Will adjuvants be needed for vaccines of the future?

Adjuvants improve the uptake of antigens by the immune system, and stimulate antigen-presenting cells (APC) to express "danger signals" such as the secretion of cytokines. The physical changes in antigen distribution can also be brought about by DNA vaccines, which provide persistent antigenic stimulation, access to the endogenous antigen processing pathway and, with the appropriate mode of injection, targeting to APC; however the question of whether DNA vaccines induce danger signals is unresolved. This presentation reviews the particular features of the adjuvant action of Al(OH)3, muramyl dipeptides and saponins, and the danger signals they induce in APC to ascertain whether they provide clues as to how DNA vaccines might be improved. Three conclusions are drawn: (i) adjuvants differ in the relative efficacy with which they stimulate Th1 and Th2 cells; (ii) IL-1 is the only identified common danger signal induced by the three adjuvants; (iii) in the case of both muramyl dipeptides and saponins there are toxic and nontoxic analogues, and the adjuvant activity can be separated from the toxicity. The basis of the difference between the toxic and non-toxic analogues is not clear.

Role of the genetic background of rats in infection by HTLV-I and HTLV-II and in the development of associated diseases.

Three aspects of the rat model of HTLV-I/II infection were investigated. (i) The efficacy of HTLV-I-transformed rat cell lines in infecting different strains of rats: WKY and Lewis HTLV-I-transformed cell lines were injected into adult WKY, Lewis and Brown Norway rats, representing syngeneic and allogeneic combinations. The HTLV-I provirus was not detected in peripheral-blood mononuclear cells (PBMC) from these rats 18 weeks after inoculation, showing that HTLV-I-transformed rat cells are not suitable for virus challenge in vaccination experiments. Rats inoculated with Lewis HTLV-I-transformed cells produced an antibody response to HTLV-I, which was higher in allogeneic (WKY and Brown Norway) than in syngeneic rats. (ii) The susceptibility of rats to HTLV-II infection: After human HTLV-II-producing cells (MO) were injected into adult WKY rats, the HTLV-II provirus was detected in PBMC 12 weeks later. Sequencing of a portion of this provirus confirmed its identity with the HTLV-II from MO cells. (iii) The role of MHC haplotype in susceptibility to neurological disease in rats inoculated as newborns with HTLV-I: The hypothesis that the RT-Ik haplotype confers susceptibility was tested by inoculating newborn OKA (RT-Ik), WKY (RT-Il), Lewis (RT-Il) and Fischer 344 (RT-I lvl) rats with human HTLV-I-producing cells (MT-2). Eighteen months later, only the WKY rats showed histological abnormality of the spinal cord, without clinical paralysis. Fischer 344 rats developed cutaneous tumors and OKA rats mammary tumors. The HTLV-I provirus was not detected in these tumors.

HTLV-I infection in squirrel monkeys (Saïmiri sciureus) using autologous, homologous, or heterologous HTLV-I-transformed cell lines.

Peripheral blood mononuclear cells (PBMC) from three adult male squirrel monkeys (Saïmiri sciureus) were transformed by human T-cell leukemia/lymphoma virus type I (HTLV-I) by cocultivation with lethally irradiated human MT-2 cells. Three permanent monkey T-cell lines producing HTLV-I were obtained and characterized. Six weeks after inoculation seroconversion was observed in three of three monkeys inoculated with autologous transformed T cells and in two of three monkeys receiving homologous cells. Proviral DNA was detected in their PBMC at various times after inoculation, with the highest proviral load and antibody titers being found in monkeys infected with homologous cells. Monkeys inoculated with heterologous MT-2 cells did not seroconvert, and HTLV-I provirus was detected only transiently in their PBMC. To determine whether in vitro and in vivo HTLV-I infection of squirrel monkey cells led to a selection of monkey-adapted viral mutants, comparative sequencing of the proviral gp21 env between ex vivo monkey HTLV-I-infected PBMC, the inoculum, and MT-2 cells was done and no significant differences were detected. The squirrel monkey, which is naturally free of simian T-cell leukemia/ lymphoma virus, thus appears to be a suitable model for evaluating HTLV-I candidate vaccines and for studying the pathogenesis of HTLV-I.

