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Liver-stage antigen 1 (LSA1) is expressed by Plasmodium falciparum only during the intrahepatic cell stage of the parasite's development. Immunoepidemiological studies in regions where malaria is endemic suggested an association between the level of LSA1-specific humoral and cell-mediated immune responses and susceptibility to clinical malaria. A recombinant LSA1 protein, FMP011, has been manufactured as a preerythrocytic vaccine to induce an immune response that would have the effect of controlling parasitemia and disease in humans. To evaluate the immunogenicity of FMP011, we analyzed the immune response of three inbred strains of mice to antigen immunization using two different adjuvant formulations, AS01B and AS02A. We report here the ability of BALB/c and A/J mice, but not C57BL/6J mice, to mount FMP011-specific humoral (antibody titer) and cellular (gamma interferon [IFN-gamma] production) responses following immunization with FMP011 formulated in AS01B or AS02A. Immunization of BALB/c and A/J mice with FMP011/AS01B induced more antigen-specific IFN-gamma-producing splenocytes than immunization with FMP011/AS02A. A slightly higher titer of antibody was induced using AS02A than AS01B in both strains. C57BL/6J mice did not respond with any detectable FMP011-specific IFN-gamma splenocytes or antibody when immunized with FMP011 in AS01B or AS02A. Intracellular staining of cells isolated from FMP011/AS01B-immunized BALB/c mice indicated that CD4(+) cells, but not CD8(+) cells, were the main IFN-gamma-producing splenocyte. However, inclusion of blocking anti-CD4(+) antibody during the in vitro restimulation ELISpot analysis failed to completely abolish IFN-gamma production, indicating that while CD4(+) T cells were the major source of IFN-gamma, other cell types also were involved.

The safety, immunogenicity, and protective efficacy of two non-replicating antigen-based vaccines and one live-attenuated virus (LAV) vaccine for dengue type-2 (dengue-2) virus were evaluated in the rhesus macaque model. The non-replicating vaccines consisted of whole, purified inactivated virus (PIV) and a recombinant subunit protein containing the amino-(N)-terminal 80% of envelope protein (r80E), each formulated with one of five different adjuvants. Each formulation was administered to three animals on a 0, 3-month schedule. Following the primary immunizations, 37 of 39 animals demonstrated dengue-2 virus neutralizing antibodies. After the booster immunizations all animals had dengue neutralizing antibodies with peak titers ranging from 1:100 to 1:9700. The highest neutralizing antibody titers were observed in the groups that received r80E antigen formulated with AS04, AS05, or AS08 adjuvant, and PIV formulated with AS05 or AS08 adjuvant. These newer adjuvants are based on alum, fraction QS-21 of saponin, and monophosphoryl lipid A (MPL). Protection was evaluated by dengue-2 virus challenge 2 months after the booster by the measurement of circulating virus (viremia) and post-challenge immune responses. Several groups exhibited nearly complete protection against viremia by bioassay, although there was evidence for challenge virus replication by Taqmantrade mark and immunological assays. None of the vaccines conferred sterile immunity.

In humans, sterile immunity against malaria can be consistently induced through exposure to the bites of thousands of irradiated infected mosquitoes. The same level of protection has yet to be achieved using subunit vaccines. Recent studies have indicated an essential function for intrahepatic parasites, the stage after the mosquito bite, and thus for antigens expressed during this stage. We report here the identification of liver-stage antigen 3, which is expressed both in the mosquito and liver-stage parasites. This Plasmodium falciparum 200-kilodalton protein is highly conserved, and showed promising antigenic and immunogenic properties. In chimpanzees (Pan troglodytes), the primates most closely related to humans and that share a similar susceptibility to P. falciparum liver-stage infection, immunization with LSA-3 induced protection against successive heterologous challenges with large numbers of P. falciparum sporozoites.

Interleukin-HP1 (HP1) is a murine T cell-derived lymphokine, originally described as a growth factor for B cell hybridomas and plasmacytomas, that was recently shown to stimulate growth and differentiation of normal B and T lymphocytes. Here, we describe a cDNA for HP1 that was isolated from a library prepared using mRNA of a murine helper T cell clone activated with a clonotypic antibody. The cDNA, which hybridizes with a mRNA of approximately 1300 bp, encodes a polypeptide consisting of 211 amino acids with a typical signal sequence of 24 residues followed by 187 amino acids, which form the mature protein (Mr = 21,710). No N-glycosylation site but several potential O-glycosylation sites were identified in the predicted sequence. Comparison of the cDNA sequence of HP1 with that of human interleukin 6 disclosed a homology of 65% at the DNA level and of 42% at the protein level with a maximum of 57% for the segment spanning residues 42-102 of mature HP1. Considering the functional homology that was previously established between these two proteins, we therefore propose that HP1 be renamed murine interleukin 6.

