Immunogenicity of soluble and particulate antigens from Leishmania donovani: effect of glucan as an adjuvant.

The protective efficacy of glucan as an adjuvant with killed promastigotes of Leishmania donovani was compared with that of soluble or particulate fractions of the parasite. When these vaccine preparations were injected either intravenously or subcutaneously in CF-1 mice, glucan potentiated resistance against L. donovani infections as reflected by significant reductions in hepatic amastigote counts relative to infected control mice. The leishmanial antigens alone afforded no protection. Serum direct agglutination titers to leishmanial antigens were highest in all groups given the vaccine intravenously, whereas the delayed-type hypersensitivity response to the antigen was positive only in groups immunized subcutaneously with glucan as an adjuvant. Some index of protection and immune response against visceral infection with the parasite was seen in groups vaccinated with glucan and soluble antigens. However, the protection afforded by glucan and particulate antigens of L. donovani more closely paralleled the resistance of mice treated with glucan and unfractionated killed promastigotes. Further antigenic analysis of particulate fractions of L. donovani may optimize effective immunization when used with appropriate adjuvants, e.g., glucan.     

Glucan-enhanced immunogenicity of killed erythrocyte stages of Plasmodium berghei.

Intravenous injections of glucan simultaneously with Formalin-killed erythrocytic stages of Plasmodium berghei elicited a greater degree of resistance in mice against subsequent infection with viable parasites than injections of killed erythrocytic stages alone. In two experiments with P. berghei strain NK 65, 100% of mice immunized with the glucan-dead parasite preparation survived challenge, whereas only 28.6% of mice receiving dead parasites alone survived. In the third experiment, using P. berghei strain NYU-2, the same proportion of mice survived after immunization with glucan and dead parasites as with dead parasites alone (i.e., 10 of 11 in each group), but mice immunized with the glucan-dead parasite preparation experienced parasitemias of significantly less intensity and shorter duration than mice which received only dead parasites before infection. Inoculation of glucan alone or with normal erythrocytes conferred no protection against challenge.     

Protective effect of glucan against visceral leishmaniasis in hamsters.

The effect of pre- or posttreatment with glucan, a reticuloendothelial stimulant, on the course of Leishmania donovani infection was assessed in highly susceptible hamsters. Intravenous administration of glucan before or after L. donovani infection significantly suppressed proliferation of amastigote-stage parasites in liver and spleen. Glucan-activated peritoneal macrophages in vitro also significantly reduced multiplication of the intracellular parasite. Ultrastructural studies revealed a well-defined hepatic granulomatous response to glucan, with hypertrophic Kupffer cells and reduced numbers of intracellular parasites compared to the control group. In additional studies, groups of hamsters were immunized by intravenous injections of glucan with Formalin-killed promastigote-stage L. donovani cells and challenged 60 days after the last immunizing injection. This treatment regimen significantly prolonged the mean survival time of those hamsters which died after infection, relative to untreated control groups. Hamsters stimulated with the glucan-killed promastigote preparation also exhibited significant reductions in splenic amastigotes on days 10 and 21 postinfection compared with all other control groups, but on day 35, splenic amastigotes did not differ significantly from those of control animals. Our composite observations provide evidence for glucan-enhanced nonspecific resistance of hamsters to visceral leishmaniasis.     

Resistance against Leishmania donovani induced with an aluminum hydroxide vaccine

Mice immunized with a subcutaneous protocol combining killed parasites and aluminum hydroxide gel exhibited significant resistance against subsequent challenge with Leishmania donovani promastigotes. Protection was greatest using 25 mg of aluminum hydroxide per injection. Resistance elicited by this killed parasite and aluminum hydroxide protocol was as effective on day 14 as that provided by immunization with a glucan and killed parasite preparation, and more effective in hepatic amastigote reduction at day 28. The effectiveness of aluminum hydroxide as an adjuvant appears to result, at least in part, from its ability to activate macrophages, thus aiding in the elimination of this intracellular parasite.     

