Reinhold Schirmbeck

MHC Class I–Restricted CTL Responses to Exogenous Antigens

A number of different modified exogenous protein antigens can efficiently prime CTL responses. The biological significance of this for priming of normal CTL responses is unknown. Processing of exogenous antigen for MHC-I presentation seems an attractive possibility to bypass the necessity that all foreign antigens have to be translated within the cytosol of professional APC in order to prime CTL responses. Many microbes have a select host cell specificity that does not include APC. They kill their host cells or severely disturb the sophisticated cellular functions that are required in APC (84xNorkin, L.C. Clin. Microbiol. Rev. 1995; 8: 293–315PubMedSee all References, 45xHaywood, A.M. J. Virol. 1994; 68: 1–5PubMedSee all References). Another scenario for a virus-specific CTL response is thus that infected cells disintegrate (possibly as apoptotic bodies) and exogenous antigen from them is transferred to APC for further processing in different cellular compartments.There are many unresolved issues regarding processing of exogenous antigens for MHC-I presentation and the priming of CTL responses. First, which are the most important uptake mechanisms? Second, is there a special (MHC-II-like) compartment for “endosomal” processing, or do all APC “leak” antigen into the cytosol and thereby into the classical MHC-I processing pathway? Third, in a proposed endosomal-like compartment, which are the proteolytic enzymes involved in processing? Do they generate a spectrum of MHC-I-bound peptides distinct from those generated in the cytosolic pathway? Finally, how do MHC-I molecules gain access to the endosomal-like compartment, and how are trimeric peptide MHC-I–β2-microglobulin complexes transported to the cell surface and their membrane expression regulated?If exogenous antigens are instrumental in priming normal CTL responses, processing in noncytosolic, vesicular compartments, distinct from the endogenous classical class I processing pathway, might be important. If so, it may be relevant to reevaluate the endogenous/MHC-I and exogenous/MHC-II processing dogma and instead classify pathways in functional terms. In CTL activation, induction (by exogenous antigen) and tar- get cell recognition (by endogenous antigen) by class I–restricted peptide presentation would result from different processing pathways. This is an important issue in terms of vaccine development, as these should primarily be expressed in the most optimal processing compartment. If exogenous antigens are of major importance in generating CTL responses, vaccines based on live, replicating vectors (and nucleic acids) induce their protective effect in a nonviable form. In this transfer process, from the infected host cell to the professional APC cell, there might be a lesson to be learned in terms of formulations for direct uptake into APC cells. The potent clinical effect of the first human recombinant vaccine licensed for clinical use, which was based on self-assembly of the hepatitis B virus small surface antigen into 22 nm subviral particles, might be a “lesson of nature” in this context (Schirmbeck et al. 1996xSchirmbeck, R, Bohm, W, and Reimann, J. Intervirology, in press. 1996; See all ReferencesSchirmbeck et al. 1996).

Multiple Effects of Codon Usage Optimization on Expression and Immunogenicity of DNA Candidate Vaccines Encoding the Human Immunodeficiency Virus Type 1 Gag Protein

ABSTRACT We have analyzed the influence of codon usage modifications on the expression levels and immunogenicity of DNA vaccines, encoding the human immunodeficiency virus type 1 (HIV-1) group-specific antigen (Gag). In the presence of Rev, an expression vector containing the wild-type (wt) gag gene flanked by essentialcis-acting sites such as the 5′-untranslated region and 3′-Rev response element supported substantial Gag protein expression and secretion in human H1299 and monkey COS-7 cells. However, only weak Gag production was observed from the murine muscle cell line C2C12. In contrast, optimization of the Gag coding sequence to that of highly expressed mammalian genes (syngag) resulted in an obvious increase in the G+C content and a Rev-independent expression and secretion of Gag in all tested mammalian cell lines, including murine C2C12 muscle cells. Mice immunized intramuscularly with thesyngag plasmid showed Th1-driven humoral and cellular responses that were substantially higher than those obtained after injection of the Rev-dependent wild-type (wt) gag vector system. In contrast, intradermal immunization of both wtgag and syngag vector systems with the particle gun induced a Th2-biased antibody response and no cytotoxic T lymphocytes. Deletion analysis demonstrated that the CpG motifs generated within syngag by codon optimization do not contribute significantly to the high immunogenicity of thesyngag plasmid. Moreover, low doses of coadministered stimulatory phosphorothioate oligodeoxynucleotides (ODNs) had only a weak effect on antibody production, whereas at higher doses immunostimulatory and nonstimulatory ODNs showed a dose-dependent suppression of humoral responses. These results suggest that increased Gag expression, rather than modulation of CpG-driven vector immunity, is responsible for the enhanced immunogenicity of thesyngag DNA vaccine.

