Immunotherapeutic activity of a conjugate of a Toll-like receptor 7 ligand

Abstract

The immunotherapeutic activity of Toll-like receptor (TLR) activators has been difficult to exploit because of side effects related to the release and systemic dispersion of proinflammatory cytokines. To overcome this barrier, we have synthesized a versatile TLR7 agonist, 4-[6-amino-8-hydroxy-2-(2-methoxyethoxy)purin-9-ylmethyl]benzaldehyde (UC-1V150), bearing a free aldehyde that could be coupled to many different auxiliary chemical entities through a linker molecule with a hydrazine or amino group without any loss of activity. UC-1V150 was covalently coupled to mouse serum albumin (MSA) at a 5:1 molar ratio to yield a stable molecule with a characteristically altered UV spectrum. Compared with the unconjugated TLR7 agonist, the UC-1V150/MSA was a 10- to 100-fold more potent inducer of cytokine productionin vitro by mouse bone marrow-derived macrophage and human peripheral blood mononuclear cells. When administrated to the lung, the conjugate induced a prolonged local release of cytokines at levels 10-fold or more higher than those found in serum. Under the same conditions, the untethered TLR7 ligand induced quick systemic cytokine release with resultant toxicity. In addition, two pulmonary infectious disease models were investigated wherein mice were pretreated with the conjugate and then challenged with either Bacillus anthracis spores or H1N1 influenza A virus. Significant delay in mortality was observed in both disease models with UC-1V150/MSA-pretreated mice, indicating the potential usefulness of the conjugate as a localized and targeted immunotherapeutic agent.

The Toll-like receptors (TLRs) are a set of conserved cellular receptors that play an important role in the recognition of microbial pathogens and in initiating the host innate immune response. These receptors recognize distinct molecular components of invading pathogens, such as cell wall structures and nucleic acids. The discovery that endogenous ligands as well as synthetic small molecules can activate certain TLR pathways has generated tremendous interest in the development of new therapeutics for diseases related to the immune response. TLR ligands control the activation of antigen-presenting cells, in particular dendritic cells, by triggering their maturation program, including up-regulation of the expression of HLA and costimulatory molecules and secretion of proinflammatory cytokines, such as TNF-α, IL-6, IL-12, and IFN-α (1). Recently, we reported that certain derivatives of guanine (2) and adenine can activate immune cells via TLR7 and can inhibit the replication of hepatitis C virus replicons in hepatocytes (3). However, the in vitro immunotherapeutic activities of TLR7 ligands have been difficult to translate in vivo, because systemic TLR activation can induce a rapid and potentially toxic cytokine syndrome (4, 5). Accordingly the major in vivo applications of TLR7 ligands have been as topically applied antiviral or antitumor agents or as immune adjuvants injected intramuscularly in small quantities (6, 7). Previously, we found that small molecule agonists of TLR7 enhanced the ability of macrophages to control Bacillus anthracis in vitro (8). However, to develop a safe and effective immunotherapeutic agent for pulmonary infection, we needed a TLR7 agonist that would activate cytokine production in the lung but without causing systemic cytokine release. One potential effective strategy involved the stable conjugation of the agent to a macromolecule, such as a protein or a polymer (9), that would restrict systemic absorption; promote local drug uptake into endosomes, where TLR7 resides (10); and perhaps deliver the immunotherapeutic to specific cells or organs (11, 12). In this regard, a recent report showed that conjugation of HIV Gag protein to a low-molecular-weight TLR7/8 agonist yielded a vaccine that elicited broad-based adaptive immunity in nonhuman primates (13). In the present study, we examined the effects of conjugation of a nonimmunogenic carrier protein to a modified adenine-based TLR7 agonist on the magnitude and duration of innate immune activation in vitro and in vivo. Because a mouse model was selected for study in vivo, a covalent conjugate of a TLR7 agonist and mouse serum albumin (MSA) connected through a versatile bifunctional linker molecule was prepared and characterized. The conjugate was 10- to 100-fold more potent than the free drug. When administrated to the lung, it induced profound local cytokine release, with minimal systemic dispersal. Mice that were pretreated by intrapulmonary delivery of the conjugate and then challenged with either B. anthracis spores or H1N1 influenza A virus had a delayed and attenuated course of infection.