Protective effect of L. donovani antigens using glucan as an adjuvant

Golden hamsters were immunized with various antigen fractions of Leishmania donovani promastigotes. Beta 1,3-glucan was used as an adjuvant in these vaccination experiments. The results indicate that immunization of animals with the microsomal fraction (subcellular fraction III) in combination with glucan confers considerable immune protection against L. donovani infection. The immune protection was confirmed by correspondingly lower parasite burden in the livers and spleens of test animals compared to controls. Additionally, the vaccinated animals showed positive skin test responsiveness after challenge, along with increased antibody titres. Immunization of animals with whole and particulate antigen fractions was also found to afford a high degree of resistance. The other subcellular and soluble antigen fractions conferred very little protection. In these experiments, glucan was found to be a potent adjuvant when injected, intraperitoneally, with Leishmania antigens. Similar doses of parasite extracts given without an adjuvant were able to confer only very little or no protection.     

Maternal transmission of immunity to white spot syndrome associated virus (WSSV) in shrimp (Penaeus monodon)

β-1,3-1,6-glucan, derived from bakers’ yeast Saccharomyces cerevisiae, was used in the present study to investigate the extent to which glucan is able to protect spawners from white spot syndrome associated virus (WSSV), and whether this protection (if any) can be passed on to hatchlings via maternal transmission of immunity. Results showed that fewer spawners in the glucan-injected groups showed the clinical symptoms of red body coloration and white spots on the shell during the 15 days between eyestalk ablation and the end of repeated spawning. This suggests that the application of glucan might lead to a slight enhancement of disease resistance in spawners, although the differences were not statistically significant within the confidence limit chosen. Challenge results showed a significant increase in relative percent survival for larvae derived from groups of glucan-injected spawners compared to those derived from groups of untreated spawners. It therefore seems that a maternally transmitted disease resistance induced by glucan, protected the larvae against a WSSV infection. Glucan immersion was not only shown to be effective for nauplii derived from spawners that were not injected with glucan, it also provided additional, cumulative protection for nauplii which already had a maternally transmitted resistance to WSSV. This is the first documented demonstration of a maternal transmission of immunity in invertebrates.     

Effect of glucan on Leishmania major infection in BALB/c mice

The effect of glucan on Leishmania major infection was studied in BALB/c mice, which are highly susceptible to leishmania infection. Glucan (0.45 mg), or isovolumetric dextrose, was administered intraperitoneally 7, 5, 3 and 1 day before infection with L. major promastigotes. At 3, 5, 6, 8 and 10 weeks after infection, animals were killed; the liver and spleen of each animal were weighed and the parasite burden was calculated. A significant (p less than 0.01) reduction in amastigote proliferation in liver and spleen of animals pretreated with glucan was demonstrated 4, 6 and 8 weeks after infection.     

Vaccine-induced immunity against cutaneous leishmaniasis in BALB/c mice.

Partially purified antigens, derived from Leishmania infantum or L. major promastigotes and isolated under reducing conditions, were used to immunize BALB/c mice. Three subcutaneous injections of the 64- to 97-kilodalton preparation in conjunction with muramyl dipeptide conferred long-lasting immunity against L. mexicana subsp. mexicana and L. major infection; they led to the development of antibodies neutralizing the infectiousness of promastigotes, induced specific delayed-hypersensitivity reactions, and generated populations of peritoneal macrophages capable of killing amastigotes. Vaccination resulted in no harmful effects, since these antigen neither exacerbated preexisting Leishmania infection nor impeded the formation of antibodies to other antigens administered concomitantly.     

Protective effect of glucan against systemic Staphylococcus aureus septicemia in normal and leukemic mice.

The reticuloendothelial stimulant glucan, a beta-1,3-polyglucose component of the cell wall of Saccharomyces cerevisiae, was evaluated for its ability to modify Staphylococcus aureus-induced lethality in normal and leukemic mice. In normal mice the intravenous injection of glucan (0.45 mg per mouse) 7 and 4 days prior to intravenous challenge with S. aureus (1.0 x 10(9)) resulted in a significantly increased survival. Histological examination of the kidneys revealed that glucan significantly inhibited renal necrosis associated with systemic staphylococcal diseases. Further studies indicated that glucan administration not only enhanced survival of leukemic mice, but also increased survival of leukemic mice with experimentally induced staphylococcal speticemia. These data denote that glucan enhances nonspecific resistance to S. aureus sepsis, promotes survival during leukemic episodes, and increases survival time of leukemic mice with experimentally induced staphylococcal infection.     

