Although it is generally recognized that the function of the immune system declines with age, the nature of the underlying defects is still poorly understood. We now demonstrate the predominance of CD8+CD28− T cell clonal expansions in elderly persons who fail to produce specific Abs following influenza vaccination. These clones express effector cell markers and are mostly CD45RA+. When isolated and put into culture, they are unable to proliferate, but produce IFN-γ (but no IL-5) upon stimulation with anti-CD3 or autoantigen. These autoreactive CD8+ type 1 effector cells seem to trigger a Th1 polarization, as CD4+ T cells from elderly persons without in vivo Ab production produce Th1, but only low amounts of Th2 cytokines upon in vitro stimulation with PHA. Therefore, the increased occurrence of CD8+CD28− clonal expansions may be decisive for the development of immune deficiency in the elderly.

Pharmacologic gene regulation is a key technology, necessary to achieve safe, long-term gene transfer. The approaches described in the scientific literature all share in common the creation of artificial transcription factors by fusing a DNA-binding domain, a drug-binding domain and a transcription activation domain. These transcription factors activate the transgene expression upon binding of the pharmacologic agent (antibiotics of the tetracycline family, insect hormone, progesterone antagonist, or immunosuppressor drug) to the drug-binding domain. The major limitations to the use of these systems for human gene and cell therapies are the toxicity of the inducer molecule and the immunogenicity of the chimeric transcription factor. Thus, the gene regulation systems should operate with clinically approved drugs with safety records that do not conflict with the therapeutic gene expression regimen. This work focuses on the characterization of the immunogenicity of a tetracycline-activated transcription factor commonly used in preclinical gene therapy, rtTA2-M2, and its impact on reporter gene expression. We demonstrate that intramuscular injection of plasmid or adenoviral vectors encoding rtTA-M2 in outbred primates generates a cellular and humoral immune response to this transcription factor. The immune response to rtTA2-M2 blunts the duration of the expression the rtTA2-M2-controlled transgene in primates, presumably by destruction of the cells that coexpress rtTA2-M2 and the reporter or therapeutic gene. This immune response may result directly from the vectors used in this study, which prompts the development of new gene transfer vectors enabling safe and efficient pharmacologic gene regulation in clinic.

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The aim of vaccination is to rapidly elicit protective immunity and generate memory for sustained protection. We studied the induction and persistence of polysaccharide (PS)-specific memory in neonatal and infant mice primed with pneumococcal conjugate (Pnc1-TT) by assessing the response to native pneumococcal PS (PPS-1), the kinetics of the PPS-1-specific IgG response to a second Pnc1-TT dose and affinity maturation. A subcutaneous (s.c.) Pnc1-TT booster induced a rapid increase in PPS-1-specific IgG, indicating efficient priming for memory by a single dose of Pnc1-TT already at 1 week of age. High levels were maintained for >12 weeks. However, a PPS-1 booster induced no response in neonatal or infant mice. The adjuvant LT-K63 significantly enhanced the IgG response and affinity to Pnc1-TT by both the s.c. and the intranasal (i.n.) route in all age groups. In neonatal and infant mice, PPS-1 and LT-K63 induced a booster response only when given i.n. following either s.c. or i.n. priming with Pnc1-TT and LT-K63. In contrast, PPS-1 with or without LT-K63 administered s.c. compromised the ongoing PPS-1-specific response elicited in neonatal mice by either s.c. or i.n. priming with Pnc1-TT and LT-K63. These results demonstrate the advantage of the mucosal route for elicitation of PS-specific memory responses in early life.

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