lipid-a and Vaccinia

Infectious intracellular and extracellular forms of vaccinia virus have different outer membrane proteins, presenting multiple targets to the immune system. We investigated the immunogenicity of soluble forms of L1, an outer membrane protein of the intracellular mature virus, and of A33 and B5, outer membrane proteins of the extracellular enveloped virus. The recombinant proteins, in 10-microg amounts mixed with a Ribi- or saponin-type adjuvant, were administered subcutaneously to mice. Antibody titers to each protein rose sharply after the first and second boosts, reaching levels that surpassed those induced by percutaneous immunization with live vaccinia virus. Immunoglobulin G1 (IgG1) antibody predominated after the protein immunizations, indicative of a T-helper cell type 2 response, whereas live vaccinia virus induced mainly IgG2a, indicative of a T-helper cell type 1 response. Mice immunized with any one of the recombinant proteins survived an intranasal challenge with 5 times the 50% lethal dose of the pathogenic WR strain of vaccinia virus. Measurements of weight loss indicated that the A33 immunization most effectively prevented disease. The superiority of protein combinations was demonstrated when the challenge virus dose was increased 20-fold. The best protection was obtained with a vaccine made by combining recombinant proteins of the outer membranes of intracellular and extracellular virus. Indeed, mice immunized with A33 plus B5 plus L1 or with A33 plus L1 were better protected than mice immunized with live vaccinia virus. Three immunizations with the three-protein combination were necessary and sufficient for complete protection. These studies suggest the feasibility of a multiprotein smallpox vaccine.

Topics: Animals; Antibodies, Viral; Cell Wall Skeleton; Cord Factors; Female; Immunoglobulin G; Lipid A; Membrane Glycoproteins; Mice; Neutralization Tests; Recombinant Proteins; Vaccination; Vaccines, Combined; Vaccines, Subunit; Vaccines, Synthetic; Vaccinia; Vaccinia virus; Viral Envelope Proteins; Viral Matrix Proteins; Viral Vaccines; Weight Loss

A chemically synthesized lipid A-subunit analogue, GLA-60, 2-deoxy-4-O-phosphono-2-[(3R)-3-hydroxytetradecanamido]-3-O-[(3R)- 3- tetradecanoyloxytetradecanoyl]-D-glucose, has many of the activities of endotoxin but has little toxicity. Then, compounds with various lengths of acyl side chain of the acyloxyacyl group at the 3-O position of GLA-60 were synthesized and evaluated for interferon (IFN)-inducing activity, natural killer (NK) cell activation and antiviral activity. The compounds with acyl side chains between C8 and C15 exhibited significant antiviral activity (inhibition of pox tail lesion formation in vaccinia virus-infected mice), serum IFN-inducing activity and NK cell activation. However, the compound carrying a C2 or a C16 acyl side chain did not exhibit these activities. The compounds with a C13 or C14 acyl side chain showed strong protective against herpes simplex virus type 1 in cyclophosphamide-immunosuppressed mice.

Topics: Acylation; Animals; Antiviral Agents; Female; Herpes Simplex; Interferons; Killer Cells, Natural; Lipid A; Mice; Simplexvirus; Vaccinia; Vaccinia virus

The C1 position of lipid A-subunit analogue GLA-27, 4-O-phosphono-D-glucosamine carrying N-3-tetradecanoyloxytetradecanoyl(C14-O-(C14)) and 3-O-tetradecanoyl (C14) groups, was S-acetylated, thiolated or phosphorylated. Enhancement of nonspecific resistance to Pseudomonas aeruginosa and vaccinia virus infections of these chemically modified compounds were investigated. Thiolation augmented the nonspecific resistance to P. aeruginosa infection. Protective activity against vaccinia virus infection was reduced by all the chemical modifications. NK cell activity was found not to be effected by S-acetylation, but to be decreased slightly by thiolation or phosphorylation. IFN-inducing activity was reduced remarkably by thiolation or S-acetylation, or completely diminished by phosphorylation, compared with that of GLA-27.

