Immunopotentiation of local and systemic humoral immune responses by ISCOMs, liposomes and FCA: role in protection against influenza A in mice

The immunogenicity and protective efficacy of an influenza A subunit vaccine preparation administered to mice in an aqueous form, or presented as immunostimulatory complexes (ISCOMs), liposomes or with Freund's complete adjuvant (FCA), were assessed in comparative studies with live infectious virus. Both intranasal and parenteral routes of administration were assessed. An enzyme-linked immunosorbent assay (ELISA) was used to measure nasal wash and serum antibody responses in groups of unprimed mice, while protection was determined by the recovery of homologous influenza virus from mouse nasal washes and lung homogenates following challenge infection by the intranasal route. The results showed that parenteral administration of the influenza antigen preparations induced variable levels of both local and systemic antibodies at weeks 3, 7 and 22 postimmunization. Although the overall greatest levels of antibody and protection were elicited in mice following live virus infection, formulation of influenza surface haemagglutinin (HA) and neuraminidase (NA) proteins into ISCOMs elicited high and persistent antibody responses and provided relatively good protection of the upper and lower respiratory tracts of these animals. The results also show a relatively poor effect of the subunit antigen preparations in promoting humoral immune responses and protection irrespective of the nature of their presentation, when given by the intranasal route.     

Gliding bacterial adjuvant stimulates feline cytokines in vitro and antigen-specific IgG in vivo

Gliding bacterial adjuvant (GBA) has been previously characterized as a potent immune modulator, stimulating the growth of murine B lymphocytes, inducing murine NK cell activity, and promoting the release of several murine cytokines. Based on these studies and our interest in potentiating the effectiveness of feline vaccines, GBA was tested for its ability to stimulate feline T cells in vitro and act as a vaccine adjuvant in vivo. In vitro, GBA stimulated feline PBLs to proliferate and release interferon (IFN) and IL-2. Unlike IFN, the release of IL-2 appeared to be unaffected by prior depletion of macrophages, indicating GBA directly stimulated feline T cells. In vivo GBA was co-administered with Keyhole Limpet Hemacyanin (KLH) and the anti-KLH antibody response was compared to cats receiving KLH emulsified in complete Freund's adjuvant (CFA). Fourteen days after the third immunization and continuing for a 30-day observation period, KLH-specific IgG titers in cats receiving GBA were significantly higher than those given CFA. However, when cats were subsequently boosted with KLH alone, those cats receiving CFA demonstrated significantly higher antibody titers throughout a second 30-day observation period. The anti-KLH antibody memory response was greatly enhanced when GBA was emulsified with incomplete Freunds adjuvant (IFA) prior to injection. Serum titers of cats given KLH in an oil-based GBA preparation were significantly higher than cats receiving KLH adjuvanted with IFA or CFA, an effect which persisted 38 days after boosting with KLH alone. Finally, GBA significantly enhanced the feline humoral response to a recombinant protein of Dirofilaria immitis, the causative agent of feline heartworm. Serum titers of cats inoculated with recombinant antigen in GBA were significantly greater than cats given recombinant antigen adjuvanted with Titermax, alum, or NAGO. These studies indicate that GBA induces T cell proliferation and the release of IL-2 and IFN in vitro and can be used to enhance the recall antibody response to both a T cell dependent antigen and an immunogen derived from Dirofilaria immitis.     

Mucosal delivery of murine interleukin-2 (IL-2) and IL-6 by recombinant strains of Lactococcus lactis coexpressing antigen and cytokine

Lactococcus lactis is a nonpathogenic and noncolonizing bacterium which is being developed as a vaccine delivery vehicle for immunization by mucosal routes. To determine whether lactococci can also deliver cytokines to the immune system, we have constructed novel constitutive expression strains of L. lactis which accumulate a test antigen, tetanus toxin fragment C (TTFC), within the cytoplasmic compartment and also secrete either murine interleukin-2 (IL-2) or IL-6. When mice were immunized intranasally with various different expression strains of L. lactis, the anti-TTFC antibody titers increased more rapidly and were substantially higher in mice immunized with the bacterial strains which secreted IL-2 or IL-6 in addition to their production of TTFC. This adjuvant effect was lost when the recombinant strains of L. lactis were killed by pretreatment with mitomycin C and could therefore be attributed to the secretion of IL-2 or IL-6 by the recombinant lactococci. These results provide the first example of the use of a cytokine-secreting, noninvasive experimental bacterial vaccine vector to enhance immune responses to a coexpressed heterologous antigen and point the way to experiments which will test the possible therapeutic efficacy of this mode of cytokine delivery.     