Results

Synthesis and Conjugation of UC-1V150 to MSA.

UC-1V150 (8) was synthesized in our laboratory in seven steps from 2,6-dichloropurine (Fig. 1). The free aldehyde group on the benzyl moiety of UC-1V150 enabled us to couple the agonist to many different auxiliary chemical entities, including proteins, oligonucleotides, aromatic molecules, lipids, viruses, and cells, through a linker molecule that contained a hydrazine or amino group. UC-1V150 was covalently coupled to MSA first modified with a succinimidyl 6-hydrazino-nicotinamide acetone hydrazone (SANH) linker to yield a stable molecule with a characteristically altered UV spectrum (Fig. 2). The UC-1V150/MSA conjugate was identified by a UV absorption peak at 342 nm due to hydrazone formation, whereas SANH alone absorbed at 322 nm. Quantification of UC-1V150 molecules conjugated per MSA was extrapolated from a standard curve of UC-1V150-SANH (Fig. 3). We consistently obtained the UC-1V150/MSA conjugates at a ratio of ≈5:1. The biological studies reported here were done by using 5:1 UC-1V150/MSA.

Fig. 1.

Synthesis of UC-1V150. Reagents and conditions were as follows: α-bromo-p-tolunitrile, K₂CO₃/dimethylformamide, 25°C (step a); NH₃-MeOH, 60°C (step b); CH₃OCH₂CH₂CH₂OH, 100°C (step c); Br₂/CH₂Cl₂, 25°C (step d); CH₃ONa/CH₃OH, reflux (step e); lithium N,N′-diemthylenediamino aluminum hydride/THF, 0°C (step f); and HCl, 25°C (step g).

Fig. 2.

Conjugation of UC-1V150 to MSA. Covalent linkage is formed via a linker, SANH, between the UC-1V150 and MSA containing an amino group.

Fig. 3.

Quantification of UC-1V150 molecules conjugated to MSA. (A) Different concentrations of UC-1V150-SANH were subjected to spectrophotometric analysis. (B) The readout of UC-1V150-SANH was used to derive a standard curve. (C) Conjugation of UC-1V150 to MSA was indicated by a UV absorption peak at 342 nm due to hydrazone formation. The concentration of UC-1V150/MSA used in this analysis at 0.25 μg was based on MSA concentration, which is equivalent to 3.63 μM MSA. The concentration of UC-1V150 in conjugate was calculated as (0.6901 − 0.0056)/0.0366 = 18.7 μM; thus, the UC-1V150:MSA ratio is 18.7/3.63 = 5.2.

Potentin vitro and in Vivo Cytokine Release in Response to UC-1V150/MSA Conjugates.

Incubation of bone-marrow-derived macrophages (BMDM) with UC-1V150 alone stimulated cytokine release (Fig. 4). When conjugated to MSA, similar or higher levels of cytokines were detected with a 10-fold lower equivalent concentration of the pharmacophore (Fig. 4). Experiments with TLR transformants, performed as described previously, confirmed that UC-1V150, similar to the compound lacking the aldehyde modification (UC-1V136), was a specific TLR7 agonist (3). After i.v. injection into mice, UC-1V150 induced serum cytokine levels that peaked at ≈2 h after injection and then quickly declined to near background levels (data not shown). Comparison of the cytokine production profiles of UC-1V150 versus the UC-1V150/MSA 2 h after i.v. injection at various dosages demonstrated that the MSA conjugate enhanced the potency by 10- to 100-fold (Fig. 5). Sera from saline or MSA control groups revealed little or no detectable cytokine levels (data not shown).

Fig. 4.