IL-12/IL-18-Dependent IFN-γ Release by Murine Dendritic Cells

Dendritic cells (DC) develop in GM-CSF-stimulated cultures from murine bone marrow progenitors in serum-free (or low serum) medium. CD11c+ myeloid DC from 7-day cultures stimulated with TNF-α, IFN-α, IFN-γ, or LPS up-regulated surface expression of CD40 and CD86 costimulator and MHC class II molecules, did not up-regulate the low “spontaneous” release of IL-18, and did not release IFN-γ. Stimulation of in vitro-generated DC with exogenous IL-12 and IL-18 (but not with IL-4 or LPS plus IL-18) induced IFN-γ expression and release in 15–20% of the DC (detectable by FACS analyses or ELISA). Endogenous IL-12 p70 produced by DC in response to ligation of CD40 stimulated IFN-γ release when exogenous IL-18 was supplied. In vivo-generated, splenic CD8α+ and CD8α− DC (from immunocompetent and immunodeficient H-2d and H-2b mice) cultured with IL-12 and IL-18 released IFN-γ. The presence of LPS during the stimulation of DC with IL-18 plus endogenous (CD40 ligation) or exogenous IL-12 did not affect their IFN-γ release. In contrast, splenic DC pretreated in vitro or in vivo by LPS strikingly down-regulated IFN-γ release in response to stimulation by IL-18 and (endogenous or exogenous) IL-12. Hence, DC are a source of early IFN-γ generated in response to a cascade of cytokine- and/or cell-derived signals that can be positively and negatively regulated.

NKT Cells Provide Help for Dendritic Cell-Dependent Priming of MHC Class I-Restricted CD8+ T Cells In Vivo

Dendritic cells (DC) are potent APCs for naive T cells in vivo. This is evident by inducing T cell responses through adoptive DC transfer. Priming specific CTL responses in vivo often requires “help”. We study alternative sources of help in DC-dependent priming of MHC class I-restricted CTL. Priming an anti-viral CTL response in naive B6 mice by adoptive transfer of antigenic peptide-pulsed DC required CD4+ T cell help. CTL priming was facilitated by providing MHC class II-dependent specific help. Furthermore, transfers of MHC class II-deficient pulsed DC into naive, normal hosts, or DC transfers into naive, CD4+ T cell-depleted hosts primed CTL inefficiently. Pretreatment of DC with immune-stimulating oligodeoxynucleotides rendered them more efficient for CD4+ T cell-independent priming of CTL. DC copresenting a Kb-binding antigenic peptide and the CD1d-binding glycolipid α-galactosyl-ceramide efficiently primed CTL in a class II-independent way. To obtain NKT cell-dependent help in CTL priming, the same DC had to present both the peptide and the glycolipid. CTL priming by adoptive DC transfer was largely NK cell-dependent. The requirement for NK cells was only partially overcome by recruiting NKT cell help into DC-dependent CTL priming. NKT cells thus are potent helper cells for DC-dependent CTL priming.

Comparison between hepatitis B surface antigen (HBsAg) particles derived from mammalian cells (CHO) and yeast cells (Hansenula polymorpha): Composition, structure and immunogenicity

