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To understand the protective mechanisms available in the mammalian immune system, we have characterized the murine protective immune response to an intracellular bacterium, Francisella tularensis LVS. This infection model has several convenient features; for example, mice that survive and clear sublethal intradermal (i.d.) infection are solidly immune to subsequent lethal infection with more than 10⁵ 50% lethal doses (LD₅₀s) given intraperitoneally (i.p.) or intravenously (i.v.) a month later (9, 10). This immunity is clearly dependent on /⁺ T cells, but either CD4⁺ or CD8⁺ T cells alone can suffice (15). As with other intracellular infections such as Listeria monocytogenes (13), a strong innate immune response mediated by macrophages and natural killer cells and dependent on gamma interferon and tumor necrosis factor alpha permits mice to survive primary infection for as long as 30 days (7). However, an unusual feature of LVS infection in mice is the rapid generation of very strong protective immunity against lethal challenge very quickly after establishment of sublethal infection: normal mice given a sublethal dose of 10³ LVS bacteria i.d. can survive a lethal i.p. or i.v. challenge of more than 10⁶ LD₅₀s given only 3 days after the initial sublethal i.d. infection (4, 5). This mechanism is of interest because of its remarkable strength, its very quick time course, and its dependence on lymphocytes, particularly B cells. The last of these properties distinguishes this mechanism from traditional innate immunity, which is mediated largely by macrophages and natural killer cells.

Since early protection in LVS infection is dependent on lymphocytes, we have questioned whether this mechanism is specific. Previous work has studied the effect of sublethal infection with either another intracellular pathogen, Salmonella typhimurium, or an extracellular bacterium, pathogenic Escherichia coli, on protection against lethal LVS challenge 3 days later. The failure to generate heterologous protection with either bacterium suggested that protection was indeed specific, as no enhanced survival could be demonstrated (5). However, in each of these infections no early protective immunity for the homologous infection could be demonstrated; i.e., sublethal infection with S. typhimurium did not protect against lethal S. typhimurium challenge 3 days later. Thus, we could not determine whether inability to demonstrate heterologous protection against LVS challenge was due to a failure of S. typhimurium to stimulate an early protective response at all or due to a requirement for antigen-specific recognition and activity. Since the nature of the innate and long-term protective immunity to L. monocytogenes is very similar to that to LVS (3, 6, 12, 13), we have reexamined these questions in this study using L. monocytogenes.

F. tularensis LVS (live vaccine strain [ATCC 29684]; American Type Culture Collection, Manassas, Va.) was cultured on modified Mueller-Hinton (MH) agar plates or in modified MH broth (Difco Laboratories, Detroit, Mich.) supplemented with ferric pyrophosphate and IsoVitaleX (Becton Dickinson, Cockeysville, Md.) as previously described (5, 10). Listeria monocytogenes EGD (ATCC 15313) was a gift from William Schwan and was cultured in brain heart infusion broth or plates (Difco). One-milliliter aliquots of bacteria frozen in broth alone at 70°C were periodically thawed for use, and viable bacteria were quantified by plating serial dilutions on MH agar plates. The number of CFU present after thawing varied less than 5% over a 6-month period. Specific-pathogen-free, male BALB/cByJ mice were purchased from Jackson Laboratories (Bar Harbor, Maine) and were used at 6 to 16 weeks of age. Male BALB/c.scid mice, as well as Igh6 (B-cell-knockout) mice and Tcrb/Tcrd (double / and / T-cell receptor [TCR]-knockout) mice on a C57BL/6J background, were also purchased from Jackson Laboratories. Male C57BL/6NCR and BALB/c.nu/nu mice were purchased from the Biological Resources Branch, Frederick Cancer Research and Development Center, National Cancer Institute (Frederick, Md.). All mice were housed in sterile microisolator cages in a barrier environment at the Center for Biologics Evaluation and Research (CBER), fed autoclaved food and water ad lib, and routinely tested for common murine pathogens by a diagnostic service provided by the Division of Veterinary Services, CBER; all such sentinel mice tested negative over the course of these experiments. In conducting the research described in this report, the investigators adhered to a protocol approved by the Animal Care and Use Committee of the CBER. Groups of three to six mice, as indicated, were given 0.5 ml i.p. or 0.1 ml i.d. of the indicated dilution of LVS or Listeria EGD; actual doses of bacteria inoculated were simultaneously determined by plate count. All materials, including bacteria, were diluted in phosphate-buffered saline (PBS) (BioWhittaker, Walkersville, Md.) containing <0.01 ng of endotoxin per ml. The mean time to death (MTD) was calculated as the arithmetic mean ± standard deviation for all mice that died within a group; surviving mice were not included in this calculation. The statistical significance of differences in MTD was assessed by Student's t test.

Initial experiments demonstrated that both BALB/cByJ and C57BL/6 mice readily survived i.d. infection with Listeria EGD. The LD₅₀ for i.d. challenge (i.d. LD₅₀) for BALB/cByJ mice was about 10⁶ bacteria (based on two determinations using groups of five mice; in one experiment the LD₅₀ was 1.1 × 10⁶ bacteria, and in the other it was 9.4 × 10⁵ bacteria). The i.d. LD₅₀ for C57BL/6 mice was greater than 10⁷ bacteria, the maximum dose that could be used to infect mice (based on two determinations using groups of five mice). The mice that survived Listeria EGD infection for more than 1 week also survived for over 2 months (data not shown). Mice given sublethal doses of Listeria EGD i.d. had no detectable bacteria in spleens, livers, and lungs 3 weeks later (data not shown) and thus were assumed to have cleared the infection. The i.p. LD₅₀ for this strain of Listeria (EGD) was about 2.5 × 10³ bacteria in BALB/cByJ mice and about 2 × 10⁴ bacteria in C57BL/6 mice (data not shown), slightly more than the previously reported i.v. LD₅₀ for this bacterial strain (12).