In vitro tumoricidal activity of resting and glucan-activated Kupffer cells

Kupffer cells compose 80-90% of fixed tissue macrophages and have been suggested to play an important role in hepatic antitumor resistance. In the present study, the ability of resting and activated Kupffer cells to lyse syngeneic mammary adenocarcinoma BW10232 cells was evaluated. Activated Kupffer cells were isolated from C57Bl/6J mice following single of multiple intravenous (IV) injections of glucan (0.45 mg/mouse), a potent macrophage-activating agent. Mice receiving 5% (w/v) dextrose served as control. Resting Kupffer cells induced significant (P less than .05) 4% and 12% specific lysis of adenocarcinoma cells at target:effector ratios of 1:10 and 1:50, respectively. Kupffer-cell-mediated tumoricidal activity was depressed on day 1 following a single IV injection of glucan. By day 3 postglucan, the antitumor activity of Kupffer cells returned to control levels and was enhanced on days 5 and 10. Following multiple IV injections of glucan on days -5, -3, and -1, Kupffer-cell-mediated cytotoxicity was elevated on days 1 and 4. These observations demonstrate that resting Kupffer cells are significantly cytotoxic to adenocarcinoma cells at T:E ratios of 1:10 and 1:50 and following a transient inhibition of Kupffer-cell-mediated tumoricidal activity, glucan was effective in significantly enhancing the antitumor activity of Kupffer cells.     

Glucan-induced enhancement of host resistance to selected infectious diseases.

We conducted studies with mice, rats, and monkeys which demonstrated the ability of glucan to induce either nonspecific or specific enhancement of host resistance to infectious diseases. Intravenous pretreatment of mice with glucan significantly enhanced the survival of mice challenged with either Venezuelan equine encephalomyelitis (VEE) virus or Rift Valley fever virus. Pretreatment was beneficial when initiated 3 days before challenge with VEE virus and 7 days before challenge with Rift Valley fever virus. Treatment of mice after VEE challenge did not increase their survival compared with controls. Glucan pretreatment of rats provided increased resistance to both intraperitoneal and low-dose aerosol challenges with virulent Francisella tularensis when the glucan was given intravenously, but not when it was administered intranasally. In contrast, intranasal glucan pretreatment enhanced the survival of mice when they were challenged by aerosol with Pseudomonas pseudomallei, whereas intravenous glucan pretreatment did not increase survival. mice given glucan combined with a marginally immunogenic dose of VEE vaccine were more resistant to homologous virus challenge than were mice given either Freund complete adjuvant plus vaccine or vaccine alone. Similarly, both primary and secondary VEE antibody titers in cynomolgus monkeys given glucan with VEE vaccine were significantly greater than titers in vaccine controls.     

Humoral responses following immunization with Leishmania infantum (ex. Oklahoma): a comparison of adjuvant efficacy in the antibody responses of Balb-C mice.

Adjuvants are commonly used in immunization protocols for the purpose of augmenting immune responses to antigens. The antigenic profile of Leishmania infantum (ex. Oklahoma) is described and the efficacies of three adjuvants delivered coincidentally with killed promastigotes are compared, by measuring relative serologic responses of Balb-C mice to specific antigenic determinants. Western blotting techniques were employed to visualize humoral responses to isolated antigens; serologic profiles were compared and contrasted. Four immunization protocols utilizing Freund's complete adjuvant, glucan adjuvant, lipovant adjuvant or phosphate-buffered saline, in conjunction with killed L. infantum (ex. Oklahoma) promastigotes were executed in parallel. All groups receiving adjuvant protocols developed enhanced serologic responsiveness. Similar profiles were observed in mice treated with glucan or lipovant. Animals receiving promastigotes in Freund's complete adjuvant also developed strong humoral responses, binding cross-reactive epitopes not recognized by other groups. Our findings indicate that glucan and lipovant present effective adjuvant alternatives, to Freund's complete adjuvant and may be of value in immunization against visceral leishmaniasis.     

Modulation of the immune response of BALB/C mice against Leishmania Major by imuvert

This study investigated the effect of Imuvert, a biological response modifier derived from Serratia marcescens, on the progression of Leishmania major infection in Balb/c mice.A single 100 μg Imuvert injection was significantly protective in Balb/c mice when challenged 28 days later in the footpad with 5 × 10⁵ stationary phase L. major promastigotes. Intraperitoneal (i.p.) and intravenous (i.v.) immunization of mice with heat-killed stationary phase L. major promastigotes significantly reduced lesion development following challenge with L. major promastigotes. Subcutaneous (s.c.) immunization had no protective effect. A single 100 μg Imuvert injection significantly reduced lesion development in s.c. immunized mice, but had a lesser effect in mice immunized by i.p. and i.v. routes. Balb/c mice receiving four Imuvert injections 14, 7, 2 and 1 day prior to footpad challenge with L. major promastigotes were not protected, but rather displayed significant exacerbation of infection.Our results suggest the possibility that Imuvert could be useful in stimulating a protective response against L. major when given along with s.c. vaccine, a realistic route for vaccinating humans in contrast to either i.v. or i.p. routes. Since the protective response in Balb/c mice against L. major is dependent on the stimulation of T_hl cells, it is suggested that the observed adjuvant effect of Imuvert given with s.c. vaccine perhaps is due to changes in immunological responses in such a direction.     

