IL-12 has also been shown to be very effective as a vaccine adjuvant for enhancement of protective antibody responses against pulmonary bacterial and viral infections, and is particularly effective when administered locally to mucosal surfaces, i.e.
, intranasally (i.n.), together with vaccine. We and others have repeatedly found that i.n. treatment of mice with IL-12 leads to increases in expression of both Th1 and Th2 antibodies not only in the serum and but particularly, in the lung. In a typical experiment, mice are inoculated i.n. with vaccine only or vaccine + IL-12. Bronchoalveolar lavage (BAL) fluids and sera are then collected on Day 35 and titers are determined by isotype-specific ELISAs. It was found that inclusion of IL-12 during vaccination induces significant levels of both IgG and IgA antibodies that are not expressed after exposure to vaccine alone. Protection against lethal viral and bacterial infections include those induced with influenza virus [41
], Streptococcus pneumoniae
], Francisella tularensis
], and Yersinia pestis
(D. Kumar and D.W. Metzger, unpublished results).
In experiments with lethal influenza virus infection [41
], it was found that all mice given only PBS 35 days before viral challenge succumbed to influenza infection by Day 9 and that i.n. immunization with only influenza subunit vaccine consisting of purified hemagglutinin (H1) and neuraminidase (N1) provided only partial protection. Combining vaccine with i.n. IL-12 inoculation, however, enhanced levels of both lung and spleen IFN-γ and IL-10, as well as serum and BAL IgG2a anti-H1N1 antibody. In addition, it enhanced expression of secretory IgA anti-H1N1 antibody in BAL fluid. Importantly, mice immunized i.n. with H1N1 and IL-12 exhibited decreased weight loss and dramatically increased survival rates after lethal challenge with live influenza virus compared to animals inoculated with vaccine in vehicle only. Protection was dependent on B cells as demonstrated using µMT KO mice, and could be transferred to naïve mice by inoculation of either serum or BAL fluid from IL-12-treated mice. IL-12 administration alone had no effect on survival.
As discussed above, the major effect of IL-12 on humoral immunity is induction of IgG2a, which happens to be the murine isotype that is most effective at mediating complement fixation and opsonization through high affinity binding to FcγRI on phagocytic cells. To examine the precise role of FcγR during IL-12-mediated protection, the influenza virus infection model was again used in BALB/c FcR γ chain KO mice. These mice have a genetic disruption in expression of the common γ chain that is shared by FcγRI and III, as well as by FcεRI and in humans, FcαR, which remains undefined in mice. WT and FcR γ chain KO mice were immunized with influenza H1N1 subunit vaccine in the presence or absence of IL-12. The mice were then challenged i.n. 5 weeks later with 2 × 103
PFU of A/PR/8/34 influenza virus and monitored daily for mortality. It was found that WT and KO mice behaved identically with regard to cytokine and antibody production, yet the KO mice were significantly more susceptible to lethal influenza infection [45
]. The results indicated for the first time that control of influenza infection by IgG2a occurs through FcγR-mediated clearance rather than simply antibody neutralization.
Similar effects were seen for pneumococcal lung infection [42
]. BALB/c mice were inoculated i.n. with 1 µg of the pneumococcal surface protein A (PspA) and IL-12. One month later, the IL-12-treated mice were found to have enhanced levels of total and IgG2a anti-PspA antibodies in both serum and BAL. Upon transfer of sera to naïve recipients and challenge with a lethal dose of S. pneumoniae
, all mice that had received normal mouse serum succumbed to infection within 5 days while mice that had received serum from animals vaccinated with PspA alone showed no significantly increased protection. Strikingly, every mouse that had received serum from animals treated with both PspA and IL-12 survived the infection. In addition, i.n. vaccination with PspA and IL-12 provided increased protection against nasopharyngeal carriage. Protection was due to passively transferred antibody since it was lost by pre-adsorption of the sera before transfer with anti-Ig-coated beads.
More recent experiments have focused on the use of IL-12 as a mucosal vaccine adjuvant for protection against biothreat agents that could be used for biowarfare and bioterrorism. F. tularensis
is a gram-negative facultative intracellular bacterium that is ingested by and multiplies within macrophages, and that causes the pulmonary form of tularemia in humans [46
]. Because of its extreme infectivity, ease of dissemination, and substantial capacity to cause illness and death, the Working Group on Civilian Biodefense considers Ft
to be a dangerous potential biological weapon and has classified it as a Category A Select Agent. Vaccination with the live vaccine strain (LVS) of F. tularensis
can partially engender protection against lethal respiratory tularemia, but use of inactivated or subunit forms of the bacterium has been found to be only poorly effective. A probable reason for the lack of efficacy observed with inactivated LVS is the fact that F. tularensis
is an intracellular pathogen. Inactivated organisms and soluble proteins typically induce Th2-like immunity, while live, attenuated organisms are more effective at inducing cell-mediated Th1-like immunity that is believed to be required for protection against intracellular pathogens. Thus, we hypothesized that establishment of a lung Th1 environment during vaccination with inactivated LVS would induce protection against challenge with fatal doses of LVS. Indeed, it was found that i.n. vaccination with inactivated LVS in the presence of IL-12 resulted in 90–100% survival after lethal challenge while mice receiving only vaccine or only IL-12 all succumbed to infection [44
]. Survival was correlated with reductions in bacterial burden, inflammation, and production of immunomodulatory cytokines (IFN-γ, TNF-α, and IL-6) in the lung as well as in the liver and spleen. While NK cells were primarily responsible for the production IFN-γ in the lungs of unvaccinated animals, vaccinated mice showed increased levels of lung IFN-γ+
CD4 T cells. These results demonstrated that i.n. vaccination with inactivated F. tularensis
LVS combined with IL-12 can result in protection from respiratory tularemia.
The role of antibodies in protection against respiratory infection with the intracellular bacterium F. tularensis
is not clear. To investigate the ability of antibodies to clear bacteria from the lungs and prevent systemic spread, studies were conducted similar to those described above for pneumococcal infection. Immune serum was passively administered i.p. to naïve mice before i.n. F. tularensis
LVS infection. It was found that immune serum treatment provided 100% protection against lethal challenge while normal serum or Ig-depleted immune serum provided no protection [47
]. Protective efficacy was correlated with increased clearance of bacteria from the lung and required expression of FcγR on phagocytes, including macrophages and neutrophils. However, complement was not required for protection. In vitro
experiments demonstrated that macrophages were more readily infected by antibody-opsonized bacteria but became highly efficient in killing upon activation by IFN-γ. Consistent with this finding, in vivo
antibody-mediated protection was found to be dependent upon IFN-γ. SCID mice that lack T cells but contain normal or even elevated numbers of NK cells were not protected by passive antibody transfer, suggesting that T cells but not NK cells serve as the primary source for IFN-γ. These data indicate that a critical interaction of humoral and cellular immune responses is necessary to provide sterilizing immunity against F. tularensis
. Of considerable interest was the finding that serum antibodies were capable of conferring protection against lethal respiratory tularemia when given 24 to 48 hr post-exposure. Thus, this study provided the first experimental evidence for the therapeutic use of antibodies in Francisella
Similar studies are now being performed using inactivated Y. pestis
for i.n. vaccination in the presence of IL-12. Curiously, it is believed that killed Y. pestis
is incapable of providing protection against the pneumonic form of plague initiated by the respiratory route of infection even though several groups are actively attempting to identify subunit targets for protection of mucosal surfaces against the pathogen [48
]. In our hands, inactivated Y. pestis
inoculated i.n. together with IL-12 can provide complete protection against pulmonary challenge with the highly virulent CO92 Y. pestis