In vitro cytokine release in response to UC-1V150/MSA conjugates. Murine BMDM were treated with UC-1V150 or UC-1V50/MSA conjugates at various concentrations as indicated. Culture supernatants were harvested 24 h later, and cytokine levels were measured by immunoassay. The results are a representative of at least two separate experiments in triplicate per treatment.

Fig. 5.

In vivo efficacy of UC-1V150/MSA conjugates. C57BL/6 mice were administered various amounts of UC-1V150 or UC-1V150/MSA via the tail vein as indicated. Sera were collected 2 h later, and cytokine levels were determined by multiplex immunoassay. Each group had four mice. The error bars indicate the SEM.

UC-1V150/MSA Conjugates Provide Prolonged and Localized Pulmonary Activity.

To ensure adequate delivery of the TLR7 agonists to the respiratory system, we initially delivered the drugs directly into the trachea. Substantial cytokine induction was found in bronchial alveolar lavage fluid (BALF) extracted from mice treated intratracheally (i.t.) with UC-1V150/MSA (Fig. 6Left), whereas serum cytokines were very low and near background levels in the same animals (Fig. 6 Right). In marked contrast, similar levels of cytokine were observed in both BALF and sera of mice injected i.t. with small-molecule TLR7 agonists, which sometimes induced behavioral changes, such as hair standing on end and shivering, suggestive of a cytokine syndrome (Table 1). Subsequent studies with UC-1V150 showed that intranasal (i.n.) delivery also induced selective cytokine production in the BALF, probably due to drug aspiration. Accordingly, i.n. administration was used to evaluate the UC-1V150 conjugates in two infectious animal models of pneumonitis. Mice pretreated i.n. with UC-1V150/MSA one day before infection with B. anthracis spores had an extended mean survival of 7.5 days compared with 5 days in control mice (P < 0.025) (Fig. 7A). In contrast, no significant difference was observed in mice treated with either saline, the equivalent amount of MSA, or with UC-1V150 alone. These data confirmed that the UC-1V150 conjugate, but not the free drug, had intrapulmonary immunotherapeutic activity. In another study, BALB/c mice were pretreated i.n. with the UC-1V150/MSA conjugate 1 day before influenza virus infection (H1N1 strain). The mean survival of the treated mice was extended to 11.5 days compared with 7 days in untreated controls (P < 0.0001) (Fig. 7B). Together these results suggest that conjugation of the TLR7 agonist to MSA enhanced its potency and reduced its toxicity after local delivery to the respiratory tract.

Fig. 6.

Sustained in vivo local activity of UC-1V150/MSA conjugates without systemic effect. C57BL/6 mice were anesthetized and administered i.t. with 3 nmol of UC-1V150/MSA. At the indicated time points, mice were killed and both BALF and sera were collected for multiplex immunoassay of cytokine levels. The data were combined from two separate experiments with at least six mice per group. The results show the mean values ± SEM.

Table 1.

Local cytokine profile in mice treated with conjugated vs. unconjugated TLR7 agonists

Cytokine	Time, h	Conjugated			Unconjugated
		BALF, ng/ml (SEM)	Serum, ng/ml (SEM)	Ratio	BALF, ng/ml (SEM)	Serum, ng/ml (SEM)	Ratio
IL-6	2	0.25 (0.09)	0.28 (0.00)	0.9	0.73 (0.40)	5.00 (1.14)	0.1
	4	1.34 (0.38)	0.06 (0.02)	22.1	1.09 (0.64)	2.93 (1.53)	0.4
	6	3.50 (1.07)	0.14 (0.05)	24.3	3.00 (0.76)	3.95 (1.24)	0.8
	24	4.06 (0.41)	0.03 (0.00)	117.2	4.39 (0.84)	1.08 (0.31)	4.1
IL-12p40	2	0.06 (0.00)	0.23 (0.03)	0.2	0.11 (0.09)	0.53 (0.53)	0.2
	4	0.11 (0.00)	0.50 (0.39)	0.2	0.06 (0.03)	0.23 (0.23)	0.3
	6	0.29 (0.04)	0.25 (0.06)	1.1	0.00 (0.00)	2.82 (0.46)	0.0
	24	5.05 (3.70)	0.11 (0.00)	45.1	0.01 (0.01)	0.49 (0.47)	0.0
TNF-α	2	1.25 (1.02)	0.08 (0.01)	16.4	2.62 (0.83)	0.52 (0.21)	5.0
	4	9.67 (1.01)	0.02 (0.01)	580.1	1.53 (0.73)	0.32 (0.16)	4.8
	6	8.28 (1.72)	0.02 (0.00)	427.5	2.03 (0.67)	0.39 (0.15)	5.2
	24	4.22 (2.80)	0.01 (0.00)	551.8	0.93 (0.47)	0.15 (0.04)	6.2

