IL-12 as an adjuvant for the enhancement of protective humoral immunity

Large numbers of microbes and microparticles enter the airways with every breath, and the respiratory tract thus represents a major portal of entry for various viral and bacterial pathogens. However, although the respiratory tract represents approximately 25% of the total 400 m² of mucosal tissue in the adult human, little is generally known about immune function in the airways, and much of our current understanding is actually based upon information obtained from studies of the gastrointestinal system. Despite our general lack of knowledge regarding the pulmonary immune system, there is continued interest in the effective induction of mucosal immunity for the prevention of respiratory infections [1]. Intranasal vaccination is an attractive, noninvasive procedure that induces both mucosal and systemic immunity. However, for inactivated or protein/polysaccharide subunit vaccines, intranasal vaccination is typically only effective when the antigen is coadministered with adjuvants. Bacterial enterotoxins, such as cholera toxin, have been found to be potent mucosal vaccine adjuvants in animals [2,3]. Unfortunately, these molecules are highly toxic to humans and variant molecules that have been engineered to cause reduced toxicity still induce significant neurological sequelae [4]. Furthermore, they primarily induce Th2 responses [5] that can exacerbate lung pathology rather than enhance protection [6]. The reasons for their effectiveness in mice are largely unknown, and this lack of knowledge concerning the requirements for a protective mucosal vaccine adjuvant has significantly hampered attempts to develop such adjuvants for use in humans.

Another adjuvant approach focuses on the use of cytokines to induce specific types of immunity. In particular, IL-12 has shown considerable promise as a mucosal adjuvant in animal models. IL-12 is a heterodimeric cytokine that has a molecular weight of 75 kDa and is composed of disulfide-bonded 40- and 35-kDa subunits. It is part of a larger family of cytokines that also comprises IL-23 and IL-27, is produced by a variety of antigen-presenting cells, including macrophages and dendritic cells, and was originally found to bind to receptors on activated T cells and natural killer cells [7,8]. Owing to its ability to stimulate IFN-γ production, IL-12 is extremely potent in enhancing cell-mediated immunity to foreign pathogens. Less well-recognized is its similar strong influence on antibody responses [9–14]. We have found that activated murine and human B cells bind IL-12 and express transcripts for both β1- and β2-chains of the IL-12 receptor [15], findings that have been confirmed by others (Gately MK, Pers. Comm.) [16,17]. Furthermore, it has been found that direct interaction of B cells with IL-12 leads to cell proliferation and differentiation [18], IFN-γ production [19,20] and production of the type 2 cytokine IL-4 [21].

It has now been demonstrated through a series of experiments in animals that IL-12 is an effective vaccine adjuvant for the enhancement of protective antibody responses against lethal pulmonary bacterial and viral infections. These include infections induced with influenza virus [22], Streptococcus pneumoniae[23–25], Francisella tularensis[26] and Yersinia pestis [Kumar D, Metzger DW, Unpublished Data]. We and others have repeatedly found that intranasal treatment of mice with IL-12 leads to increases in expression of both Th1- and Th2-associated antibodies in the serum [9–14] as well as in the lung [22–27]. In a typical experiment, mice are inoculated intranasally with vaccine only or vaccine plus IL-12. Lung fluids are collected on day 35 and titers determined by isotype-specific enzyme-linked immunosorbent assays. It has been found that the inclusion of IL-12 during intranasal vaccination induces significant levels of both pulmonary IgG and IgA antibodies that are not expressed after exposure to vaccine alone. These increases are partially IFN-γ dependent and IFN-γ independent [28,29]. In experiments with influenza virus infection, it was found that all mice given only phosphate buffered saline before viral challenge succumbed to influenza, and that intranasal immunization with influenza subunit vaccine alone provided only partial protection. However, combining the vaccine with intranasal IL-12 followed 35 days later by virus challenge resulted in 100% protection [22]. IL-12 administration alone had no effect on survival. Protection did not occur in vaccinated B-cell-deficient mice [22], nor in mice lacking Fc receptor expression [30].

