Insect cells as a new substrate for vaccine production.

Vaccine antigens can be expressed in several expression systems, of which expression in eukaryotic cell lines is a well-known example. Whereas for most current applications cell substrates such as Vero, MRC-5 or chicken embryo fibroblasts are used, cell lines derived from insects present an alternative for the future production of vaccines. Since such substrates are not yet extensively used, knowledge about their feasibility and safety for vaccine antigen production is limited. In the current example, an insect-derived cell line Hi-5, used for the expression of the human papilloma virus L1 antigen, was extensively characterized, in particular for aspects of viral safety. A comprehensive QC testing programme, complemented with additional characterization tests relevant to the insect nature of the cell line, was developed. Current results indicate the satisfactory quality and safety of the Hi-5 cell line. The proposed testing programme could stand as an example for the release testing and further characterization which would be required for insect cell lines.

In vitro cellular immune responses to complex and newly defined recombinant antigens of Mycobacterium tuberculosis.

The immunological diagnosis and development of new antituberculosis vaccines require the characterization of Mycobacterium tuberculosis antigens inducing cell-mediated immune responses. In this study, we have tested peripheral blood mononuclear cells (PBMC) from tuberculosis (TB) patients (n = 43) and Bacille Calmette-Guérin (BCG)-vaccinated healthy subjects (n = 24) for in vitro cellular immune responses, as indicated by antigen-induced proliferation and interferon (IFN)-gamma secretion, in response to a panel of complex (culture filtrate and cell wall preparations) and single recombinant antigens (Mtb8.4, Mtb9.8, Mtb9.9, Mtb32A, Mtb39A, Mtb40, Mtb41 and Ag85B) of M. tuberculosis. The results of cellular responses showed that the majority (ranging from 70 to 98%) of TB patients and healthy donors responded to the complex antigens in antigen-induced proliferation and IFN-gamma secretion assays. However, when PBMC from the same groups of patients and healthy donors were tested with the recombinant antigens, TB patients showed strong recognition (>50% responders) of Mtb9.8 and Mtb39A in proliferation assays (median SI = 6.2 and 6.4, respectively) and of Mtb9.8, Mtb39A, Mtb40 and Ag85B in IFN-gamma assays (median delta IFN-gamma= 15.5, 10.8, 7.8 and 8.1 U/ml, respectively). BCG-vaccinated healthy donors showed weak (<30% responders) to moderate (31-50% responders) responses to all of the recombinant antigens in both assays. When PBMC of a subset of TB patients (n = 11) were tested for secretion of protective Th1 cytokines [IFN-gamma, tumour necrosis factor (TNF)-alpha and interleukin (IL)-12] and the immunosuppressive cytokine IL-10, the complex CF and CW antigens as well as the recombinant Mtb9.8, Mtb9.9, Mtb40 and Ag85B induced the secretion of both types of cytokines. On the other hand, Mtb41 induced only IL-10, while Mtb8.4, Mtb32Aand Mtb39A induced the secretion of one or more of Th1 cytokines, but not IL-10. In conclusion, the recombinant antigens inducing the secretion of Th1 cytokines could be useful as subunit vaccine candidates against TB.

Prospects for a better vaccine against tuberculosis.

There have been many new promising approaches to developing human vaccines against tuberculosis (TB). Advances in gene and antigen identification, availability of genome sequences, a greater understanding of immune mechanisms in resistance to TB, the development of adjuvants and delivery systems to stimulate T-cell immunity, and increased funding from public and private agencies are some of the reasons for progress in this area. Dozens of vaccine candidates have been tested in animal models in recent years, and several of these are poised to move into clinical trials in the next several years. Thus, there is renewed optimism for the potential of developing new and improved TB vaccines.

Improved immunogenicity and protective efficacy of a tuberculosis DNA vaccine encoding Ag85 by protein boosting.

C57BL/6 mice were vaccinated with plasmid DNA encoding Ag85 from Mycobacterium tuberculosis, with Ag85 protein in adjuvant, or with a combined DNA prime-protein boost regimen. While DNA immunization, as previously described, induced robust Th1-type cytokine responses, protein-in-adjuvant vaccination elicited very poor cytokine responses, which were 10-fold lower than those observed with DNA immunization alone. Injection of Ag85 DNA-primed mice with 30 to 100 microg of purified Ag85 protein in adjuvant increased the interleukin-2 and gamma interferon (IFN-gamma) response in spleen two- to fourfold. Further, intracellular cytokine analysis by flow cytometry also showed an increase in IFN-gamma-producing CD4(+) T cells in DNA-primed-protein-boosted animals, compared to those that received only the DNA vaccination. Moreover, these responses appeared to be better sustained over time. Antibodies were readily produced by all three methods of immunization but were exclusively of the immunoglobulin G1 (IgG1) isotype following protein immunization in adjuvant and preferentially of the IgG2a isotype following DNA and DNA prime-protein boost vaccination. Finally, protein boosting increased the protective efficacy of the DNA vaccine against an intravenous M. tuberculosis H37Rv challenge infection, as measured by CFU or relative light unit counts in lungs 1 and 2 months after infection. The capacity of exogenously given protein to boost the DNA-primed vaccination effect underlines the dominant role of Th1-type CD4(+) helper T cells in mediating protection.

