Synergistic adjuvant activity of immunostimulatory DNA and oil/water emulsions for immunization with HIV p55 gag antigen.

A synthetic oligonucleotide containing a previously identified adjuvant active CpG DNA sequence was evaluated for its ability to augment antibody and CTL responses to p55 gag from HIV-1 in mice. Surprisingly, the CpG oligonucleotide, although, it had previously been described as the most potent adjuvant sequence in mice for the particulate HbsAg, was ineffective when used in a simple combination with urea-solubilized p55 antigen. However, a potent adjuvant effect was observed with the CpG sequence when it was formulated with emulsions. Enhancement of antibody titer by CpG emulsion formulations was observed with urea-solubilized p55 antigen, however, significantly higher titers were obtained with p55 bound to polylactide-co-glycolide microparticles. In both cases IgG2a was enhanced in the presence of CpG. It appears likely that presentation of CpG with emulsions and particulate antigens enhances their delivery into antigen presenting cells (APC) and results in more effective presentation of antigen and adjuvant. To support this hypothesis, preliminary in vitro studies were undertaken to show upregulation of CD86 on mouse bone marrow-derived dendritic cells (BMDC) in vitro, following incubation with CpG formulations.

Induction of potent immune responses by cationic microparticles with adsorbed human immunodeficiency virus DNA vaccines.

The effectiveness of cationic microparticles with adsorbed DNA at inducing immune responses was investigated in mice, guinea pigs, and rhesus macaques. Plasmid DNA vaccines encoding human immunodeficiency virus (HIV) Gag and Env adsorbed onto the surface of cationic poly(lactide-coglycolide) (PLG) microparticles were shown to be substantially more potent than corresponding naked DNA vaccines. In mice immunized with HIV gag DNA, adsorption onto PLG increased CD8(+) T-cell and antibody responses by approximately 100- and approximately 1,000-fold, respectively. In guinea pigs immunized with HIV env DNA adsorbed onto PLG, antibody responses showed a more rapid onset and achieved markedly higher enzyme-linked immunosorbent assay and neutralizing titers than in animals immunized with naked DNA. Further enhancement of antibody responses was observed in animals vaccinated with PLG/DNA microparticles formulated with aluminum phosphate. The magnitude of anti-Env antibody responses induced by PLG/DNA particles was equivalent to that induced by recombinant gp120 protein formulated with a strong adjuvant, MF-59. In guinea pigs immunized with a combination vaccine containing HIV env and HIV gag DNA plasmids on PLG microparticles, substantially superior antibody responses were induced against both components, as measured by onset, duration, and titer. Furthermore, PLG formulation overcame an apparent hyporesponsiveness of the env DNA component in the combination vaccine. Finally, preliminary data in rhesus macaques demonstrated a substantial enhancement of immune responses afforded by PLG/DNA. Therefore, formulation of DNA vaccines by adsorption onto PLG microparticles is a powerful means of increasing vaccine potency.

Potency of a genetically detoxified mucosal adjuvant derived from the heat-labile enterotoxin of Escherichia coli (LTK63) is not adversely affected by the presence of preexisting immunity to the adjuvant.

The objective of the current studies was to evaluate whether the potency of a genetically detoxified mucosal adjuvant, derived from heat-labile enterotoxin of Escherichia coli (LTK63), was adversely affected by preexisting immunity. Studies of mice and pigs have involved consecutive intranasal immunization with LTK63 and 2 different vaccines (influenza virus hemagglutinin and a protein-polysaccharide conjugate of Neisseria meningitidis group C). The antibody responses to the vaccines plus LTK63 in naive animals were compared with the responses achieved in animals that previously had been immunized with the alternate vaccine plus LTK63. The data showed that the responses of both animal models to intranasal immunization were not adversely affected by the presence of preexisting immunity to the LTK63 adjuvant.

Genetically detoxified mutants of heat-labile enterotoxin from Escherichia coli are effective adjuvants for induction of cytotoxic T-cell responses against HIV-1 gag-p55.