The composition, structure and immunogenicity of hepatitis B surface antigen (HBsAg) particles derived from Chinese hamster ovary (CHO) cells and from cells of the yeast Hansenula polymorpha were compared. The particles were similar in size distribution (mean 20-33 nm), in shape (spherical), in gross composition (protein to lipid weight ratio of 60:40), and in types of lipids (phospholipids > > sterols = sterol esters = triacylglycerols). Differences related to genetic engineering and type of host cells were found in peptide and lipid compositions. CHO-HBsAg has three peptides: S, M and L, each in two forms of glycosylation, while the Hansenula-HBsAg has only the nonglycosylated S peptide. The electrical surface potential at the lipid/water interface of HBsAg derived from Hansenula is more negative than that of HBsAg derived from CHO, which was close to neutrality. Although the numbers of cysteine residues (all in the S peptides) are identical (14), 11 of them are free thiols in the CHO-HBsAg, compared with three to four in the Hansenula-HBsAg. The fact that 85% of the phospholipids are hydrolyzed by phospholipase C and that all the aminophospholipids react with trinitrobenzenesulfate suggests that the particles derived from both cell types are either leaky vesicles or have a lipoprotein-like structure. Subcutaneous injection into mice of fluorescein-isothiocyanate-labeled HBsAg particles from both sources resulted in their accumulation in the marginal sinus of lymph nodes. The humoral responses to subcutaneous injection into mice of CHO- and Hansenula-HBsAg were similar: however, the cytotoxic T lymphocyte response to CHO-HBsAg was lower.

Activating immunity in the liver. I. Liver dendritic cells (but not hepatocytes) are potent activators of IFN-gamma release by liver NKT cells.

A prominent subset of the hepatic innate immune system is α-galactosylceramide (αGalCer)-reactive, (CD4+ and CD4−CD8−) CD1d-restricted NKT cells. We investigated in C57BL/6 (B6) mice which hepatic cell type stimulates hepatic NKT cell activation. Surface expression of CD1d but not CD40, CD80, or CD86 costimulator molecules was detected in hepatocytes. Pulsed in vitro or in vivo with αGalCer, hepatocytes triggered IL-4 release by liver NKT cells but required exogenous IL-12 to trigger IFN-γ release by NKT cells. Liver dendritic cells (DC) isolated from nontreated mice showed low surface expression of MHC, CD1d, and CD40, CD80, or CD86 costimulator molecules that were strikingly up-regulated after αGalCer injection. Although liver CD11c+ DC displayed lower CD1d surface expression than hepatocytes, they were potent stimulators of IFN-γ and IL-4 release by liver NKT when pulsed with αGalCer in vitro or in vivo. Liver DC are thus potent stimulators of proinflammatory cytokine release by NKT cells, are activated themselves in the process of NKT cell activation, and express an activated phenotype after the NKT cell population is eliminated following αGalCer stimulation.

Alternative pathways for processing exogenous and endogenous antigens that can generate peptides for MHC class I-restricted presentation.

Summary: The concept of distinct endogenous and exogenous pathways for generating peptides for MHC‐I and MHC‐II‐restricted presemation to CD4+ or CD8+ T cells fits well with the bulk of experimental data. Nevertheless, evidence is emerging for alternative processing pathways that generate peptides for MHC‐I‐restricted presentation. Using a well characterized, particulate viral antigen of prominent medical importance (the hepatitis B surface antigen), we summarize our evidence that the efficient, endolysosomal processing of exogenous antigens can lead to peptide‐loaded MHC‐I molecules. In addition, we describe evidence for endolysosomal processing of mutant, stress protein‐bound, endogenous antigens that liberate peptides binding to (and presented by) MHC‐I molecules. The putative biological role of alternative processing of antigens generating cytotoxic T lymphocyte‐stimulating epitopes is discussed.

Priming Th1 Immunity to Viral Core Particles Is Facilitated by Trace Amounts of RNA Bound to Its Arginine-Rich Domain