C57BL/6 mice were administered i.t. UC-1V150/MSA conjugate or unconjugated UC-1V136 at 3 nmole or 500 nmole per mouse, respectively. BALF and sera were collected at the indicated time points, and cytokine levels were determined by multiplex immunoassay. Mean values from at least three to five mice per group are shown ± SEM.

Fig. 7.

Preclinical efficacy of UC-1V150/MSA in pulmonary infectious diseases. (A) Age-matched female A/J mice were administered i.n. saline only or saline containing MSA (amount equivalent to UC-1V150/MSA), UC-1V150, or UC-1V150/MSA at 0.75 nmol per mouse 1 day before B. anthracis infection, and survival was followed for 13 days. (B) BALB/c mice were administered i.n. saline or UC-1V-150/MSA at 5 nmol per mouse 1 day before influenza infection. Survival followed up to 21 days. In each model, Kaplan–Meier survival curves and log-rank tests were performed to determine significance. At least eight mice were tested in each group.

Discussion

The compound UC-1V150 is one of the most potent and versatile synthetic small-molecule TLR7 ligands yet discovered because (i) it is active at nanomolar concentrations; (ii) it can be coupled to a variety of macromolecules with enhancement of activity in some cases; and (iii) its pharmacokinetic properties can be changed by modification of the auxiliary groups. The TLR7-protein conjugate UC-1V150/MSA was characterized as having approximately five small molecules covalently linked to each MSA protein molecule. The conjugate retained TLR7 agonist activity and indeed was both more potent and had a longer duration of action, compared with the free monomeric drug. Moreover, we showed that this conjugate could be delivered effectively to the respiratory system by i.n. or i.t. administration. Drug delivery by i.n. proved to be effective in two mouse models of infectious disease, a bacterial infection and a viral infection. When considering delivery to the respiratory system, a potentially important advantage of preparing the TLR7 agonists as conjugates of macromolecules is that systemic side effects may be avoided by confining the immunostimulatory activity to the local mucosal environment.

The macromolecular conjugate would be expected to be absorbed into the systemic circulation more slowly than the free drug and, indeed, may be avidly scavenged by resident macrophages and dendritic cells expressing TLR7. Accordingly, the conjugate should mitigate the type of severe side effects that have been associated with systemic delivery of TLR7/8 agonists.^‖ The UC-1V150/MSA conjugate may also provide beneficial immunotherapeutic activity when administered to mucosal sites, such as the genitourinary and gastrointestinal tracts, for the control of infectious, allergic, or malignant diseases. The macromolecular carrier of the TLR7 agonist may also provide an improved approach for selective delivery of the immunotherapeutic to a specific organ or tissue. For example, the lipid conjugates of UC-1V150 can be incorporated into liposomes of different size and composition, whereas protein conjugates of the TLR7 agonist may target different dendritic cell subsets. Differences in the intracellular trafficking of the UC-1V150 conjugate may induce distinct patterns of cytokine production, analogous to the effects observed with TLR9-activating oligonucleotides (14).

One potential problem that has been observed with drugs conjugated to proteins is the development of antibodies against the low-molecular-weight hapten-like portion of the molecule. However, UC-1V150, unlike the TLR7/8 vaccine conjugates studied earlier, has a simple adenine-like structure that is unlikely to induce hypersensitivity reactions. Indeed, we have not observed anti-UC-1V150 antibodies after administration of the protein conjugates, except after repeated administration of a keyhole limpet hemocyanin carrier in complete Freud's adjuvant (unpublished data).

