B7-2) and CD154 (CD40L) has been shown to enhance immune responses in several murine models. The role of specific costimulatory molecules in non-rodent species remains incompletely characterized. In these studies, we demonstrate that the co-administration of CD80 and CD86, but not CD154, to an existing candidate subunit DNA vaccine (ESAT-6) against bovine tuberculosis, enhances protection after aerosol challenge with virulent Mycobacterium bovis. Additionally, we have shown that vaccination with M. bovis BCG is protective against tuberculosis following aerosol challenge in cattle. Two independent trials were conducted in cattle to determine the adjuvant effect of encoded antigen + CD80/CD86 and directly compare the adjuvant activities of CD80/CD86 to those of CD154. Co-administration of either CD80/CD86 or CD154 enhanced ESAT-6-specific IFN-gamma responses as compared to animals vaccinated with ESAT-6 DNA alone. However, following aerosol challenge, only animals vaccinated with CD80/CD86 possessed decreased pathology of the lungs and associated lymph nodes, as measured by gross examination, radiographic lesion morphometry and bacterial recovery. Collectively, these results demonstrate that the co-administration of costimulatory molecules with a protective antigen target enhances bovine immune responses to DNA vaccination, and that CD80/CD86 is superior to CD154 in augmenting DNA vaccine-induced protection in experimental bovine tuberculosis.

Granulysin and NK-lysin are antimicrobial proteins found in the granules of human and swine cytotoxic lymphocytes. A murine counterpart to granulysin has not been identified to date, indicating the importance of additional models to fully characterize the role of granulysin-like molecules in the immune response to infectious disease. Two partial nucleotide sequences corresponding to the complete functional domain of granulysin and NK-lysin were amplified from bovine PBMC mRNA. Following stimulation with phorbol ester and calcium ionophore, expression of the bovine gene was detected in CD3(+) T cells, CD4(+) T cells, CD8(+) T cells, WC1(+) gammadelta T cells, and PBMC depleted of CD3(+) T cells, but was absent in CD21(+) cells and CD14(+) cells. Intracellular flow cytometry and immunoblotting confirmed the presence of protein corresponding to the bovine granulysin homologue in activated T lymphocytes and PBMC. Synthetic human, bovine, and swine peptides corresponding to the C terminus of helix 2 through helix 3 region of granulysin displayed potent antimicrobial activity against Escherichia coli, Salmonella enteritidis, Staphylococcus aureus, and Mycobacterium bovis bacillus Calmette-Guérin. Human and bovine peptides corresponding to helix 2 displayed antimycobacterial activity against M. bovis bacillus Calmette-Guérin. Expression of the bovine gene was detected in laser microscopy-dissected lymph node lesions from an M. bovis-infected animal. The identification of a biologically active bovine homologue to granulysin demonstrates the potential of the bovine model in characterizing the role of granulysin in the immune response to a variety of infectious agents.

Setting: 1,25-dihydroxyvitamin D3 (1,25(OH)(2)D(3)) is a potent modulator of immune responses and may be beneficial in the treatment of tuberculosis. Recent evidence suggest that 1,25(OH)(2)D(3) may affect T-dependent responses in cattle; however, mechanisms by which this vitamin modulates activation of bovine T cells are unclear., Objective: Determine the effects of 1,25(OH)(2)D(3) on the expression of CD25, CD44, and CD62L by bovine T cell subsets proliferating in response to antigen stimulation., Design: Antigen-specific recall responses of Mycobacterium bovis bacille Calmette-Guerin (BCG) vaccinated cattle were used as a model system to evaluate effects of 1,25(OH)(2)D(3) on the proliferation and activation of bovine T cell subsets., Results: CD4(+) and gamma delta TCR(+) cells were the predominant T cell subsets responding to soluble crude M. bovis-derived antigens (i.e., purified protein derivative and a BCG whole cell sonicate) by proliferation and activation-induced alterations in phenotype. These subsets exhibited increased CD25 and CD44 mean fluorescence intensity (mfi) and decreased CD62L mfi upon antigen stimulation. Addition of 1,25(OH)(2)D(3) inhibited proliferation of CD4(+) cells and decreased the expression of CD44 on responding (i.e., proliferating) CD4(+) and gamma delta TCR(+) cells., Conclusion: These findings suggest that the production of 1,25(OH)(2)D(3) by macrophages within tuberculous lesions would inhibit proliferation and CD44 expression by co-localized CD4(+) and gamma delta TCR(+) cells.