CpG DNA is a potent enhancer of specific immunity in mice immunized with recombinant hepatitis B surface antigen

Unmethylated CpG dinucleotides in bacterial DNA or synthetic oligodeoxynucleotides (ODN) cause B cell proliferation and Ig secretion, monocyte cytokine secretion, and activation of NK cell lytic activity and IFN-gamma secretion in vivo and in vitro. The potent immune activation by CpG ODN suggests possible utility for enhancing immune responses to vaccines. Mice immunized with recombinant hepatitis B virus surface Ag and a CpG ODN as an immune enhancer have titers of Abs against HBsAg (anti-HBs) that are five times higher than those of mice immunized with HBsAg and the standard adjuvant, aluminum hydroxide (alum). Ab titers in mice immunized with HBsAg and both CpG ODN plus alum were 35 times higher than the titers in mice immunized with alum alone, indicating a strong synergistic interaction between the CpG ODN and alum. ODN without CpG motifs had little or no immune-enhancing activity at the doses used herein. Alum induces a Th2 humoral response (mostly IgG1) and no CTL. In contrast, CpG ODN gives a strong Thl response with predominantly IgG2a Abs and CTL, even when mixed with alum. In vitro studies to determine possible mechanisms of CpG immune-enhancing effects show that CpG ODN induce expression of costimulatory molecules on Ag-presenting cells and drive B cell isotype switching in the appropriate cytokine milieu. These studies demonstrate that CpG ODN are promising new immune enhancers for vaccination applications.     

Aqueous-based microencapsulation enhances virus-specific humoral immune responses in mice after parenteral inoculation

Vaccines are commonly administered by the parenteral route. Therefore, adjuvant strategies which include parenteral immunization may improve the efficacy of a number of current vaccines. The capacity of aqueous-based microencapsulation to enhance virus-specific IgG responses in mice inoculated intramuscularly with small quantities of antigen was evaluated. Mice were inoculated with either 10(4), 10(3), or 10(2) p.f.u. of microencapsulated rotavirus (bovine strain WC3), placebo microcapsules plus free virus, or virus alone. Mice were subsequently bled 1, 2, 4, 6, and 9 months after inoculation. Microencapsulation of rotavirus enhanced virus-specific humoral immune responses. In addition, virus-containing microcapsules composed of spermine-chondroitin sulfate induced levels of virus-specific antibodies greater than those found after inoculation with virus-containing microcapsules composed of spermine-alginate. Mechanisms by which microencapsulation may enhance virus-specific humoral immunity are discussed.     

IL-12 is a potent neonatal vaccine adjuvant

Neonatal animals show generally poor responsiveness to foreign antigens and are known to display polarized expression of Th2-like cytokines and antibody responses. We now report that newborn mice display a reduction in peripheral expression of the Th1-inducing cytokine, IL-12. Attempts to overcome this decrease by immunization and treatment with IL-12 within 24 h of birth resulted in elevated levels of IFN-gamma and IL-10 mRNA in the spleens of mice compared to animals exposed to antigen only. Moreover, such animals showed dramatic enhancement of IgG2a and IgG2b antibody levels upon adult challenge compared to mice primed with antigen alone. These effects appeared to be due to induction of neonatal B cell memory. IgG1 antibody levels, a measure of Th2 activity, were unaffected or even somewhat enhanced by neonatal IL-12 treatment. Taken together, these results provide evidence that IL-12 administration induces a Th1-like cytokine response in newborns and causes priming for heightened memory antibody responses in vivo. Our findings suggest the use of IL-12 as a vaccine adjuvant in neonates for inducing protection against common childhood pathogens.     