Intra-pinna anti-tumor vaccination with self-replicating infectious RNA or with DNA encoding a model tumor antigen and a cytokine.

To optimize polynucleotide vaccinations for protective antitumor immunity we used a self-replicating RNA vaccine in which Semliki Forest virus replicase drives RNA expression of the lacZ gene coding for beta-galactosidase as model tumor-associated antigen (TAA). This was compared with replicase-deficient control RNA and with lacZ DNA plasmids with respect to gene expression in vitro and in vivo and for vaccination using the mouse ear pinna as an optimal immunization site. In vitro, the highest expression was observed with self-replicating RNA. Gene expression following pinna inoculation of either non-replicating DNA plasmids or self-replicating RNA was similar, lasting for 2-3 weeks. Higher antibody responses were obtained with RNA than with DNA. beta-Gal peptide specific CTL memory responses to lacZ DNA or RNA lasted for more than 6 weeks while respective responses induced by lacZ-transfected tumor cells lasted for only 2 weeks. To achieve a protective response against lacZ tumor cells with self-replicating RNA about a 100-fold lower dose of polynucleotide was sufficient in comparison to DNA. The extent of protective antitumor immunity not only depended on the gene dose used for vaccination, but also on the aggressiveness of the lacZ-transfected tumor line used for challenge. In comparison to lacZ-transfected tumor cells as vaccines, polynucleotide vaccination also demonstrated superiority with regard to cross-protection. Protective antitumor immunity could be strongly increased upon co-inoculation of lacZ DNA with IL-2 DNA or IL-12 RNA. IL-2 DNA, but not IL-12 RNA, also augmented the CTL response while IL-12 RNA, but not IL-2 DNA, reduced the antibody response. These results demonstrate efficient protective antitumor immunity after intra-pinna lacZ TAA polynucleotide vaccination and show additional immunomodulatory effects by co-administration of cytokine polynucleotides.

Molecular characterization and human T-cell responses to a member of a novel Mycobacterium tuberculosis mtb39 gene family.

We have used expression screening of a genomic Mycobacterium tuberculosis library with tuberculosis (TB) patient sera to identify novel genes that may be used diagnostically or in the development of a TB vaccine. Using this strategy, we have cloned a novel gene, termed mtb39a, that encodes a 39-kDa protein. Molecular characterization revealed that mtb39a is a member of a family of three highly related genes that are conserved among strains of M. tuberculosis and Mycobacterium bovis BCG but not in other mycobacterial species tested. Immunoblot analysis demonstrated the presence of Mtb39A in M. tuberculosis lysate but not in culture filtrate proteins (CFP), indicating that it is not a secreted antigen. This conclusion is strengthened by the observation that a human T-cell clone specific for purified recombinant Mtb39A protein recognized autologous dendritic cells infected with TB or pulsed with purified protein derivative (PPD) but did not respond to M. tuberculosis CFP. Purified recombinant Mtb39A elicited strong T-cell proliferative and gamma interferon responses in peripheral blood mononuclear cells from 9 of 12 PPD-positive individuals tested, and overlapping peptides were used to identify a minimum of 10 distinct T-cell epitopes. Additionally, mice immunized with mtb39a DNA have shown increased protection from M. tuberculosis challenge, as indicated by a reduction of bacterial load. The human T-cell responses and initial animal studies provide support for further evaluation of this antigen as a possible component of a subunit vaccine for M.tuberculosis.

Superiority of the ear pinna over muscle tissue as site for DNA vaccination.

Three different vaccination sites were compared for efficiency of immunization with naked DNA. Using the bacterial lacZ gene as a model, all three sites of the mouse (skeletal muscle, dermis of abdominal skin or of the ear pinna) could express the gene product beta-gal but varied in expression time with muscle tissue showing the longest expression. Expression time, however, did not correlate with immune response intensity. The ear pinna was by far the most effective and muscle the least effective priming site for specific humoral and cytotoxic T cell-mediated immune responses. Following intra-pinna DNA inoculation, beta-gal expressing cells were detectable around the injection site and in the major draining lymph node. Efficiency of immunization was also dependent on the promoter and expression vector used. The cytomegalus virus promoter driven pCMV beta vector was superior to the Moloney murine leukemia virus LTR driven BAG vector. LacZ DNA immunization was also compared with cell-based vaccination with lacZ-transfected tumor cells, in which case again the pinna was the best site for inducing strong immune responses. Tumor-specific T cell responses could also be well induced in the pinna, leading to cytotoxic T lymphocyte induction and protective antitumor immunity. Thus, the pinna was found to be a privileged site for induction of antitumor responses and for genetic immunization, an important finding of immediate practical and potential future clinical implications.