Immunization against chlamydial genital infection in guinea pigs with UV-inactivated and viable chlamydiae administered by different routes.

Female guinea pigs were immunized with viable or UV light-inactivated chlamydiae (agent of guinea pig inclusion conjunctivitis), belonging to the species Chlamydia psittaci, by intravenous, subcutaneous, oral, or ocular routes. All animals were then inoculated vaginally with viable chlamydiae to determine the extent of protection against challenge infection induced by the various regimens. The course of genital infection was significantly reduced in intensity in all groups of animals except the unimmunized controls and those animals immunized orally with inactivated antigen. Guinea pigs immunized with viable antigen were more likely to develop resistance to challenge infection and, in general, had a significantly greater degree of protection than animals immunized with inactivated antigen. No one route seemed superior in producing a protective response. Animals in all groups demonstrating protection developed serum and secretion immunoglobulin G antibody responses to chlamydiae. Lymphocyte proliferative reactions to chlamydial antigen were variable among groups. Immunoblot analysis of serum and secretions indicated a wide range of antibody specificities, but most protected animals produced antibodies to the major outer membrane protein, lipopolysaccharide, and the 61-kilodalton protein. No definitive associations could be made between the increased ability of immunization with viable organisms to produce resistance to challenge infection and a particular immune parameter. These data indicate that viable chlamydiae given by various routes are able to induce a strong immune response which can provide resistance against reinfection in some cases or at least reduce the degree of infection to a greater degree than inactivated antigen. However, complete resistance to genital tract infection may be difficult to obtain and alternate immunizations strategies may have to be developed.     

Modification of post-operative C. albicans sepsis by glucan immunostimulation

Glucan, a beta-1,3 polyglucose, was evaluated for its ability to enhance resistance of post-operative mice to experimentally induced C. albicans sepsis. Male C57BL/6J mice were injected i.v. with glucan (0.45 mg/mouse) on days 10,7,4 and 1 prior to midline laparotomy and intravenous challenge with 3 X 10(6) C. albicans. The detrimental effect of surgery on survival following C. albicans infection was manifested by a 47% survival in the non-surgery-infected group in contrast to 20% in the surgery-infected group. Protection against C. albicans was observed in the glucan-treated groups. The glucan-treated non-operated mice manifested 100% survival while the surgery group had a 73% survival. Glucan significantly enhanced macrophage phagocytic function in control and operated mice. Laparotomy alone did not significantly depress macrophage phagocytosis. Histopathological studies revealed that glucan markedly inhibited the renal pathology associated with C. albicans challenge both in the presence and absence of laparotomy. These data indicate that glucan increased survival and reduced renal pathology associated with C. albicans challenge in the post-operative period. These observations suggest that Biologic Response Modifiers such as glucan may be effectively employed in patients who are at risk for post-operative infections.     

Leishmania pifanoi amastigote antigens protect mice against cutaneous leishmaniasis.

In the search for a leishmaniasis vaccine, extensive studies have been carried out with promastigote (insect stage) molecules. Information in this regard on amastigote (mammalian host stage) molecules is limited. To investigate host immune responses to Leishmania amastigote antigens, we purified three stage-specific antigens (A2, P4, and P8) from in vitro-cultivated amastigotes of Leishmania pifanoi by using immunoaffinity chromatography. We found that with Corynebacterium parvum as an adjuvant, three intraperitoneal injections of 5 micrograms of P4 or P8 antigen provided partial to complete protection of BALB/c mice challenged with 10(5) to 10(7) L. pifanoi promastigotes. These immunized mice developed significantly smaller or no lesions and exhibited a 39- to 1.6 x 10(5)-fold reduction of lesion parasite burden after 15 to 20 weeks of infection. In addition, P8 immunization resulted in complete protection against L. amazonensis infection of CBA/J mice and partial protection of BALB/c mice, suggesting that this antigen provided cross-species protection of mice with different H-2 haplotypes. At different stages during infection, vaccinated mice exhibited profound proliferative responses to parasite antigens and increased levels of gamma interferon production, suggesting that a Th1 cell-mediated immune response is associated with the resistance in these mice. Taken together, the data in this report indicate the vaccine potential of amastigote-derived antigens.     