Genetic control of immune response to recombinant antigens carried by an attenuated Salmonella typhimurium vaccine strain: Nramp1 influences T-helper subset responses and protection against leishmanial challenge

Attenuated strains of Salmonella typhimurium have been widely used as vehicles for delivery and expression of vaccine antigens in murine models of infectious disease. In mice, early bacterial replication following infection with S. typhimurium is controlled by the gene (Nramp1, formerly Ity/Lsh/Bcg) encoding the natural-resistance-associated macrophage protein (Nramp1). Nramp1 regulates macrophage activation and has multiple pleiotropic effects, including regulation of tumor necrosis factor alpha, interleukin 1beta (IL-1beta), and major histocompatibility complex class II molecules, all of which influence antigen processing and presentation. Nramp1 also has a direct effect on antigen processing, possibly by regulating the activity of proteases in the late endosomal compartment. Hence, there are multiple ways (regulation of bacterial load or recombinant antigen dose, class II molecule expression, costimulatory or adjuvant activity, and antigen processing) that Nramp1 might influence responses to recombinant salmonella vaccines. To test the hypothesis that Nramp1 influences responses to vaccination, congenic mouse strains have been used to analyze immune responses to recombinant antigens (tetanus toxoid antigen and leishmanial gp63) carried by live attenuated S. typhimurium aroA aroD mutants. Results show that congenic mice carrying the wild-type (S. typhimurium resistance) Nramp1 allele mount a predominantly T-helper-1 (IL-2 and gamma interferon) response to vaccination and show enhanced resolution of lesions following challenge infection with Leishmania major. In contrast, mice carrying mutant (S. typhimurium susceptibility) Nramp1 mount a T-helper-2 (immunoglobulin E and IL-4) response and show exacerbated lesion growth upon challenge.     

Tissue microarrays for high-throughput molecular profiling of tumor specimens

The impact of oral treatment with insulin on disease development was studied in diabetes prone BB rats. Because of the positive outcome of a prior study in non obese diabetic (NOD) mice, BB rats received insulin in combination with a bacterial adjuvant. Porcine insulin was given orally twice weekly from 35-100 days of age, the E. coli preparation OM-89 was fed on alternate days. Other groups received vehicle, the bacterial adjuvant, or insulin alone. Both insulin containing oral dosing regimens induced a transient non significant delay in diabetes onset. Insulin alone, however did not decrease the final diabetes incidence. Oral dosing with insulin plus adjuvant caused exacerbation of disease development as judged from the decreased survival rate in comparison with the insulin treated group (p < 0.05). Intra-islet infiltration also increased (p < 0.005) compared with the insulin or vehicle treated groups. The effect correlated with enhanced interferon gamma (IFNgamma) and decreased interleukin 10 (IL-10) gene expression in the gut suggesting a shift towards proinflammatory T helper 1 (Th1) reactivity (p < 0.01). Although treatment with adjuvant alone also increased the degree of insulitis, an enhanced incidence of diabetes and a shift in cytokine expression was only seen in the group receiving insulin plus adjuvant. Taken together, the data suggest that treatment with a bacterial adjuvant and oral insulin may alter the gut immunoregulatory state such that disease promoting rather than protective immune responses are induced.     

Inactivation of glycoprotein gE and thymidine kinase or the US3-encoded protein kinase synergistically decreases in vivo replication of pseudorabies virus and the induction of protective immunity

To evaluate the contribution of glycoprotein E (gE), thymidine kinase (TK), and the US3-encoded protein kinase (PK) in the induction of protective immunity to pseudorabies virus (PRV), we intranasally inoculated pigs, the natural host of this virus, with mutant PRV strains in which the genes encoding these proteins were inactivated. Both single and double mutants were constructed. Of these proteins, gE has previously been demonstrated to induce antibodies (in mice and pigs), which require complement to neutralize the virus, and helper T cell responses (in mice). PK and TK have thus far not been reported to induce B or T cell responses. All mutants had a strongly reduced virulence for pigs in comparison with wild-type (wt) PRV. After primary infection, most virus was excreted by wt PRV-inoculated animals. Animals inoculated with gE-PK- and gE-TK- double mutants excreted less virus than animals inoculated with gE-, PK-, and TK- single mutants. After challenge infection with the virulent PRV strain NIA-3, no virus was excreted by wt PRV- and PK- mutant-immunized animals, indicating complete protective immunity. Only one of seven gE- and two of seven TK- mutant-immunized animals excreted virus after the challenge inoculation. In contrast, most animals immunized with the gE-PK- or gE-TK- double mutants excreted virus after the challenge inoculation. Daily mean virus excretion after challenge infection was inversely correlated with daily mean virus excretion after primary infection. In most animals, lack of virus excretion was associated with lack of secondary antibody responses, probably attributable to inadequate stimulation of memory B cells as a consequence of early elimination of viral antigen. Thus, inactivation of gE, TK, and PK all affected the immunogenicity of PRV and the effect of gE and TK and gE and PK inactivation appeared synergistic. We found no simple correlation between in vitro growth properties of the mutants and their immunogenic capacity. Strains lacking PK reached lower end titers in vitro than the other mutants. The most likely explanation for the lower protective capacity of some of the mutants appears their reduced replicative capacity in some cells or tissues in vivo, rather than a loss of particular epitopes.     