There is an urgent need for prophylactic and therapeutic vaccines against human immunodeficiency virus (HIV). Mucosal immunization strategies have great potential to elicit both mucosal and systemic cellular immunity required to protect against HIV-induced acquired immune deficiency syndrome (AIDS). However, mucosal immunizations with soluble protein antigens generally require adjuvants. In this study, we tested two mutants of the heat-labile enterotoxin (LT) from Escherichia coli, LTK63: with no measurable ADP-ribosyltransferase activity, and LTR72: with residual ADP-ribosyltransferase activity, as mucosal adjuvants for induction of cytotoxic T lymphocyte (CTL) responses to coadministered HIV gag p55 protein. We found that intranasal (i.n.) immunizations with HIV gag p55 protein coadministered with LTK63 or LTR72 induced systemic CTL responses comparable to that obtained following intramuscular (i. m.) immunizations with the same adjuvants. Moreover, oral coadministration of LTR72, but not LTK63, resulted in local as well as systemic p55-specific CTL responses in mesenteric lymph nodes (MLN) and spleens (SP) of the immunized mice. These data have important implications for current efforts to develop a safe vaccine against HIV.

Microparticles in MF59, a potent adjuvant combination for a recombinant protein vaccine against HIV-1.

Novel adjuvant formulations involving PLG microparticles with entrapped recombinant protein antigens (env gp120 and p24 gag) from human immunodeficiency virus type-1 (HIV-1), dispersed in the emulsion adjuvant MF59 were evaluated as potential HIV-1 vaccine candidates in mice and baboons. In mice, the adjuvant combination induced significantly enhanced antibody responses in comparison to either adjuvant used alone. In addition, the polylactide co-glycolide polymer (PLG) microparticles and MF59 combination induced CTL activity against HIV-1 p24 gag. In baboons, the adjuvant combination induced significantly enhanced antibody titers after a single dose of gp120, but the responses were comparable to gp120 in MF59 alone after boosting. Both MF59+gp120 alone and PLG/gp120 in MF59 induced neutralizing antibodies against a T cell line-adapted (TCLA) strain and a primary isolate of HIV-1. In contrast to the observations with gp120, immunization in baboons with PLG/p24 in MF59 induced significantly enhanced antibody responses after boosting, in comparison to immunization with MF59 alone + p24.

The comparison of the effect of LTR72 and MF59 adjuvants on mouse humoral response to intranasal immunisation with human papillomavirus type 6b (HPV-6b) virus-like particles.

Infections with genital human papillomaviruses (HPV) are likely to be neutralised more efficiently if a mucosal immune response can be elicited at the viral entry site. Local IgA antibodies are highly induced when antigens are co-administered with mucosal adjuvants, such as cholera toxin (CT) and Escherichia coli heat labile enterotoxin (LT) which, however, are not expected to have wide application because of their pronounced toxicity. We have immunised mice intranasally with HPV-6b virus-like particles (VLPs) and a genetically modified LT-derived molecule with only residual toxicity, LTR72, and compared the humoral responses with those obtained following systemic immunisation with VLPs and the MF59 adjuvant. Titration of anti-HPV antibodies in sera and vaginal secretions established that LTR72 was able to elicit higher serum and mucosal IgA titers, in addition to IgG serum levels, comparable to those obtained by parenteral immunisation. These results confirm the potential of toxin-derived adjuvants and extend their use in combination with HPV antigens.

Intranasal immunization with recombinant gD2 reduces disease severity and mortality following genital challenge with herpes simplex virus type 2 in guinea pigs.

The ability of a genetically detoxified mutant of heat labile enterotoxin (LTK63) to act as a mucosal adjuvant following intranasal immunization with recombinant gD2 has previously been reported in mice [Ugozzoli M, O'Hagan DT, Ott GS. Intranasal immunization of mice with herpes simplex virus type 2 recombinant gD2: the effect of adjuvants on mucosal and serum antibody responses. Immunol 1998;93:563-571.]. In the current studies, these observations were extended to the guinea pig model. Immunized guinea pigs were subsequently challenged intravaginally with HSV-2. Intranasal immunization with gD2 and LTK63 induced a significant reduction in disease severity and a reduction in mortality. However, only intramuscular immunization with a potent adjuvant (MF59) induced protection against the incidence of disease.

A comparison of biodegradable microparticles and MF59 as systemic adjuvants for recombinant gD from HSV-2.