We have recently purified and partially sequenced a new T cell-derived lymphokine with growth factor activity for B cell hybridomas and plasmacytomas, which we named interleukin HP1 (HP1). Here we show that, in response to viral infection or after treatment with poly(rI).poly(rC), L cells produce a factor that is capable of supporting the in vitro growth and survival of HP1-dependent cell lines. Serologic and structural evidence is presented in favor of the identity between the fibroblast factor and HP1, demonstrating that non-T cells can make HP1-related molecules.

A T-cell-derived lymphokine was identified by its ability to support the growth of a subset of B-cell hybridomas. Hybrids that failed to survive in the absence of this molecule represented a major proportion of rat-mouse hybridomas but were very rare among mouse-mouse B-cell hybrids. Stable factor-dependent B-cell hybridomas were used to monitor the purification of the growth factor from the supernatant of a clonotypically stimulated mouse helper T-cell clone. Sequential fractionation using gel filtration, anion-exchange chromatography, and reversed-phase HPLC resolved the factor from other B-cell growth factors and yielded a single-chain protein characterized by a major charge (pI = 5-7) and molecular mass (22- to 29-kDa) heterogeneity, probably due to variations in glycosylation. The NH2-terminal amino acid sequence of this protein, which is active on B-cell hybridomas in the 0.1 pM range, showed no significant homology with that of known lymphokines. Because the purified factor also supported the growth and survival in vitro of murine plasmacytomas (to be published elsewhere), it was provisionally designated interleukin-HP1 (where H stands for hybridoma and P stands for plasmacytoma).

Serum of lipopolysaccharide (LPS)-treated mice was found to support the growth of interleukin-HP1 (HP1)-dependent hybridoma and plasmacytoma cell lines. This growth-promoting activity, which was undetectable in normal serum, rose more than 1000-fold within 2 h after i.v. injection of the toxin and disappeared in less than 1 day. It could be traced to a single component, which behaved like HP1 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse phase high performance liquid chromatography, and was completely inhibited by a rabbit anti-HP1 antiserum. The LPS-induced factor was apparently not of T cell origin, as indicated by the strong activity found in the serum of LPS-treated nude mice. In vitro, LPS also considerably enhanced the production by macrophages of a factor with similar characteristics.

The human "26-kd protein' is a secreted glycoprotein expressed, for example, in (blood) leukocytes, in epithelial cells treated with various inducers, but most strongly in interleukin-1 (IL-1)-treated fibroblasts. After finding it has antiviral and 2-5A synthetase-inducing activity, one group of authors called this protein IFN-beta 2. However, recently the full-length 26-kd cDNA sequence was shown to be identical with that of a B-cell-differentiating lymphokine called BSF-2, and another report suggested that the 26-kd protein could support the growth of some transformed murine B cell lines. To define its biological activities, we expressed the recombinant 26-kd protein by translating in Xenopus laevis oocytes a pure, synthetic chimeric mRNA containing the 26-kd protein coding region surrounded by Xenopus laevis beta-globin untranslated regions. A similar construction, but containing the HuIFN-beta cDNA coding region, was used to produce HuIFN-beta by the same procedure. Both recombinant glycoproteins were secreted, glycosylated, and their amounts were measured by [35S]methionine incorporation by the oocyte. Here we show that the recombinant 26-kd protein exhibits a high growth factor activity when assayed on an IL-HP1-dependent murine B cell hybridoma (sp. act. approximately 2 X 10(8) U/mg) as well as a potent differentiating activity on human CESS cells (sp. act. approximately 5 X 10(7) U/mg). While rHuIFN-beta was inactive in the latter two assays, it had the expected antiviral activity of 1-5 X 10(8) U/mg. The parallel recombinant 26-kd protein preparations had no detectable antiviral activity (i.e. a maximal specific activity of 1-3 X 10(2) U/mg, if any). The 26-kd protein is thus clearly an interleukin, and considering the confusing nomenclature now in use, this factor may better be renamed "interleukin 6'.

Human fibroblast cultures, when stimulated with interleukin-1 (IL-1) produce a growth factor for B-cell hybridoma and plasmocytoma cell lines. The availability of both a fast-growing and high-producer cell line (MG-63 osteosarcoma cells) and of a highly sensitive and specific assay system for this hybridoma growth factor (HGF) allowed us to obtain analytically pure preparations. Crude HGF from MG-63 cells was processed through a five-step concentration and purification schedule. Sequential adsorption to controlled pore glass (CPG) beads, antibody affinity chromatography and gel filtration resulted in a 10,000-fold purification to a specific activity of 10(9) units/mg HGF. Electrophoretically pure HGF was obtained after additional purification by cation-exchange chromatography and reversed-phase HPLC. The purification procedure revealed two distinct biologically active HGF components. The amino-terminal sequence of one of the two components was determined and found to correspond to that already predicted from cDNA clones of a protein alternatively called 26-kDa protein, interferon-beta 2 (IFN-beta 2) or B-cell stimulating factor-2 (BSF-2). The first two designations (26-kDa protein and IFN-beta 2) refer to a postulated fibroblast secretory protein with so far no unambiguously defined function; the latter designation (BSF-2) refers to a T-cell product possessing differentiation stimulatory effect on B-cell lines. The reported results firmly establish that the protein is secreted by fibroblasts and reveal that it possesses B-cell growth stimulatory activity. The new designation interleukin-6 (IL-6) is proposed to resolve prescribing nomenclature confusion.