Similar effects have been seen for pneumococcal lung infection. For example, upon transfer of sera to naive recipients and intranasal challenge with a lethal dose of S. pneumoniae, all mice that received normal mouse serum succumbed to infection within 5 days, and mice receiving serum from animals vaccinated with pneumococcal surface protein A (PspA) alone showed no significantly increased protection [31]. Strikingly, every mouse that received serum from animals treated with both PspA and IL-12 survived the infection. Protection was due to transferred antibody since it was lost by pre-adsorption of the sera before transfer with anti-Ig-coated beads. In addition, intranasal vaccination with PspA and IL-12 provided increased protection against nasopharyngeal carriage. The aforementioned results were obtained using a T-dependent pneumococcal protein antigen as the vaccine but, importantly, similar results have been observed using pneumococcal and meningococcal polysaccharide vaccines, given either as T-dependent conjugate vaccines, for example Prevnar^®, or as T-independent nonconjugated vaccines, such as Pneumovax^®[23–25,28,32]. In both cases, it was found that IL-12 treatment at the time of immunization induced significantly elevated levels of IgG2a and IgG3 anti-polysaccharide antibodies in serum, as well as IgA and IgG antibodies in the lung. In keeping with previous results using T-dependent protein antigens, IL-12 did not inhibit but rather enhanced total IgM and IgG1 serum antibody levels. Mice lacking T cells (β^-δ^--knockout mice) or T and natural killer cells (CD3ε-transgenic mice) still showed enhancement of antibody responses to T-independent forms of polysaccharide antigens, demonstrating that such cells were not necessary for the observed IL-12 effects [28]. Furthermore, the use of IFN-γ-deficient mice showed that stimulation of T-independent antibody responses by IL-12 was only partially dependent on IFN-γ.

Of interest are experiments demonstrating the ability of IL-12 to serve as an effective mucosal vaccine adjuvant in neonates. S. pneumoniae is the leading bacterial cause of acute otitis media (OM) in young children and often produces invasive disease. Typical intramuscular routes of vaccination are poorly protective against development of OM. To test intranasal vaccination efficacy, neonatal 1-week-old mice were inoculated with pneumococcal polysaccharide conjugate vaccine and IL-12 as a mucosal adjuvant. The protective efficacy of this treatment was then tested by challenging immunized infant (3-week-old) mice with bacteria to induce OM and invasive disease. Intranasal vaccination was found to enhance levels of specific antibodies, mostly IgA antibodies, in middle ear (ME) washes and sera [33]. Immunization in the presence of IL-12 resulted in enhanced clearance of S. pneumoniae from the ME, and opsonization of bacteria with ME wash fluids or sera from immunized mice caused increased bacterial clearance from the ME of naive mice. In addition, immunized mice demonstrated 89% survival after OM-induced invasive pneumococcal infection, compared with 22% survival in unvaccinated mice. These results indicate that intranasal vaccination of neonatal mice in the presence of IL-12 is able to enhance ME mucosal and systemic immune responses to pneumococci and efficiently protect against both OM and invasive infection.

Of considerable concern regarding the use of any vaccine adjuvant in humans is the issue of potential toxicity. This is particularly relevant for IL-12 since, in human trials, IL-12 was found to be highly toxic when administered parenterally, limiting its usefulness for in vivo therapy. Indeed, IL-12 has been reported in one human pneumococcal vaccination study to induce unacceptable levels of side effects when administered subcutaneously, without concomitant induction of protective antibody levels [34]. We thus conducted a detailed study to compare the toxicity of IL-12 administered directly to a mucosal site versus subcutaneous administration [35]. It was found that IL-12 administered subcutaneously for 6 consecutive days had an LD₅₀ of between 0.125 and 0.25 µg/day compared with intranasal IL-12, which had a four- to eight-fold higher LD₅₀ (1 µg/day). Furthermore, when delivered intranasally, IL-12 induced less systemic IFN-γ and fewer pathological changes, yet was efficacious, as indicated by enhanced levels of IgG2a. Others have similarly seen a lack of IL-12 toxicity when administered intranasally, even after the administration of very high doses of IL-12, and have also observed no neurological inflammation, which is a significant problem with the use of bacterial enterotoxins as adjuvants [Boyaka P, McGhee J, Pers. Comm.].

Thus, the findings to date indicate that IL-12 enhances both protein and polysaccharide vaccine antibody responses, and that introduction of IL-12 intranasally is an effective and safe route of inoculation. Nonetheless, when combined with vaccination, IL-12 may induce sufficient local inflammation to allow for IgG transudation, especially during an inflammatory infection process. Under noninflammatory conditions, such as occurs during bacterial nasopharyngeal colonization, it is likely that secretory IgA, which is also induced by IL-12 administration, is most critical for protection [25]. Future work will provide additional insight into the efficacy of IL-12 as a vaccine adjuvant and its immunomodulatory activity in the lung environment.

Financial & competing interests disclosure

The work was supported by grant #RO1 AI41715 from the National Institutes of Health. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.