Improving vaccine performance with adjuvants.

New vaccines are presently under development and in testing for the control of infectious diseases, including human immunodeficiency virus (HIV) and tuberculosis. Several of these vaccines are composed of synthetic, recombinant, or highly purified subunit antigens. Subunit vaccines are designed to include only the antigens required for protective immunization and to be safer than whole-inactivated or live-attenuated vaccines. However, the purity of the subunit antigens and the absence of the self-adjuvanting immunomodulatory components associated with attenuated or killed vaccines often result in weaker immunogenicity. Immunologic adjuvants are agents that enhance specific immune responses to vaccines. Formulation of vaccines with potent adjuvants is an attractive approach for improving the performance of vaccines composed of subunit antigens. Adjuvants have diverse mechanisms of action and should be selected for use on the basis of the route of administration and the type of immune response (antibody, cell-mediated, or mucosal immunity) that is desired for a particular vaccine.

Adjuvants in perspective.

Many vaccines currently under development and testing are composed of synthetic, recombinant, or highly purified subunit antigens. Vaccines composed of these subunit antigens are often considered to be safer than whole-inactivated or live-attenuated vaccines. However, vaccines containing purified subunit antigens are often less immunogenic than traditional vaccines. Immunological adjuvants are agents that enhance specific immune responses to vaccines. Formulation of vaccines with potent adjuvants is an attractive approach for enhancing immune responses to subunit antigens. Adjuvants have diverse mechanisms of action and should be selected for use based on the route of administration and the type of immune response (antibody, cell-mediated, or mucosal) desired for a particular vaccine. Adjuvant mechanisms of action include: (i) increasing the biological or immunological half-life of vaccine antigens; (ii) improving antigen delivery and presentation; and (iii) inducing the production of immunomodulatory cytokines. Through modulation of cytokine responses, adjuvant formulations can be designed that favour the development of Th1 (type 1) or Th2 (type 2) immune responses to vaccine antigens. Novel adjuvants are currently undergoing preclinical and clinical testing with experimental vaccines, including vaccines against HIV-1. Standardized preclinical adjuvant safety tests are also being developed.

Adsorption to aluminum hydroxide promotes the activity of IL-12 as an adjuvant for antibody as well as type 1 cytokine responses to HIV-1 gp120.

A series of protocols were tested to examine the adjuvant effects of IL-12 on humoral and type 1 cytokine responses elicited in mice by recombinant gp120 envelope protein from HIV-1. This Ag fails to induce detectable Ab responses when administered s.c. alone, but stimulates low Ab levels when combined with aluminum hydroxide (alum). Moreover, when i.p. injected rIL-12 was included in the immunization, no increase in Ab production was observed. Importantly, optimal gp120 Ab responses were achieved by immunizing mice s.c. with gp120 and rIL-12 simultaneously coadsorbed to alum. These animals displayed a highly polarized, type 1 cytokine profile, with the emergence of anti-gp120 Ig belonging to the IgG2 and IgG3 isotypes. In addition, a major increase occurred in Ab of the IgG1 subclass. The superior adjuvant activity of alum-adsorbed IL-12 compared with that of the free cytokine correlated with the prolonged detection of IFN-gamma in the sera of animals immunized using the former procedure. In related experiments, in vitro neutralization of IL-12 was shown to inhibit IFN-gamma production by spleen cells from mice immunized with gp120 plus alum, but not by splenocytes from mice primed in the presence of IL-12, suggesting that the latter protocol induces a stable type 1 phenotype. These studies demonstrate that presentation of IL-12 on alum enhances its immunomodulatory effects and establish a protocol for the use of the cytokine as an adjuvant for simultaneously promoting both humoral Ab and type 1 cytokine responses.

Nucleic acid vaccines.

The use of nucleic acid-based vaccines is a novel approach to immunization that elicits immune responses similar to those induced by live, attenuated vaccines. Administration of nucleic acid vaccines results in the endogenous generation of viral proteins with native conformation, glycosylation profiles, and other posttranslational modifications that mimic antigen produced during natural viral infection. Nucleic acid vaccines have been shown to elicit both antibody and cytotoxic T-lymphocyte responses to diverse protein antigens. Advantages of nucleic acid-based vaccines include the simplicity of the vector, the ease of delivery, the duration of expression, and, to date, the lack of evidence of integration. Further studies are needed to assess the feasibility, safety, and efficacy of this new and promising technology.

Immunologic adjuvants for modern vaccine formulations.

Optimization of the immunogenicity of many new-generation vaccine formulations, including combination vaccines, will require the use of immunologic adjuvants other than the aluminum compounds in today's licensed vaccines. The selection of adjuvants for use in vaccine formulation may be as critical as the choice of the vaccine antigens themselves in providing optimal efficacy for the target populations, vaccine compliance, and cost. Adjuvants have diverse mechanisms of action and must be selected for use based on the immune responses desired for a particular candidate vaccine. Recent advances in the number and variety of adjuvants available for clinical evaluation coupled with the increased understanding of their mechanisms of action encourage the inclusion of adjuvants as part of rational vaccine design. Finally, the proposed standardized methods to evaluate adjuvant safety should be implemented for human candidate vaccines formulated with novel adjuvants.