New agents for the prevention and treatment of influenza virus infections are being sought, particularly with the spread of highly pathogenic strains from Asia. Morbidity and mortality from commonly circulating strains is high each year. Treatment of the infection can be accomplished by approved antiviral drugs, which are moderately effective if started early. Enhancement of the immune system is also being investigated as a strategy that could accelerate protective antiviral responses, especially in immune compromised hosts. Along these lines, the immunomodulatory agent UC-1V150/MSA conjugate was tested against an influenza A (H1N1) virus infection in mice. It has been our experience that treatment of influenza virus and bacterial infections with immune modulators is often not effective. It is possible that systemic immune activation via TLR signaling does not create a local cytokine and chemokine gradient required to mobilize immune cells to the site of infection. In support of this hypothesis, the unconjugated UC-1V150, which is rapidly absorbed through the mucosa, failed to protect mice from B. anthracis infection, whereas the UC-1V150 conjugate was effective.

B. anthracis has become an agent of bioterrorism. A rapid response against microbial pathogens is critical for effective biodefense. In general an antibody or cellular immune response may protect against these pathogens; however, generating these protective responses quickly requires prior exposure to specific antigens for each organism. Although it is known that influenza virus engages TLR7 (15), bacterial anthrax most likely can engage TLR2, TLR4, and TLR9. In addition to being a common signaling intermediary for the TLRs, MyD88 has also been shown to be necessary for resistance to infection in a mouse model of anthrax (16). Because the UC-1V150 conjugate works effectively as an adjuvant against infections that use different pathways, it can be applied as a biodefense strategy that would not need be specific to the antigens of a particular microbe and that would be useful in mixed as well as single-agent attacks.

Materials and Methods

Chemistry of UC-1V150.

The synthesis of UC-1V150 is depicted in Fig. 1, and the preparation of the indicated compounds 2–8 was as follows.

Compound 2: 4-(2,6-dichloropurin-9-ylmethyl)benzonitrile.

Sixteen millimoles of 2,6-dichloro-9H-purine (compound 1) was dissolved in 50 ml of dimethylformamide with potassium 50 mmol of carbonate added, and the mixture was stirred at ambient temperature for 16 h after adding 22 mmol of α-bromo-p-tolunitrile. After filtration to remove insoluble inorganic salts, the filtrate was poured into 1,500 ml of water and extracted with ethyl acetate (2 × 400 ml), dried over magnesium sulfate, and evaporated to yield a residue that was subjected to flash silica gel chromatography using 1:2:10 ethyl acetate/acetone/hexanes. [Yield, 3.33 g (69%).] UV, NMR, and MS were consistent with structure assignment.

Compound 3: 4-(6-amino-2-chloropurin-9-ylmethylbenzonitrile.

Compound 2 (1.9 g) was placed in a steel reaction vessel, and 80 ml of 7 M methanolic ammonia was added. The sealed vessel was heated at 60°C for 12 h and cooled in ice, and the solid product was filtered off. (Yield, 1.09 g.) UV, NMR, and MS were consistent with assigned structure.

Compound 4: 4-[6-amino-2-(2-methoxyethoxy)purin-9-ylmethyl]benzonitrile.

The sodium salt of 2-methoxyethanol was first generated by dissolving 81 mg of sodium metal in 30 ml of 2-methoxyethanol with heat, and then 1.0 g of compound 3 dissolved in 300 ml of methoxyethanol was added with heat. The reaction mixture was heated for 8 h at 115°C bath temperature and concentrated in vacuo to near dryness; the residue was then partitioned between ethyl acetate and water. Flash silica gel chromatography of the organic layer by using 5% methanol in dichloromethane gave 763 mg of product. NMR was consistent with structure assignment.