Differential induction of cellular responses by live and dead Leishmania promastigotes in healthy donors

The most effective protection against human leishmaniasis has been achieved following vaccination with live promastigotes. Killed promastigotes + BCG can protect, albeit to a lower degree. To explore what mechanisms may be involved in these differences, the ability of live and dead promastigotes to induce immune responses were evaluated in vitro. The data showed that live and dead promastigotes differ in their ability to induce proliferation and cytokine production. Cytokine gene expression of Th1 related cytokines (IL-12, IFNgamma and TNFalpha) in adult PBMC was more evident to live than to heat killed promastigotes. This was coupled with significantly higher number of IFNgamma secreting cells induced by live than killed promastigotes. However, alpha-IL-12 antibodies did not block the IFNgamma response induced by live promastigotes. Proliferative responses were variable. In contrast to adult PBMC no IFNgamma secreting MNC could be detected in cord blood. However, in these cells the live promastigotes consistently induced higher proliferative response compared to dead. Implications of these findings are discussed.     

Anti-infective effect of poly-beta 1-6-glucotriosyl-beta 1-3-glucopyranose glucan in vivo.

Mice challenged with Escherichia coli or Staphylococcus aureus were protected against lethal peritonitis by the intravenous administration of 10 micrograms of poly-beta 1-6-glucotriosyl-beta 1-3-glucopyranose (PGG) glucan per animal 4 to 6 h prior to bacterial challenge. Subsequent studies with the rat model for intra-abdominal sepsis indicated that intramuscular doses of 10 to 100 micrograms per animal 24 and 4 h prior to surgical implantation of the bacterial inoculum reduced the early mortality associated with the peritonitis phase of this experimental disease process. Quantitative cultures of blood obtained from challenged rats showed that significantly fewer organisms were present in the blood of PGG glucan-treated animals than in that of untreated animals. Quantitative studies of leukocytes of rats and mice following a single injection of PGG glucan showed a modest transient increase in the total leukocyte count. The possible mechanisms by which protection occurs in the animal model system are discussed.     

Effects of muramyl dipeptide and trehalose dimycolate on resistance of mice to Toxoplasma gondii and Acanthamoeba culbertsoni infections

The effects of synthetic muramyl dipeptide (MDP) and natural trehalose dimycolate (TDM) against parasitic infections by intracellular Toxoplasma gondii and free-living Acanthamoeba culbertsoni were studied. Significant resistance against oral T. gondii infection was induced by intraperitoneal pretreatment with TDM but not with MDP. The protective effect of TDM against T. gondii was corroborated by a significant reduction in the number of cysts in brains of pretreated animals and elevated serum antibody levels. Partial protection against lethal intranasal A. culbertsoni infection was conferred by specific immunization with viable trophozoites of nonpathogenic Acanthamoeba lugdunensis. The nonspecific resistance induced by intravenous pretreatment with MDP was similar to, whereas that stimulated by TDM was lesser than the protection conferred by A. lugdunensis. The Fc receptor-mediated phagocytosis of 51Cr-labeled sheep red blood cells by alveolar macrophages was enhanced by MDP. The phagocytic activity of peritoneal macrophages was increased by lower doses of TDM.     

Establishment of an appropriate inoculum dose of Leishmania donovani promastigotes required to establish a visceral infection in laboratory animal rodent models

Syrian hamsters and BALB/c mice were inoculated intraperitoneally with various doses of stationary phase Leishmania donovani promastigotes derived from primary, secondary and tertiary cultures. Axenic derived amastigotes from a tertiary culture and mass-culture derived promastigotes from primary, secondary, and tertiary cultures were also used. Animals were sacrificed after 30 days incubation period and parasite-loads quantified from Giemsa stained spleen smears. A primary inoculum dose of 1 x10(8) was found to be the most appropriate in effecting a visceral infection. This parasite dose resulted in a spleen parasite-load of 10-20 amastigotes per field of microscope view at x1,000 magnification. Those involved in candidate vaccine molecules or experimental drugs against kala-azar will find these results useful.