The role of adjuvants in retroviral vaccines.

The global HIV epidemic continues unchecked. Reports to the World Health Organization's Global Programme on AIDS indicate that more than 14 million persons have become infected with HIV and more than two million have died with AIDS. The spread of AIDS has generated a worldwide mandate for the development of safe and effective vaccines against HIV. Vaccines have been the most effective defense against other viral diseases such as polio and smallpox. However, the development of a vaccine against HIV-1 is a formidable task due to the variation of the virus, inadequate animal models of HIV disease, and the lack of correlates of protective immunity. Several candidate HIV vaccines are composed of synthetic, recombinant, or highly purified subunit antigens. Vaccines composed of subunit antigens generally are considered to be safer than traditional whole-killed or live-attenuated vaccines. However, purified subunit vaccines often are inherently less immunogenic than traditional vaccines. Immunologic adjuvants are agents that act generally to enhance specific immune responses to vaccine antigens. Formulation of experimental HIV vaccines with potent immunologic adjuvants is an attractive approach for amplifying and directing immune responses to highly purified antigens. Alum adjuvants, consisting of aluminum salts, first described in the 1920s, remain the only adjuvants in U.S.-licensed vaccine formulations. Novel adjuvants now undergoing preclinical and clinical testing with experimental subunit vaccine include detoxified lipid A, adjuvant emulsions, liposomes, biodegradable microspheres, muramyl peptides, and saponins. Adjuvants have been shown to elicit cytotoxic T-cell responses as well as antibody to subunit antigens.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of the immunosuppressive activity of CKS-17, a synthetic retroviral envelope peptide, by muramyl dipeptide.

CKS-17, a heptadecapeptide corresponding to a region highly conserved in retroviral transmembrane proteins is known to be immunosuppressive both in vitro and in vivo when conjugated to a carrier protein. Here we examined the effect of the synthetic adjuvant muramyl dipeptide (MDP) on the immunosuppressive properties of CKS-17-BSA in vitro. MDP was found to abrogate CKS-17-BSA-induced inhibition of both IgM plaque-forming cell responses and antitetanus toxin IgG secretion by BALB/c mouse spleen cells immunized in vivo and in vitro by sheep red blood cells and tetanus toxoid, respectively. In contrast, the CKS-17-BSA suppression of concanavalin A-induced splenocyte proliferation was not abrogated by MDP. The data suggest that muramyl peptides could be useful as immunoadjuvants for vaccines against retrovirus-associated immunosuppressive diseases.

Modulation of carrier-induced epitopic suppression by Bordetella pertussis components and muramyl peptide.

Synthetic antigens employed in experimental synthetic vaccines are generally small haptenic peptides. Therefore, effective immunization with these antigens usually requires the use of an immunogenic carrier. Tetanus toxoid has been proposed for use as a carrier in future synthetic vaccines due to its high immunogenicity and acceptance for human use. Previous studies employing standard hapten/carrier systems such as DNP/KLH have demonstrated, however, that an epitope-specific suppression occurs when mice previously primed with carrier are subsequently immunized with an haptenic epitope conjugated to the same carrier. These same studies have shown that Bordetella pertussis vaccine administered at the time of carrier priming abrogates epitopic suppression. In the present investigation, epitopic suppression was studied in a synthetic vaccine model employing tetanus toxoid as a carrier. Results from these studies indicated that mice primed with tetanus toxoid 1 month before immunization with a peptide-tetanus toxoid conjugate exhibited enhanced secondary anti-tetanus toxin responses but decreased anti-peptide responses. Furthermore, injection of pertussis vaccine or purified B. pertussis toxin or endotoxin at the time of carrier priming could block the establishment of epitopic suppression. Administration of B. pertussis components enhanced antibody responses to both the carrier and the synthetic peptides as compared with responses of control animals. In addition, administration of an adjuvant-active nonpyrogenic derivative of muramyl dipeptide. Murabutide, with carrier priming reduced epitopic suppression of anti-peptide responses. B. pertussis toxin or endotoxin administered to mice previously suppressed by carrier priming with the first injection of carrier-peptide conjugate overcame epitopic suppression with resultant titers of anti-peptide antibody equal to or greater than nonsuppressed controls. These results suggest that the use of adjuvants with future synthetic vaccines may contribute the additional advantage of overcoming epitopic suppression, thus permitting the use of common, well-tolerated carrier systems such as tetanus toxoid in synthetic vaccine preparations.

Epitopic suppression in synthetic vaccine models: analysis of the effector mechanisms.