Compound 5: 4-[6-amino-8-bromo-2-(2-methoxyethoxy)purin-9-ylmethyl]benzonitrile.

Compound 4 (700 mg) was dissolved in dichloromethane (400 ml) and bromine (7 ml) was added dropwise. The mixture was stirred overnight at room temperature and extracted first with 2 liters of a 0.1 M aqueous sodium thiosulfate solution, then with 500 ml of aqueous sodium bicarbonate (saturated). The residue from the organic layer was chromatographed on silica gel by using 3% methanol in dichloromethane to yield 460 mg of bromo product. NMR, UV, and MS were consistent with structure assignment.

Compound 6: 4-[6-amino-8-methoxy-2-(2-methoxyethoxy)purin-9-ylmethyl]benzonitrile.

Sodium methoxide was generated by reaction of 81 mg of sodium metal in 30 ml of dry methanol and combined with a 700-mg solution of compound 5 dissolved in dry dimethoxyethane, and the temperature raised to 100°C. After overnight reaction, the mixture was concentrated in vacuo, and the residue was chromatographed on silica by using 5% methanol in dichloromethane. (Yield, 120 mg.) NMR was consistent with structure assignment.

Compound 7: 4-[6-amino-8-methoxy-2-(2-methoxyethoxy)-purin-9-ylmethyl]benzaldehyde.

Compound 6 (100 mg) was dissolved in 3 ml of dry THF and cooled to 0°C under argon. The reducing agent, lithium N,N′-(dimethylethylenediamino)-aluminum hydride, used to convert the nitrile to the aldehyde function was prepared essentially as previously described (17). A 0.5 M solution in dry THF was prepared, and 0.72 ml of it was added to the reaction flask. The mixture was stirred at 0–5°C for 1 h, quenched by addition of 3 M HCl, extracted with ethyl acetate followed by dichloromethane, and then concentrated in vacuo to yield 85 mg. NMR was consistent with structure assignment.

Compound 8: 4-[6-amino-8-hydroxy-2-(2-methoxyethoxy)purin-9-ylmethyl]benzaldehyde (UC-1V150).

Compound 7 (800 mg) was combined with 504 mg of sodium iodide and 40 ml of acetonitrile, and 0.5 ml of chlorotrimethylsilane was slowly added. The mixture was heated at 70°C for 3.5 h, cooled, and filtered. The solid product was washed with water and then ether to yield 406 mg. NMR, UV, and MS were consistent with structure assignment.

Conjugation of UC-1V150 to MSA.

The benzaldehyde group on UC-1V150 enabled facile coupling to a variety of auxiliary molecules with free amino or hydrazine groups by using well established conjugation chemistry. To prepare a conjugate that could be administrated to mice and conveniently monitored spectrophotometrically, we chose SANH as a linker and MSA (Sigma, St. Louis, MO) as the auxiliary molecule (Fig. 2). Initially, the amino groups on MSA derivatized with SANH according to standard procedures (18). To remove excess SANH, the reaction mixture was loaded on a NAP-10 column equilibrated with PBS and eluted with PBS. Then the UC-1V150 was added to the modified proteins at a 40-fold molar excess in dimethylformamide and incubated at 20°C overnight. Unreacted UC-1V150 was removed by size-exclusion gel filtration as described above. The lyophilized conjugate was stable for at least 6 months at −20°C.

Mice.

Female C57BL/6 mice (5–6 wk of age) were obtained from Harlan West Coast (Germantown, CA), and female A/J mice (6–8 wk of age) were purchased from The Jackson Laboratories (Bar Harbor, ME). A/J mice were used for infection with the Sterne strain of B. anthracis (19). The mice were bred and maintained under standard conditions in the University of California at San Diego Animal Facility, which is accredited by the American Association for Accreditation of Laboratory Animal Care. All animal protocols received prior approval by the Institutional Review Board. For the H1N1 influenza study, female BALB/c mice (16–18 g) were obtained from Charles River Laboratories (Wilmington, MA) and maintained in the American Association for Accreditation of Laboratory Animal Care-accredited Laboratory Animal Research Center of Utah State University.