To determine whether the early protective immune mechanism operates in L. monocytogenes infection, BALB/cByJ mice were given a sublethal priming dose of 10⁵ Listeria EGD bacteria i.d. and then lethally challenged with 2.5 × 10⁵ Listeria EGD bacteria i.p., or 100 LD₅₀s, 3 days later; these mice survived both infections (Table 1). Further, 80% of mice challenged with 1,000 LD₅₀s survived, although mice given 10,000 LD₅₀s (2.5 × 10⁷ bacteria) did not survive. As shown previously (5), mice primed with 10⁴ LVS bacteria i.d. survived a challenge with more than 10⁵, but not 10⁷, LD₅₀s of LVS (Table 1). When BALB/cByJ mice were primed with a sublethal infection of 10⁴ LVS bacteria i.d. and then challenged with heterologous L. monocytogenes lethal infection, survival rates were almost as good as those seen with homologous L. monocytogenes priming (Table 1). Conversely, mice primed with 10⁵ Listeria EGD bacteria and challenged with various doses of LVS survived almost as well as mice primed with the homologous LVS infection (Table 1). Taken together, these data indicate that the early protective mechanism (sublethal infection followed quickly by secondary lethal infection) can readily be elicited in L. monocytogenes infection. As in LVS infection, early protection in Listeria infection is relatively strong and able to protect against a lethal challenge of about 1,000 LD₅₀s, which is a large absolute number of bacteria in Listeria infection. Further, heterologous protection can easily be demonstrated as well, indicating that this protective mechanism is nonspecific.

To determine the cellular basis of early protection in L. monocytogenes infection, various immunodeficient mice were primed with 10⁵ Listeria EGD bacteria i.d. and challenged with a maximal lethal infection of 2.5 × 10⁶ Listeria EGD bacteria (1,000 LD₅₀s) i.p. 3 days later. Normal BALB/cByJ and C57BL/6J mice readily survived priming and challenge (Table 2). However, lymphocyte-deficient scid mice, T-cell-deficient nu/nu mice, double TCR-knockout mice, and Igh6 (B-cell-knockout) mice were quite compromised in their ability to survive secondary lethal infection (Table 2). In combined experiments, 100% (14 of 14) of double TCR-knockout mice and 64% (9 of 14) of B-cell-knockout mice given this 1,000-LD₅₀ challenge dose died in less than 10 days, indicating that early protection involves both B cells and T cells. Thus, early protection in LVS infection is similar to that in Listeria infection in being heavily dependent on B cells, but unlike LVS, early protection in Listeria infection also requires T cells. Clearly, this lymphocyte-dependent early protection is distinct from traditional innate immunity, which is a function primarily of neutrophils, macrophages, and natural killer cells (13).

This nonspecific protection is similar to observations previously described by Killar and Eisenstein (11). In those studies, mice given an avirulent vaccine strain of S. typhimurium, SL3235, were protected against challenge with 1,000 LD₅₀s of a virulent S. typhimurium challenge given 3 days, but not 1 day, after immunization. However, mice given Salmonella SL3235 were also protected against challenge with 100 LD₅₀s of L. monocytogenes given 6, but not 3 days after immunization. This nonspecific cross-protection waned by 21 to 30 days. Similarly, we have observed no reciprocal protection between LVS and Listeria EGD in mice given 10 LD₅₀s of bacteria as a heterologous challenge 35 days after priming (data not shown). The Salmonella SL3235 studies attributed protection primarily to macrophages, since adherent cells but not T cells were able to adoptively transfer protection (11); nonetheless, evidence for T-cell responsiveness in proliferation assays was presented, and cross-protection was not tested in immunodeficient mice. It is possible, as originally suggested by Mackaness many years ago, that early T-cell activity is responsible for macrophage activation that leads to control of infection. Further, comparison of the present study with the earlier report (11) suggests a possible explanation for our previous inability to demonstrate early protection in Salmonella infection: our previous results may have been due to differences in the bacterial strains used, not to inherent lack of early protection in Salmonella.

The bacterial components responsible for generation of early protective immunity are currently under study. Another report demonstrating nonspecific protection in these intracellular infections, by using a different vaccine strain of F. tularensis and challenging 2 weeks later with the NCTC 7973 strain of L. monocytogenes, attempted to isolate bacterial cell surface proteins responsible for stimulating cross protection and failed to do so (2). In contrast, we have obtained evidence that both LVS chromosomal DNA and LVS lipopolysaccharide, which is neither endotoxic nor a traditional B-cell mitogen or macrophage activator (1, 14), can stimulate early protection (8). Thus, future studies will continue to investigate the cellular and molecular basis for this nonspecific, strong, early protective mechanism, and consider situations in which this rapid protection might be exploited in immunotherapies against infections.