Expression and immunogenicity in rats of recombinant adenovirus 5 DNA plasmids and vaccinia virus containing the HTLV-I env gene.

The complete human T-cell leukemia virus type I (HTLV-I) env gene was inserted into an expression cassette containing the adenovirus 5 major late promoter (Ad5-MLP). Recombinant Ad5-HTLV-I-env was obtained by homologous recombination in 293 cells simultaneously transfected by the expression cassette and the genomic DNA of Ad5. In vitro expression of the HTLV-I-env gene in the recombinant vector was detected by immunofluorescence and Western blotting. Functional expression of HTLV-I-env was confirmed by syncitium formation specifically in HeLa cells infected with Ad5-HTLV-I-env. Two immunization regimens against HTLV-I were tested in WKY and Fischer F-344 rats. The first involved WKY rats primed with Ad5-HTLV-I-env or naked DNA plasmids containing the HTLV-I-env gene and boosted with Ad5 containing the HTLV-I-env gp46 gene or with baculovirus-derived recombinant gp46. No antibody against HTLV-I was detected, while HTLV-I-specific cytotoxic T lymphocytes were recovered from all immunized groups but not from controls. The second approach involved Fischer F-344 rats primed and boosted with recombinant vaccinia virus containing the HTLV-I-env gene. Such rats developed antibodies against the HTLV-I env gp21 and gp46 (non-neutralizing). After challenge with human HTLV-I-producing cells (MT-2), both immunization regimens were found to induce partial protection.

Infection of rats with human T-cell leukemia virus type-I: susceptibility of inbred strains, antibody response and provirus location.

The susceptibilities of different strains of inbred rats to infection with the human T-cell leukemia virus (HTLV-I) after inoculation of human HTLV-I producer cell lines were compared. The Fisher F344 and Brown Norway strains developed the highest antibody response to HTLV-I, while the Lewis and BB strains were low responders. Antibodies against the HTLV-I gag proteins, and env gp21 but not env gp46, were detected in Western blots with sera from HTLV-I-infected Fischer F344 and Brown Norway rats. These sera were inactive in an in vitro syncytium-formation inhibition test. The HTLV-I provirus was detected by polymerase chain reaction in all Fischer F344, and some Lewis and Brown Norway rats, but not in the BB, which lack CD8+ T lymphocytes. The most frequent locations of the HTLV-I provirus in the Fischer F344, Lewis and Brown Norway rats at 12 weeks after infection were the peripheral blood mononuclear cells (PBMC) and spinal cord. In a second experiment in Brown Norway rats, the provirus was again detected in the PBMC of rats at 12 weeks, but not at 22 weeks, and among the other organs tested at 22 weeks the sympathetic nerve ganglia were positive. It is concluded that HTLV-I infection occurs in adult rats, but is suppressed with time.

Human T cell lymphotropic virus: necessity for and feasibility of a vaccine.

Human T cell lymphotropic virus types I and II (HTLV-I/II) are endemic in certain areas of the world. They cause two life-threatening diseases, adult T cell leukaemia/lymphoma and tropical spastic paraparesis. A vaccine is needed because in developing countries there are no other feasible preventive interventions against these diseases and in Western countries intravenous drug users at high risk for HTLV-I and HTLV-II infections and the health workers in contact with such populations must be protected. We have developed a rat model in which we observed variations of susceptibility to viral infection between inbred strains, the most susceptible being the Fischer F344, and the possibility of viral latency in the nervous system. We have prepared a recombinant adenovirus vector that expresses the HTLV-I envelope glycoprotein env in HeLa cells. A target human population in French Guyana, in which the prevalence rate reaches 5.6% in one ethnic group (Bonis), has been identified for possible intervention.