Particulate hepatitis B core Ag (C protein) (HBcAg) and soluble hepatitis B precore Ag (E protein) (HBeAg) of the hepatitis B virus share >70% of their amino acid sequence and most T and B cell-defined epitopes. When injected at low doses into mice, HBcAg particles prime Th1 immunity while HBeAg protein primes Th2 immunity. HBcAg contains 5–20 ng RNA/μg protein while nucleotide binding to HBeAg is not detectable. Deletion of the C-terminal arginine-rich domain of HBcAg generates HBcAg-144 or HBcAg-149 particles (in which >98% of RNA binding is lost) that prime Th2-biased immunity. HBcAg particles, but not truncated HBcAg-144 or -149 particles stimulate IL-12 p70 release by dendritic cells and IFN-γ release by nonimmune spleen cells. The injection of HBeAg protein or HBcAg-149 particles into mice primes Th1 immunity only when high doses of RNA (i.e., 20–100 μg/mouse) are codelivered with the Ag. Particle-incorporated RNA has thus a 1000-fold higher potency as a Th1-inducing adjuvant than free RNA mixed to a protein Ag. Disrupting the particulate structure of HBcAg releases RNA and abolishes its Th1 immunity inducing potency. Using DNA vaccines delivered intradermally with the gene gun, inoculation of 1 μg HBcAg-encoding pCI/C plasmid DNA primes Th1 immunity while inoculation of 1 μg HBeAg-encoding pCI/E plasmid DNA or HBcAg-149-encoding pCI/C-149 plasmid DNA primes Th2 immunity. Expression data show eukaryotic RNA associated with HBcAg, but not HBeAg, expressed by the DNA vaccine. Hence, codelivery of an efficient, intrinsic adjuvant (i.e., nanogram amounts of prokaryotic or eukaryotic RNA bound to arginine-rich sequences) by HBcAg nucleocapsids facilitates priming of anti-viral Th1 immunity.

Contributions of host and symbiont pigments to the coloration of reef corals

For a variety of coral species, we have studied the molecular origin of their coloration to assess the contributions of host and symbiont pigments. For the corals Catalaphyllia jardinei and an orange‐emitting color morph of Lobophyllia hemprichii, the pigments belong to a particular class of green fluorescent protein‐like proteins that change their color from green to red upon irradiation with ∼400 nm light. The optical absorption and emission properties of these proteins were characterized in detail. Their spectra were found to be similar to those of phycoerythrin from cyanobacterial symbionts. To unambiguously determine the molecular origin of the coloration, we performed immunochemical studies using double diffusion in gel analysis on tissue extracts, including also a third coral species, Montastrea cavernosa, which allowed us to attribute the red fluorescent coloration to green‐to‐red photoconvertible fluorescent proteins. The red fluorescent proteins are localized mainly in the ectodermal tissue and contribute up to 7.0% of the total soluble cellular proteins in these species. Distinct spatial distributions of green and cyan fluorescent proteins were observed for the tissues of M. cavernosa. This observation may suggest that differently colored green fluorescent protein‐like proteins have different, specific functions. In addition to green fluorescent protein‐like proteins, the pigments of zooxanthellae have a strong effect on the visual appearance of the latter species.

Immunostimulatory CpG Motifs Trigger a T Helper-1 Immune Response to Human Immunodeficiency Virus Type-1 (HIV-1) gp160 Envelope Proteins

Abstract Bacterial DNA sequences containing unmethylated CpG motifs have recently been proposed to exhibit immunostimulatory effects on B-, T- and NK cells, leading to the induction of humoral and cell-mediated immune responses. In the present study we investigated the immunomodulatory effects of a CpG-containing oligodeoxynucleotide (CpG ODN) to the HIV-1 gp160 envelope (Env) protein in the BALB/c mouse model. Priming and boosting of mice with gp160 adsorbed to aluminium hydroxide (Alum) induced a typical T helper-2 (Th2)-dominated immune response with high titers of gp160-specific immunoglobulin (Ig)G1 isotypes but a weak IgG2a response. Specifically re-stimulated splenocytes from these mice predominantly secreted interleukin (IL)-5 but only minute amounts of interferon-γ (IFN-γ) upon specific re-stimulation. In contrast, a boost immunisation of gp160/Alum primed mice with a gp160/Alum/CpG combination resulted in a seven times higher production of IgG2a antibodies, without affecting the titers of IgG1 isotypes. Furthermore, approximately 10-fold increased levels of IFN-γ, but significantly reduced amounts of IL-5, were secreted from gp160-restimulated splenic cells. A further greater than 30-fold increase in the levels of specific IgG2a responses and a substantially elevated secretion of IFN-γ were observed when the mice received gp160/Alum/CpG combinations for priming and boost injections. Thus, CpG ODNs are useful as an adjuvant to induce a typical Th0/Th1 response to HIV gp160 proteins. However, despite the induction of a more Th1-like immune response, gp160/Alum/CpG combinations were not sufficient to prime an Env-specific cytotoxic T-cell (CTL) response.