A recombinant form of glycoprotein D from herpes simplex virus type-2 (gD2) was encapsulated into polylactide-co-glycolide (PLG) microparticles using a previously established solvent evaporation technique. The mean size of the microparticles was about 1 micron and high encapsulation efficiency of the antigen was achieved (70-80%). The microparticles were administered intramuscularly to Balb/C mice and the immune responses were compared with those obtained with the oil in water adjuvant MF59. The serum IgG response to gD2 induced by the microparticles was comparable with that induced by MF59. The serum neutralization titres were also comparable for microparticles and the emulsion. However, the microparticles induced a higher IgG2a isotype response and a more potent serum IFN-gamma response than MF59, suggesting a more Th1 type of response. The MF59 induced higher levels of serum IL-4 and IL-5 cytokines, suggesting a more Th2 type of response.

Intranasal immunization of mice with herpes simplex virus type 2 recombinant gD2: the effect of adjuvants on mucosal and serum antibody responses.

Mucosal immunization offers the potential for inducing IgA antibody responses in the vagina, the site of infection for many viruses, including herpes simplex type 2 (HSV-2). To investigate this possibility, mice were immunized intranasally with 10 micrograms glycoprotein D2 (gD2) from HSV combined with a series of adjuvants of proven efficacy; the oil in water emulsion MF59, poly(D,L-lactide-co-glycolide) microparticles (PLG) (encapsulated or co-administered), immune-stimulating complexes (iscoms) (incorporated or co-administered with iscomatrix) and the genetically detoxified enterotoxin from Escherichia coli, LT-K63. Encapsulation of gD2 into PLG microparticles, incorporation of gD2 into iscoms and co-administration of gD2 with LT-K63 induced mucosal IgA antibody responses (nasal wash, saliva and vaginal wash) which were greater than those induced by intramuscular administration of gD2 with MF59. Intranasal immunization with these formulations also induced substantial levels of serum IgG and neutralizing antibodies. These studies demonstrated that intranasal immunization with potent adjuvants is an effective means to induce mucosal antibody responses, even in the lower genital tract.

Dendritic cells internalize vaccine adjuvant after intramuscular injection.

Vaccine adjuvants help antigens elicit rapid, potent, and long-lasting immune responses. The lack of understanding of the immunological mechanism of action of adjuvants has limited the rational development of vaccines for human use. In particular, little is known about how the immune system processes adjuvants. The goal of the present study was to determine the fate of the vaccine adjuvant MF59, labeled with the fluorescent dye Dil, after injection with fluorescein-labeled gD2 antigen from type 2 herpes simplex virus. At 3 h after intramuscular injection into BALB/c mice, most of the MF59 was still in the form of extracellular droplets in the muscle, but a detectable fraction of the MF59 was in cells in the subcapsular sinus of draining inguinal lymph nodes. At 48 h, most of the MF59 at the site of injection was inside cells that were immunoreactive for the dendritic cell markers DEC-205 and MHC class II molecules, reflecting the interaction of MF59 with antigen presenting cells. At this time, intracellular MF59 was also abundant in the paracortical (T cell) region of lymph nodes. The gD2 antigen was also intracellular in muscle and colocalized MF59 at 48 h, and the presence of MF59 increased the amount of intracellular antigen. Similarly, serological antibody titers to gD2 were 207-fold higher after two injections when MF59 was administered with the antigen. These findings suggest that MF59 interacts with antigen presenting cells at the site of injection and then moves to the draining lymph nodes, where it increases the efficiency of antigen presentation to T cells.

Separation and purification of high molecular weight glycoproteins using agarose gel electrophoresis.

Several components of the extracellular matrix in the molecular weight range of 220 kDa to 150 kDa were purified by preparative electrophoresis on 2.5% Pro-Sieve agarose gels. These high molecular weight glycoproteins, separated under reducing conditions, were recovered in solution by extraction of individual agarose gel slices and analyzed on sodium dodecyl sulfate polyacrylamide gels and Western blots. This simple method permitted the separation and recovery of the laminin B chains (220 kDa and 205 kDa) and entactin (150 kDa) and may prove useful for the purification of other high molecular weight species.