Human T cell lymphotropic virus: necessity for and feasibility of a vaccine

Human T cell lymphotropic virus types I and II (HTLV-I/II) are endemic in certain areas of the world. The cause two life-threatening diseases, adult T cell leukaemia/lymphoma and tropical spastic paraparesis. A vaccine is needed because in developing countries there are no other feasible preventive interventions against these diseases and in Western countries intravenous drug users at high risk for HTLV-I and HTLV-II infections and the health workers in contact with such populations must be protected. We have developed a rat model in which we observed variations of susceptibility to viral infection between inbred strains, the most susceptible being Fischer F344, and the possibility of viral latency in the nervous system. We have prepared a recombinant adenovirus vector that expresses the HTLV-I envelope glycoprotein env in HeLa cells. A target human population in French Guyana, in which the prevalence rate reaches 5.6 percent in one ethnic group (Bonis), has been identified for possible intervention (AU)

An HTLV-I vaccine: why, how, for whom?

Endemic infection with the human T cell leukemia/lymphoma viruses I and II (HTLV-I/II) is now recognized to be worldwide, and is becoming epidemic among intravenous drug abusers (IVDAs) in the United States and Europe. The number of people around the world infected with HTLV-I can be estimated as between 10 and 20 million (Table 1). HTLV-I causes a rapidly progressing adult T cell leukemia/lymphoma (ATLL), and an incurable progressive neuromyelopathy named tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM), as well as a number of less well-studied syndromes. There is evidence that coinfection with HTLV-I or -II accelerates progression to AIDS. The cumulative lifetime risk of developing ATLL or TSP/HAM is around 5%, which, in terms of the induction of serious diseases, places HTLV-I in the same category of viruses for which efficient vaccines are made and used. Furthermore, there are factors favoring the feasibility of a vaccine against HTLV-I, in that the virus displays relatively low antigenic variability, natural immunity occurs in humans, and experimental vaccination with the envelope (Env) antigen is successful in animal models. A vaccine against HTLV-I would be of significant public health value in the fields of oncology, neurology, and AIDS, and it would serve as a pathfinder for a vaccine against HIV.

The control of the antibody isotype response to recombinant human immunodeficiency virus gp120 antigen by adjuvants.

Both saponin and muramyl dipeptide (MDP) formulated with a squalane-in-water emulsion of large particle size prepared with a vortex mixer were superior to Al(OH)3 as adjuvants for HIV gp120 in mice. All the adjuvants induced IgG1 antibody, but saponin elicited the highest titers of IgG2a. The secretion of interleukin-5 (IL-5) and interferon gamma (IFN gamma) by lymph node cells cultured in vitro with gp120 was studied. All the cultures secreted IL-5, but only those from saponin-immunized mice produced IFN gamma, suggesting that saponin is capable of activating both the Th1 and TH2 T-cell subsets. The titers of neutralizing antibodies were low with both MDP and saponin, and they occurred in mice which were also positive for antibodies against a V3 loop peptide. Glucosaminylmuramyl dipeptide (GMDP) which is less pyrogenic than MDP and a nonpyrogenic analog GMDPA, displayed equivalent adjuvant activity to MDP. The level and isotype composition of antibodies induced by GMDP in combination with squalane emulsions depended on the dimension of the emulsion particles. With a large (2500 nm) particle size the response was confined to IgG1 in Balb/c mice, but when this was reduced to 150 nm by sonication the antibody response was increased and relatively high levels of IgG2a appeared in some mice.

Adjuvanticity and ISCOM formation by structurally diverse saponins.

Adjuvant activity and immunostimulating complex (ISCOM) formation by a series of saponins and glycoalkaloids differing in the structures of their aglycones and sugar chains were examined. The only two saponins apart from Quillaia that were adjuvant-active were Gypsophila and Saponaria, which resemble Quillaia in that they contain saponins with branched sugar chains attached to positions 3 and 28 of the aglycone. Glycoalkaloids with a branched sugar chain lacked adjuvant activity. Saponaria saponins formed irregular ISCOM-like structures, and Gypsophila produced a sheet of joined pore-like structures. The alfalfa hederagenin saponin and Quinoa also formed pore-sheets, despite lacking adjuvanticity.