CD8-deficient mice exhibit augmented mucosal immune responses and intact adjuvant effects to cholera toxin

We used normal, CD4 and CD8 gene-targeted mice to investigate the role of CD4+ and CD8+ T cells in the regulation of gut mucosal immune responses following oral immunizations with cholera toxin (CT) adjuvant. Phenotypic analysis of mucosa-associated tissues revealed normal CD3+ T-cell frequencies in CD4-/- and CD8-/- mice such that in CD4-/- mice the CD8+ and double-negative (DN) T cells were increased. In CD8-/- mice the CD4+ T cells were increased, with the exception that in the intraepithelial compartment the CD3+ T cells were predominantly DN gamma delta T-cell receptor (TCR)+ T cells. All mice, normal and deficient, failed to respond to oral immunization with the antigen, keyhole limpet haemocyanin (KLH), alone. In the presence of CT adjuvant, however, CD8-/- mice consistently exhibited three- to fivefold stronger gut mucosal responses compared to normal C57B1/6 mice. By contrast, no difference was observed for systemic responses between CD8-/- and normal mice. Thus the up-regulation selectively affected mucosal responses, suggesting that, contrary to the CD8-/- mouse gut, the normal gut mucosa may host CD8+ T cells that exert a local suppressive effect on T- and B-cell responses. The magnitude of the enhancing effect of CT was comparable in CD8-/- and normal mice, clearly demonstrating that the adjuvant mechanism of CT does not require CD8+ T cells. On the other hand, the adjuvant effect of CT required CD4+ T cells, because no or poor anti-KLH responses were observed in CD4-/- mice. In both normal and CD8-/- mice CT adjuvant promoted KLH-specific CD4+ T-cell printing without any selective effect on the differentiation towards a T-helper type-1 (Th1) or Th2 dominance. Furthermore, CT adjuvant increased the frequency of CD4+ T cells expressing a memory phenotype, i.e. CD44high, LECAM-1low and CD45RBlow. We have shown, using gene-targeted mice, that CD8+ T cells are not required for the adjuvant effect of CT, and that CD8+ T cells may exert local mucosal down-regulation of intestinal immune responses.     

The role of macrophages in the induction and regulation of immunity elicited by exogenous antigens

Different delivery vehicles may target to different antigen presenting cells (APC) because of their composition, size and/or physical properties. In this study, we examined the priming of cytotoxic T lymphocyte (CTL) responses to a soluble exogenous protein in vivo, using various delivery vehicles. In addition, we determined the role of macrophages as APC in vivo for each of these delivery vehicles by comparing the induction of antigen-specific CTL and serum antibodies in normal and macrophage-depleted mice. Influenza A virus-derived virosomes, liposomes and monophosphoryl lipid A/squalene (MPLSQ) efficiently induced antigen-specific CTL as well as antibody responses, of which virosomes proved to be the most efficient inducers. In mice that were immunized with cell-associated antigen, strong CTL responses but no antigen-specific antibodies were detectable, while aluminium hydroxide and aluminium phosphate elicited antigen-specific antibodies but no CTL responses. Elimination of macrophages in vivo before immunization abrogated CTL responses induced with liposomes and MPL/SQ, but did not affect induction of antigen-specific CTL with virosomes or cell-associated antigen. Importantly, serum antibody levels were not altered after macrophage depletion, regardless of the delivery vehicle used, suggesting that in the absence of macrophages, other APC may phagocytose the exogenous antigens for major histocompatibility complex (MHC) class II processing and presentation. These results suggest that soluble exogenous antigens delivered in different carrier systems may be processed differently by different APC in vivo for MHC class I- or class II-restricted presentation.     