Topics: Animals; Female; Immunity, Innate; Interferons; Killer Cells, Natural; Lipid A; Mice; Mice, Inbred ICR; Pseudomonas Infections; Structure-Activity Relationship; Vaccinia

Non-specific protective activities against vaccinia virus (VV) and Pseudomonas aeruginosa infections as well as interferon (IFN)-inducing, natural killer (NK) cell and macrophage activation activities of chemically synthesized lipid A-subunit analogs were investigated. The analogs are 4-O-phosphono-D-glucosamine derivatives carrying different 2-N- and 3-O-linked acyl substituents such as (R)-3-tetradecanoyloxytetradecanoyl (C14-O-(C14)), (R)-3-hydroxytetradecanoyl (C14-OH) and tetradecanoyl (C14) groups. Compounds GLA-59 and GLA-60, which possess C14-OH and C14-O-(C14) groups as their acyl substituents, showed stronger IFN-inducing and anti-vaccinia virus activities than GLA-27 and GLA-68, which possess a C14 group instead of the C14-OH group in GLA-59 and GLA-60, although NK cell activation activity was similarly high in all of these compounds. In protective activity against P. aeruginosa infection and macrophage activation activity, GLA-60 and GLA-68, which carry a C14-O-(C14) group at the 3-O-position, expressed higher activities than GLA-27 and GLA-59, which carry the acyloxyacyl group at the 2-N-position. These results indicate that the acyl substituent (whether the counterpart of the C14-O-(C14) group is a C14 or a C14-OH group) and the binding position of the acyloxyacyl group at the 2-N- or the 3-O-position strongly influence the manifestation of antimicrobial and immunomodulating activities in different ways depending on the activity. Among the compounds, GLA-60 satisfied the structure requirements for protection against both VV and P. aeruginosa infections. This compound is a hopeful immunomodulator for prevention against broad microbial infections.

Topics: Adjuvants, Immunologic; Animals; Drug Resistance; Glucosamine; Lipid A; Mice; Mice, Inbred ICR; Pseudomonas Infections; Structure-Activity Relationship; Vaccinia

Protection against vaccinia virus infection and induction of interferon (IFN) were investigated in Propionibacterium acnes-primed mice following treatment with chemically synthesized lipid A-subunit derivatives. The antiviral activity was based on the reduction of numbers of tail lesions in mice injected intravenously with the test compounds 1 day before virus infection. GLA-27, a 4-O-phosphono-D-glucosamine carrying 3-O-tetradecanoyl (C14) and N-3-tetradecanoyloxytetradecanoyl [C14-O-(C14)] groups, offered significant antiviral activity. Chemical modifications at the C1 position of GLA-27, e.g. phosphorylation, replacement of OH by an SH, did not cause a significant change in antiviral activity. GLA-57 carrying an N-3-dodecanoyloxytetradecanoyl group showed stronger activity than GLA-27, but GLA-58 carrying an N-3-hexadecanoyloxytetradecanoyl group did not exhibit significant activity. GLA-59 carrying 3-O-3-hydroxytetradecanoyl and N-C14-O-(C14) groups was more active than GLA-27 and GLA-57. GLA-60 possessing the same fatty acid substituents as GLA-59 but in the reversed order was the most active of all compounds tested. This suggests that the nature and position of the acyl substituents are important for achieving the antiviral effects. The (R) isomers of GLA-59 and GLA-60 possessed stronger IFN-inducing activity than the (S) isomers, but no significant difference in antiviral activity was seen between the isomers.

Topics: Animals; Cell Line; Female; HeLa Cells; Humans; Interferons; Lipid A; Mice; Mice, Inbred ICR; Molecular Structure; Vaccinia; Vaccinia virus; Viral Plaque Assay

Activities for enhancing nonspecific host resistance against microbial infections and growth inhibition against tumors by a lipid A-subunit analogue GLA-27 were investigated. GLA-27, a 4-O-phosphono-D-glucosamine derivative with 2-N-3-tetradecanoyloxytetradecanoyl and 3-O-tetradecanoyl group, inhibited significantly the growth of solid-type tumors RL male 1 lymphoma and Meth A fibrosarcoma. In the case of RL male 1 lymphoma, 2.5 micrograms of GLA-27 was needed to exhibit the activity, while for Meth A fibrosarcoma, 250 micrograms of GLA-27 was needed. On the other hand, tail lesions induced by intravenous inoculation with vaccinia virus were effectively suppressed in mice injected with 1 microgram of GLA-27 1 day before the viral injection. The antiviral activity of GLA-27 was 100 times higher than that of MDP by ED50. GLA-27 also showed nonspecific protective activity against Pseudomonas aeruginosa infection at a dose of 30 micrograms/mouse, although the activity was far less than that induced by lipid A. GLA-27 did not exhibit toxic activities such as pyrogenicity and Shwartzman reaction.

Topics: Adjuvants, Immunologic; Animals; Anti-Bacterial Agents; Antineoplastic Agents; Antiviral Agents; Drug Evaluation, Preclinical; Lipid A; Male; Mice; Mice, Inbred Strains; Neoplasms, Experimental; Pseudomonas Infections; Vaccinia