The processing of interleukin-1 beta studied with antibodies raised against synthetic peptides from the precursor N-terminal region.

The exact sequence of events during processing of human interleukin-1 beta (IL-1 beta) and the fate of the N-terminal region are unknown. We have used anti-peptide sera specific for the precursor and mature regions of IL-1 beta to study biosynthesis. These were raised against peptides corresponding to amino acids 1-15, 17-32, and 43-54 of the precursor and a peptide corresponding to the C-terminal 33 amino acids of mature human IL-1 beta. Antiserum to the mature region peptide immunoprecipitated the 35-kD precursor from cell lysates and 17-kD mature IL-1 beta and a 31-kD protein from the culture supernatants from radiolabeled human peripheral blood monocytes stimulated with lipopolysaccharide (LPS). Antisera to peptides from the precursor region also immunoprecipitated the 35-kD IL-1 beta precursor but not the 31-kD or 17-kD forms. Of the precursor-specific sera, only antiserum to amino acids 1-15 specifically recognized any other proteins; a peptide of 18 kD and a low molecular weight peptide, both of which accumulated in the medium. The 18-kD protein was not recognized by any of the other antisera and is unlikely to be the N-terminal region of the precursor removed during processing. Pulse-chase experiments indicated that the 31-kD protein could be a processing intermediate and also that it was itself an end product along with full-length precursor. Only 17-kD mature IL-1 beta had biological activity.

Ultrastructural localization of interleukin 1 in human peripheral blood monocytes; evidence for IL-1 beta in mitochondria.

The pathway of interleukin 1 (IL-1) secretion from the cell remains unclear. IL-1 beta is the major form produced by human monocytes, and is synthesized as a precursor of 35 kDa which is processed to the extracellular biologically active 17 kDa form. We have examined the intracellular localization of IL-1 beta in lipopolysaccharide (LPS)-stimulated human peripheral blood monocytes, by immunocytochemistry and immunoprecipitation of subcellular fractions. LPS treatment slightly damaged the cells. Unstimulated cells showed very little immunolabelling. In contrast, there was heavy immunolabelling on LPS stimulated cells. Immunolabelling occurred within the cytoplasm, nucleus and mitochondria. There was no immunolabelling on the membranous secretory organelles and the plasma membrane. Blebs of cytoplasm budding from the cell surface were immunolabeled, suggesting an alternative route of secretion of IL-1 beta from the cell. Immunoprecipitation studies confirmed these results.

Enhancement of bovine growth hormone activity by antibodies against growth hormone peptides.

The biological activity of growth hormones can be enhanced by complexing with monoclonal antibodies of appropriate specificity. In order to define the regions associated with the phenomenon, site-directed antisera to ovine GH (oGH) were prepared by vaccination of sheep with synthetic peptides. Peptides from six distinct regions of the oGH molecule raised antibodies which recognized the hormone in solid-phase radioimmunoassay; however, only one peptide elicited high-affinity antibody as determined by liquid-phase assay. This peptide, corresponding to amino acid sequence 35-53, resulted in circulating hormone antibody in the majority of vaccinated sheep. Immunoglobulin prepared from the serum of immunized animals produced an enhancement of the somatotrophic activity of exogenously administered GH in dwarf mice as determined by the incorporation of [35S]sulphate into costal cartilage. The identification of an antigenic peptide sequence from oGH/bovine GH which elicits enhancing antisera, raises the possibility of a growth-promotion vaccine.

3-Deazaadenosine--an inhibitor of interleukin 1 production by human peripheral blood monocytes.