Novel intranasal immunization techniques for antibody induction and protection of mice against gastric Helicobacter felis infection

Intranasal (i.n.) delivery of antigen can be highly effective for generating circulating and secretory antibody responses. Mice were immunized i.n. with two antigens, human IgA, and Helicobacter pylori urease in the presence or absence of mucosal adjuvant. To restrict antigen delivery to the upper airways, protein solutions were administered in a small volume without anesthesia. Repeated daily i.n. administration of antigen without adjuvant elicited high levels of specific IgG in serum and IgA in serum, saliva, and feces. Once weekly i.n. immunization with co-administration of cholera toxin or Escherichia coli heat-labile toxin as adjuvant elicited somewhat lower levels of antibody to urease. When challenged with Helicobacter felis, only mice immunized with urease in the presence of adjuvant were protected against gastric infection.     

Kinetics, localization and cytokine profile of T cell responses to immune stimulating complexes (iscoms) containing human influenza virus envelope glycoproteins

Immune stimulating complexes (iscoms) are 40 nm particles combining adjuvant-active Quillaja saponins and multimeric presentation of antigens. The distribution in mice of influenza virus iscoms and the resulting T cell responses in the lymph nodes (LN) and spleen were characterized. After a single subcutaneous injection, iscoms were delivered to the draining LN where they induced a transient population of LN cells which responded with proliferation and secretion of interleukin-2 (IL-2), gamma-interferon (IFN-gamma) and interleukin-4 (IL-4) after restimulation. The response in the spleen developed more slowly, sustained for 12 weeks and was characterized by cells producing in particular IL-2 and IFN-gamma but also IL-4. A booster resulted in a dramatic enhancement of the production of IFN-gamma, indicating that iscoms efficiently recruit cells with Th1 properties. Comparisons of T cell responses to iscoms and to influenza virus antigen in Freund's complete adjuvant demonstrate that these adjuvants affect both the localization and cytokine profile of T cell responses.     

Induction of cytotoxic T lymphocytes by recombinant canarypox (ALVAC) and attenuated vaccinia (NYVAC) viruses expressing the HIV-1 envelope glycoprotein

Successful immunization against many viruses, including retroviruses such as HIV-1, is thought to depend upon the roles of both antibody and cytotoxic T-lymphocyte responses. With safety a major concern, we developed two poxvirus recombinants expressing the envelope glycoprotein of HIV-1 IIIB. Canarypox (ALVaC), which is not known to replicate in mammalian cells, and a highly attenuated vaccinia (NYVAC) virus deleted of 18 open reading frames associated with virulence and host range were used as vectors. Upon inoculation into BALB/c mice, both the ALVAC and NYVAC recombinants were capable of inducing antibody responses to HIV gp120 and provoking remarkable levels of primary and memory Thy1.2+, CD4-, CD8+ cytotoxic T-lymphocyte responses to the hypervariable V3 loop of the HIV-1 envelope glycoprotein.     

Effectiveness of liposomes possessing surface-linked recombinant B subunit of cholera toxin as an oral antigen delivery system

Liposomes appear to be a promising oral antigen delivery system for the development of vaccines against infectious diseases, although their uptake efficiency by Peyer's patches in the gut and the subsequent induction of mucosal immunoglobulin A (IgA) responses remain a major concern. Aiming at targeted delivery of liposomal immunogens, we have previously reported the conjugation via a thioether bond of the GM1 ganglioside-binding subunit of cholera toxin (CTB) to the liposomal outer surface. In the present study, we have investigated the effectiveness of liposomes containing the saliva-binding region (SBR) of Streptococcus mutans AgI/II adhesin and possessing surface-linked recombinant CTB (rCTB) in generating mucosal (salivary, vaginal, and intestinal) IgA as well as serum IgG responses to the parent molecule, AgI/II. Responses in mice given a single oral dose of the rCTB-conjugated liposomes were compared to those in mice given one of the following unconjugated liposome preparations: (i) empty liposomes, (ii) liposomes containing SBR, (iii) liposomes containing SBR and coadministered with rCTB, and (iv) liposomes containing SBR plus rCTB. Three weeks after the primary immunization, significantly higher levels of mucosal IgA and serum IgG antibodies to AgI/II were observed in the rCTB-conjugated group than in mice given the unconjugated liposome preparations, although the latter mice received a booster dose at week 9. The antibody responses in mice immunized with rCTB-conjugated liposomes persisted at high levels for at least 6 months, at which time (week 26) a recall immunization significantly augmented the responses. In general, mice given unconjugated liposome preparations required one or two booster immunizations to develop a substantial anti-AgI/II antibody response, which was more prominent in the group given coencapsulated SBR and rCTB. These data indicate that conjugation of rCTB to liposomes greatly enhances their effectiveness as an antigen delivery system. This oral immunization strategy should be applicable for the development of vaccines against oral, intestinal, or sexually transmitted diseases.     