In Vitro Stimulation of BMDM.

BMDM were isolated from various strains of mice and grown as previously described (20) and were seeded in 96-well plates at a density of 5 × 10⁴ cells per well. Compounds were added to 10-day-old cultures at a final concentration ranging from 0.01 to 10 μM or as otherwise indicated. After 24 h of incubation, culture supernatants were collected and assayed for cytokine inductions by either sandwich ELISA (BD Pharmingen, San Diego, CA) (20) or multiplex Luminex (Austin, TX) assay using the Beadlyte Mouse MultiCytokine customized kit (Upstate, Charlottesville, VA, and eBiosciences, San Diego, CA), according to the manufacturer's instructions.

Administration of Compounds to Mice.

Female age-matched C57BL/6 mice were injected with 100 μl of saline solution containing UC-1V150 or UC-1V150/MSA, each containing the equivalent of 0.38–38 nmol of the pharmacore via the tail vein. For intrapulmonary administration, mice were anesthetized with i.p. Avertin solution and shaved around the neck area. The trachea were exposed with a small incision and injected with 50 μl of saline solution containing various amounts of UC-1V150/MSA or the unconjugated drug. After recovery and at different time points, serum and BALF were collected and analyzed for IL-6, IL-12p40, IFN-γ, RANTES, and MCP-1 by Luminex assay. In other experiments, mice were anesthetized with an intramuscular ketamine/xylene solution and administered the same amount of UC-1V150/MSA in i.t. doses of 50 μl or i.n. doses of 20 μl. Because similar cytokine levels were observed in the BALF 24 h after administration by either method, the more convenient i.n. route was used in infectious model studies.

Infection of A/J Mice with B. anthracis Spores.

Spores were prepared from the Sterne strain of B. anthracis (pXO1⁺pXO2⁻) as previously described (8, 21). Purified spores were stored in PBS at 1 × 10⁸ to 4 × 10⁸ cfu/ml at 4°C. Before infection, the spores were heated to 65°C for 30 min to initiate germination. A/J mice were anesthetized intramuscularly with ketamine/xylene solution and administered i.n. with 0.75 nmol of UC-1V150 or UC-1V150/MSA per mouse 1 day before anthrax infection. Control mice received saline only or saline containing MSA at equivalent amounts as in UC-1V150/MSA. Infection was carried out i.n. with 2 × 10⁵ to 8 × 10⁵ spores of B. anthracis in a 20-μl volume. Survival was observed for 13 days, because the majority of the saline-treated mice died within 3–6 days. Results were obtained from eight mice per group.

Infection of BALB/c Mice with Influenza Virus.

Influenza A/New Caledonia/20/99 (H1N1) virus was obtained from the Centers for Disease Control and Prevention (Atlanta, GA). The virus was propagated twice in Madin Darby canine kidney cells, further passaged seven times in mice to make it virulent, followed by another passage in cell culture to amplify it. Mice were anesthetized i.p. with 100 mg/kg ketamine and infected i.n. with virus at ≈10^5.0 cell culture infectious doses per mouse in a 50-μl inoculum volume. A single 75-μl i.n. dose of either saline alone or saline containing UC-1V150/MSA to 5 nmol per mouse was given 24 h before virus exposure. Ten infected mice per treated group and 20 placebo control animals were followed for survival for 21 days.

Statistics.

Cytokine levels were compared by the Mann–Whitney U test with P < 0.05 to determine statistical significance. Kaplan–Meier survival curves and log-rank tests were performed by using Prism software version 4.0c (GraphPad, San Diego, CA) to compare differences in survival.

Abbreviations

TLR

Toll-like receptor

MSA

mouse serum albumin

BMDM

bone-marrow-derived macrophages

BALF

bronchial alveolar lavage fluid

i.t.

intratracheal(ly)

i.n.

intranasal(ly)

SANH

succinimidyl 6-hydrazino-nicotinamide acetone hydrazone.