We have recently described the purification and NH2-terminal amino acid sequence of a T cell-derived hybridoma growth factor that was provisionally designated interleukin-HP1 (IL-HP1). Here we report that a T cell supernatant containing high titers of this hybridoma growth factor considerably facilitated the establishment of primary cultures of murine plasmacytomas. Most plasmacytoma cell lines derived from such cultures remained permanently dependent on IL-HP1-containing T cell supernatant for both survival and growth in vitro. These cell lines, however, retained their ability to form tumors in irradiated pristane-treated mice. Analytical fractionation of a T cell supernatant rich in IL-HP1 by either gel filtration, isoelectric focusing, or reversed-phase HPLC revealed the existence of only one plasmacytoma growth factor activity that strictly copurified with IL-HP1, strongly suggesting the identity of both factors. This conclusion was further supported by the finding that IL-HP1 purified to homogeneity supported the growth of both B cell hybridomas and plasmacytomas. For half-maximal growth, plasmacytomas, however, required a concentration of IL-HP1 of approximately 30 pM, which is approximately 200 times higher than that required by B cell hybridomas. A clear difference in the specificity of IL-HP1 and B cell stimulatory factor 1 (BSF-1) was demonstrated by the finding that IL-HP1-dependent plasmacytomas did not survive in the presence of BSF-1, whereas helper T cell lines that proliferated in the presence of BSF-1 failed to respond to IL-HP1.

The human 26-kDa glycoprotein (26K) is a cytokine produced by lymphoid as well as nonlymphoid cells. So far it is active as (a) a potent hybridoma and plasmacytoma growth factor on mouse cells, (b) a B cell differentiating factor on human cells, and (c) (for some authors) an interferon (IFN). Internally labeled recombinant human 26K, obtained by translation of mRNA in Xenopus oocytes, was used to investigate the presence of specific receptors for this new cytokine and to analyze its binding to responsive cells. The results indicate that (a) the 26K-responsive human lymphoblastoid CESS cells express about 1500 high-affinity (Kd = 30 pM) binding sites for this cytokine, (b) this binding is not competed for by interleukin (IL)1, IL2, tumor necrosis factor (TNF), IFN-alpha 2, IFN-beta or IFN-gamma, and (c) these 26K-binding sites are different from the classical type I (alpha-beta) IFN receptors by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Cultures of normal diploid fibroblasts and of a human osteosarcoma cell line (MG-63) are shown to be able to produce a factor which promotes the growth of B cell hybridomas (hybridoma growth factor, HGF). The induction is stimulated by treatment of the cells with interleukin 1 (IL 1) (alpha or beta) or polyriboinosinic-polyribocytidylic acid [poly(rI).poly(rC)]. Combined treatment with cycloheximide and actinomycin D also stimulates production and enhances production induced by IL 1 or poly(rI).poly(rC). Extremely small doses of IL 1 (0.1 units/ml) are active as inducer of HGF. Also, under optimal conditions the yield of HGF can attain as much as 10(4) units/ml. Tumor necrosis factor (TNF-alpha), which otherwise shares various properties with IL 1, is a weak inducer of HGF. Although there is a superficial resemblance between induction of HGF and that of interferon-beta, the two activities are serologically distinct and conditions for their induction are quite different. In fact, conditions for induction of HGF are indistinguishable from those described for the induction of the mRNA of the so-called 26-kDa protein (also known as interferon-beta 2). Finally, the HGF derived from IL 1- or poly(rI).poly(rC)-treated fibroblasts is serologically not distinguishable from that produced by mitogen-stimulated peripheral blood leukocytes.

Mouse plasmacytoma FLOPC21 was adapted to culture in the presence of a mouse Th cell supernatant. A stable factor-dependent cell line was derived from this culture and the factor responsible for its growth was identified as granulocyte-macrophage colony-stimulating factor.

A factor that promotes the growth of certain B cell hybridomas and of plasmacytomas is shown to be produced by normal human fibroblasts and by a line of human osteosarcoma cells (MG-63) after treatment with IL-1 or TNF. The hybridoma-plasmacytoma growth factor (HPGF) is identified with a 26 kD protein whose mRNA was previously shown to be induced in the same cells by the same inducers. First, poly(A)-rich RNA extracted from IL-1-treated cells could be enriched in HPGF-mRNA content by hybridization to 26 kD cDNA. Second, MG-63-derived HPGF purified to electrophoretic homogeneity was subjected to amino acid sequence analysis, whereby the NH2-terminal sequence was found to match the nucleotide sequence of a 26 kD cDNA clone.