Biodegradable microparticles with an entrapped branched octameric peptide as a controlled-release HIV-1 vaccine

Polyactide-co-glycolide microparticles, with an entrapped branched octameric peptide from human immunodeficiency virus (HIV-1), were prepared by a solvent evaporation method. The microparticles were characterized for size distribution, antigen loading level, and integrity. Mice in one group were each immunized with a single dose of a controlled-release microparticle formulation containing 300 micrograms of peptide and the serum IgG responses to the antigen were compared with those of mice from a second group that were immunized at 0, 4, and 26 weeks with 100-microgram doses of the same peptide immunogen adsorbed to alum. The controlled-release microparticles induced an antibody response comparable to that from the alum-immunized group. The subcutaneous and the intramuscular routes of administration were compared in additional groups of mice for the microparticles, and both routes induced similar responses. A suspending vehicle for the microparticles was also evaluated and did not affect the immunogenicity of the controlled-release formulation containing both small and large microparticles, although the immunogenicity of smaller microparticles immunized alone was affected.     

Cytokines as adjuvants for ruminant vaccines

Successful vaccination against any potential pathogen is critically dependent on inducing an appropriate immune response. The pivotal role of cytokines in the immune response to vaccination suggests that non-protective responses or responses that exacerbate disease subsequent to infectious challenge may be the result of limiting or preferential production of one or a number of these mediators. This suggests that the use of recombinant cytokines as vaccine adjuvants may offer a mechanism whereby the magnitude and phenotype of the immune response to vaccination can be specifically modified. In mice, recombinant cytokines have been used extensively as therapeutics, while studies describing vaccine adjuvant activity are more limited. Recombinant (r) interleukin (IL)-1, IL-2 and interferon (IFN) gamma have been used primarily to enhance humoral responses with enhanced protection assessed where appropriate. Cytokine adjuvant studies in ruminants have been restricted to recombinant ovine (rov) and bovine (rbov) IL-1 and IL-2. In sheep, their application has been optimised with respect to dose, route of delivery and formulation, for induction of humoral and cell mediated immunity (DTH and cytotoxicity) to the model protein antigen (Ag) avidin. The level of adjuvant activity of IL-1 in particular compares favourably to that of a variety of other traditional and new chemical adjuvants and detailed analysis has indicated no adverse local or systemic side-effects. Recent studies in our laboratory demonstrating the effectiveness of rovIL-1 as an adjuvant in single and multi-component bacterial toxoid vaccines, and studies from other laboratories demonstrating the application of rbovIL-1 as an adjuvant for the response in cattle to live attenuated viral vaccines, suggest that rIL-1 may become the adjuvant of choice for diseases where protection is mediated by high levels of circulating antibody (Ab). With respect to helminth parasites, IL-1 may prove useful as a component of vaccines based on "hidden Ags" which rely on induction of Ab. Based on analysis in mouse models of helminth infection, other cytokines such as IL-4 and IL-10 may be appropriate for vaccines based on induction of mechanisms involved in natural immunity.     