3-Deazaadenosine (c3Ado) has been reported to have properties of an immunosuppressive and anti-inflammatory agent. This study was designed to investigate whether c3Ado might exert anti-inflammatory activities through inhibiting Interleukin 1 (IL-1). c3Ado was found to be a potent inhibitor of IL-1 production by LPS stimulated human peripheral blood monocytes and acted at the level of synthesis rather than secretion. c3Ado also had direct effects on IL-1 biological activity in two separate assays; thymocyte proliferation and induction of prostaglandin release. Further experiments indicated that c3Ado also inhibited growth factor dependent proliferation driven by both Interleukin-2 and Interleukin-3 as well as the proliferation of a number of non-growth factor dependent cells. However, short term exposure to c3Ado resulted in no inhibition of 3H-thymidine incorporation by cells but a significant inhibition of 3H-uridine uptake into TCA precipitable material. These results suggest that c3Ado is a selective inhibitor of RNA synthesis and inhibits IL-1 production and activity by blocking new messenger RNA production induced by LPS or IL-1. General inhibition of RNA synthesis would also account for its anti-proliferative activity.

Adjuvants for anti-parasite vaccines.

To date the most successful human vaccines use attenuated living pathogens, but the advent of techniques in genetic engineering has meant that pure antigen can be provided in quantity. This has allowed the development of combined vaccines that use only the parasite antigens that convey protective immunity. However, isolated antigens lose immunogenicity so to regain potency, living attenuated carriers like Vaccinia or Salmonella can be used. To avoid the attendant drawbacks of carriers as immunopotentiating agents, adjuvants are under investigation as alternatives for use in vaccines against parasitic infections. In this review, Robert Bomford describes the adjuvants currently being examined for use in vaccines for both protozoan and helminth infections including Leishmania, malaria and Schistosoma. He also points out the drawbacks of using adjuvants and the dilemma of needing to stimulate cell'-mediated immunity while avoiding the immunopathological consequences of doing so.

Antibodies to interleukin-1 raised with synthetic peptides: identification of external sites and analysis of interleukin-1 synthesis in stimulated human peripheral blood monocytes.

Rabbit antibodies against peptides corresponding to amino acids 1-18, 45-65 and 71-90 of mature human interleukin-1 beta (IL-1 beta) precipitated 125I-labelled IL-1 beta, showing that these sites are accessible to antibody and located externally. Immunoprecipitation of 35S-methionine-labelled LPS-stimulated human peripheral blood monocytes followed by SDS-PAGE revealed the expected major bands of molecular weights 35,000 and 17,500. The 35,000 protein was found in the cell lysate and extracellularly in the medium, but the 17,500 protein was exclusively in the medium. A previously undescribed 31,000 band was also detected in the medium. These results are most simply explained by the hypothesis that the 35,000 IL-1 beta precursor is released from the cell and processed extracellularly to the 17,500 mature form. The 31,000 molecule may represent a processing intermediate.

Enhancement of bovine growth hormone activity in vivo by monoclonal antibodies.

Monoclonal antibodies (MAbs) prepared against ovine growth hormone (oGH) and recognizing several distinct or related specificities on bovine growth hormone (bGH), have been shown to strikingly enhance the growth promoting properties of the hormone in vivo as determined by 35SO2-4 incorporation in cartilage. The phenomenon is dependent on both hormone and antibody dose and is saturable in the presence of excess antibody. Competition binding analysis between pairs of antibodies has shown that they define distinct antigenic regions on bGH of which one is represented by four MAbs. The most potent growth enhancement was associated with a group of three MAbs (OA11, OA12 and OA13) binding to topographically closely related sites. Another MAb (OA14) with a specificity similar to OA11 and OA13 as defined by competition assay failed to enhance bGH activity. Two antibodies binding to a further two sites (OA15 and OA16) demonstrated modest growth enhancement activity when in complex with bGH, whereas the binding of another antibody (OA17) did not significantly affect hormonal activity. Univalent antibody fragments (Fab) derived from MAb OA11 were equi-potent to the bivalent form of the antibody in the enhancement of bGH activity. Determination of the effects of the different MAbs on the binding of 125I-bGH to liver microsome receptors revealed substantial increase in specific binding (3.5-fold) and is associated with some growth enhancing MAbs. However, not all growth enhancing MAbs increased receptor binding and in the case of one, OA16, clear cut inhibition of receptor binding was observed. Tentative conclusions have been drawn on the possible underlying mechanisms of the MAb mediated enhancement phenomenon.