The induction of an immune response against synthetic peptides usually requires the use of an immunogenic carrier. The use of tetanus toxoid (TT) has been proposed for this purpose as it is highly immunogenic and has been used extensively in humans. Previous studies have demonstrated that an epitope-specific suppression of IgG antibody responses occurs when mice previously primed with TT are subsequently immunized with SODP, a haptenic epitope linked to TT. In the present investigation, we characterized the effector populations which regulate anti-SODP antibody responses in TT/TT-SODP immunized mice. In vitro studies showed that epitopic suppression did not arise due to nonspecific suppressor phenomena. Coculture experiments demonstrated that epitopic suppression was partially mediated by suppressor T cells which specifically inhibited the anti-hapten but not the anti-carrier antibody response. The majority of these T cells were shown to possess the Lyt-2+ phenotype. Apart from the T suppressor population we demonstrated a deficiency at the B-cell level which contributed to the total suppressive effect. Epitopic suppression, therefore, resulted from the effects of dual specific suppressor mechanisms.

Synthetic immunomodulators and synthetic vaccines.

Efforts have been made for several years to obtain well-defined, nontoxic adjuvants and antigens which could be used for human vaccination and immunostimulation. Among synthetic adjuvants, MDP represents one of the most studied family of compounds. This molecule is the minimal active structure of whole Mycobacteria and has been shown to be endowed with numerous biological activities. MDP is adjuvant active, increases nonspecific resistance against infectious challenges and, under certain conditions, increases resistance against tumor grafts. Moreover, MDP has other pharmacological properties such as pyrogenic and somnogenic activities. Several hundred MDP derivatives have been synthetized and some of the biological activities have been dissociated. One MDP derivative presently under clinical trials has been shown to be adjuvant active but is devoid of pyrogenicity. The mechanisms of activity of these MDP and derivative molecules will be discussed. More recently, synthetic antigens which are copies of natural epitopes, have been shown to induce protective antibodies against bacterial or viral pathogens. These synthetic antigens conjugated to synthetic carriers or to synthetic adjuvants such as MDP, may permit the preparation of totally synthetic vaccines.

Immune suppression and induction of gamma interferon by pertussis toxin.

It has been suggested that pertussis toxin is a virulence factor of Bordetella pertussis. Although extracts enriched in pertussis toxin activity have been reported to enhance immune responsiveness, other studies have demonstrated a suppressive ability, suggesting that the toxin may contribute to the virulence of B. pertussis through mechanisms involving immune suppression. We report that purified pertussis toxin suppressed the in vitro immunoglobulin M antibody response of mouse splenocytes to sheep erythrocytes. At submitogenic doses, the toxin also suppressed [3H]thymidine incorporation by splenocytes, suggesting that it interfered with antibody formation by inhibiting lymphocyte proliferation. Antiviral activity was detected in culture supernatants obtained from pertussis toxin-suppressed splenocyte cultures by using a cytopathic effect inhibition assay. This antiviral activity was virus nonspecific, sensitive to pH 2.0 treatment, stable to heating at 56 degrees C, and neutralized by anti-gamma interferon antiserum. Finally, the fractionation of splenocytes by anti-immunoglobulin panning techniques suggested that Lyt2+ lymphocytes proliferated in response to pertussis toxin and produced interferon. Our results suggest that pertussis toxin may contribute to the virulence of B. pertussis through stimulation of Lyt2+ lymphocytes, resulting in the induction of gamma interferon and the subsequent inhibition of the primary antibody response.

Cryptococcal capsular polysaccharide-induced modulation of murine immune responses.

Cryptococcus neoformans, an opportunistic fungal pathogen, often causes serious and life-threatening infections in immunocompromised hosts as well as in normal individuals. In the present study, purified cryptococcal capsular polysaccharide antigen was examined for its effect on several parameters of immune response and its ability to induce immune response to itself. Injection of the antigen into mice resulted in a dose-related specific antibody response which was detected at the individual antibody plaque-forming-cell level by a hemolytic assay in gel. Relatively low doses of cryptococci induced a maximal response, whereas higher doses resulted in a markedly depressed response. The antibody response to the cryptococcal capsular polysaccharide antigen appeared to be T cell independent and regulated by suppressor T cells, since mice injected with antilymphocyte serum or antithymocyte serum showed specific antibody responses to the antigen that were higher than those of untreated mice. It also markedly affected the in vitro mixed-lymphocyte reaction when added to cultures of mouse spleen cells being challenged in vitro with mitomycin C-treated allogeneic cells. The lower doses stimulated the response, whereas higher doses suppressed it. The macrophage response to yeast cells but not opsonized sheep erythrocytes was also modulated by the cryptococcal antigen.

Detection of antibodies to Legionella pneumophila in immune guinea pig serum by solid-phase immunofluorescence.

A semiautomated solid-phase immunofluorescence apparatus (FIAX; International Diagnostic Technology, Santa Clara, Calif.) was utilized to develop a rapid method for detection of antibody to Legionella pneumophila. The sera from guinea pigs immunized with a mixture of killed L. pneumophila and Freund complete adjuvant displayed markedly enhanced antibody activity as measured by FIAX when compared with that obtained from adjuvant-injected or unimmunized animals. A correlation was observed between FIAX net fluorescence units and microagglutination titers of serum samples obtained from immunized animals. Within-run and between-run coefficients of variation performed on selected immune serum samples were low. These results demonstrated that the FIAX method could readily and reproducibly detect Legionella-specific antibodies in the sera of actively immunized animals and suggest the possibility of a broader application of FIAX in the serological detection of exposure to L. pneumophila antigen.