Immunological memory after priming with a thymus independent antigen, NIP-ficoll. 4-hydroxy-5-iodo-3-nitrophenylacetyl coupled to polymer of sucrose and epichlorhydrin

The capacity of mouse spleen fragments to mount an anti-NIP (4-hydroxy-5-iodo-3-nitrophenylacetyl) response in vitro was studied. The fragments came from unprimed, NIP-Ficoll (polymer of sucrose and epichlorhydrin) or NIP-CG (chicken globulin) primed mice. Unprimed spleen fragments from C57BL/6 mice gave a good anti-NIP response to NIP-Ficoll, whereas CBA fragments did not. Priming with NIP-Ficoll made CBA fragments responsive and enhanced slightly the response of C57BL/6 fragments when stimulated with the same antigen. This memory effect could be seen only after a small priming dose. Priming the mice with NIP-Ficoll made their spleen fragments responsive to a protein conjugate of NIP (NIP-CG), but this effect was seen only after priming with a high dose. The antibody class distribution and the kinetics of the appearance of different immunoglobulin classes were similar in the primary and secondary responses in vitro. The peak responses of IgM, IgA and IgG were reached on day eight and the relative amount of IgG was the same in the primary and in the secondary responses. Spleen fragments derived from NIP-CG primed mice produced more IgG anti-NIP antibodies than fragments derived from untreated mice when immunized in vitro with NIP-Ficoll. The amount of IgG was, however, much higher when these fragments were challenged with the homologous antigen, NIP-CG.     

Studies on experimental adjuvanted influenza vaccines: comparison of immune stimulating complexes (Iscoms) and oil-in-water vaccines

Detergent-disrupted influenza virus vaccines, formulated as Iscoms, or oil-in-water (o/w) emulsions, were administered parenterally to mice and evaluated for immunogenicity and protective efficacy. Both formulations enhanced both primary and secondary serum antibody responses. The magnitude of these responses with o/w emulsions was further enhanced by the addition of the non-ionic block copolymer L121 in the emulsion. Four weeks after primary immunization, mice were challenged by exposure to an aerosol containing infectious virus. Resistance to challenge in terms of survival rate and weight change correlated well with serum antibody titre for all formulations. Two major differences were observed between the adjuvant formulations. Iscom vaccines, formulated with Quil-A or the less toxic Quillaia saponin preparation Iscoprep 703, induced specific cytotoxic T-lymphocyte responses, whereas the o/w-based vaccines did not. In addition, dose-site reactivity studies in sheep showed that Iscom vaccines were less reactive than o/w-based vaccines, the degree of reactivity of the latter increasing sharply with increasing L121 concentration. On the basis of these studies, Iscoms were chosen for development as a potential adjuvant for human influenza vaccines.     

Induction of protective host immunity to carcinoembryonic antigen (CEA), a self-antigen in CEA transgenic mice, by immunizing with a recombinant vaccinia-CEA virus

Human carcinoembryonic antigen (CEA) is a well-characterized oncofetal glycoprotein whose overexpression by human carcinomas has been a target for cancer immunotherapy. Transgenic mice that express CEA as a self-antigen with a tissue distribution similar to that of humans have been developed. This study investigates: (a) the responsiveness of the CEA transgenic (CEA.Tg) mice to endogenous CEA or CEA administered as a whole protein in adjuvant; and (b) whether the presentation of CEA as a recombinant vaccinia virus could generate CEA-specific host immunity. By and large, the CEA.Tg mice were unresponsive to CEA, as shown by the lack of detectable CEA-specific serum antibodies and the inability to prime an in vitro splenic T-cell response to CEA. Furthermore, the administration of whole CEA protein in adjuvant to CEA.Tg mice failed to elicit either anti-CEA IgG titers or CEA-specific T-cell responses. Only weak anti-CEA IgM antibody titers were found in those mice. In contrast, CEA.Tg mice immunized with recombinant vaccinia virus expressing CEA generated relatively strong anti-CEA IgG antibody titers and demonstrated evidence of immunoglobulin class switching. These mice also developed T(H)1-type CEA-specific CD4+ responses and CEA peptide-specific cytotoxicity. The ability to generate CEA-specific host immunity correlated with protection of the CEA.Tg mice against a challenge with CEA-expressing tumor cells. Protection against tumor growth was accomplished with no apparent immune response directed at CEA-positive normal tissue. The results demonstrate the ability to generate an effective antitumor immune response to a tumor self-antigen by immunization with a recombinant vaccinia virus. CEA.Tg mice should be an excellent experimental model to study the effects of more aggressive immunization schemes directed at established tumors with the possible development of accompanying autoimmune responses involving normal tissues.