A change in gating mode leading to increased intrinsic Cl- channel activity compensates for defective processing in a cystic fibrosis mutant corresponding to a mild form of the disease.

The effects of the mild cystic fibrosis (CF) mutation P574H were analysed and compared with those of three severe ones (delta I507, delta F508 and R560T). Immunochemical and functional analyses indicate that the rank order of CFTR expression at the cell surface is: wild type CFTR > P574H >> delta F508 >> R560T approximately 0. Patch-clamp analysis indicates that the open probability of P574H Cl- channels is almost twice as high as that of the wild type CFTR-Cl- channel. This increased intrinsic activity of individual P574H CFTR-Cl- channels compensates for the lower number of P574H CFTR-Cl- channels reaching the cell surface, and probably explains the milder form of CF associated with the P574H mutation. NS004, a recently described activator, restores near normal CFTR activity in cells expressing the P574H-CFTR channel. The P574H mutation modifies the gating mode of the channel with a large increase (approximately x 7) in the mean channel open time. Proline 574 might play an important role in the process connecting ATP hydrolysis at the nucleotide binding domain and opening and closing events of the CFTR-Cl- channel.

Autocrine mitogen IgEGF cooperates with c-myc or with the Hcs locus during hepatocarcinogenesis in transgenic mice.

Hepatocarcinogenesis is deterministic in transgenic mice expressing in the liver gene construct Alb-DS4 that encodes autocrine growth factor IgEGF (D Stern et al. (1987), Science 235: 321-324), causing their death within 7.1 months. Hepatic expression of construct AAT-myc encoding murine c-myc causes liver cancer in 44% of the mice at 14.8 months. Cooperation of these genes was evident in CD2F1 transgenics bearing Alb-DS4 plus AAT-myc, in which accelerated hepatocellular carcinoma (HCC) formation caused death of all mice within 4.4 months. Alb-DS4 also cooperates with the Hcs locus, which in C3H/HeJ mice mediates high susceptibility to spontaneous hepatocarcinogenesis, causing accelerated formation of HCC to which mice succumbed at 5.1 months. Thus, genes that predispose to HCC formation cooperate in transgenic mice and their interaction is a key to understand mechanisms that cause liver cancer.

Immunohistochemical localization of cystic fibrosis transmembrane conductance regulator in human fetal airway and digestive mucosa.

The cellular distribution of the cystic fibrosis transmembrane conductance regulator (CFTR) in human fetal digestive and respiratory mucosa has been studied by immunohistochemistry. The streptavidin-biotin immunoperoxidase method was applied to paraffin-embedded specimens collected from normal fetuses ranging from 7 to 39 wk of gestation. By the 7th wk, CFTR protein was strongly detected in the yolk sack; in contrast, the staining was weak in the undifferentiated epithelium of the intestine and the airways. At 12 wk, the intestine showed strongly and diffusely stained enterocytes and a basal cytoplasmic reactivity in the first secretory cells. During development, only slight changes could be detected in the digestive epithelial distribution of CFTR. In the airways, the CFTR distribution followed the cephalocaudal maturation. In the tracheal ciliated cells, the CFTR protein was diffusely detected in the cytoplasm as early as 7 wk. After 24-25 wk, CFTR was localized at the apical domain of the ciliated cells and was also present in the collecting ducts and in the glands of the airways, predominantly in the periphery of the acini. Our data suggest that the CFTR is present as early as 7 wk during organogenesis and probably plays an important role during fetal life. There is an evolution in the CFTR distribution during airway development, whereas, in the intestine, CFTR is highly expressed through the epithelium as early as 12 wk and keeps the same distribution until birth.

From in vitro to in vivo. Progress in the use of cultured cells for human therapy.

The use of cultured cells with the ultimate goal of using the cells or their products for human therapy has experienced an exponential growth during the last decade. Stable cell cultures have been established and genetically modified to obtain high quality products for protein replacement therapy or vaccines. Cells have also been directly isolated from the human organism and, after their expansion in vitro, been retransferred as skin grafts for treatment of burns or for cancer therapy by activated lymphocytes. With the explosive development of molecular biology techniques, it is now possible to genetically modify ex vivo, cells derived from the human body. These modifications should allow targeted expression of therapeutic genes into specific cells which will, upon retransfer to the body, exert their therapeutic action in a diseased organism.

Low-conductance chloride channels in IEC-6 and CF nasal cells expressing CFTR.

The properties of the cystic fibrosis gene product (CFTR) were studied by expression of cloned cDNA in different cell systems. Infection of both simian fibroblast (Vero) cells and immortalized CF nasal polyp cells (NCF3A) with a vaccinia virus encoding CFTR induced forskolin-induced Cl- permeability and low-conductance (8 pS) Cl- channels. By stable transfection of the rat intestinal crypt-derived cell line IEC-6 we have isolated a clone, IEC-CF7, which expresses CFTR mRNA and antigen. IEC-CF7 cells, but not IEC-6, display forskolin-induced Cl- permeability and multiple linear low-conductance (+/- 8 pS) Cl- channels in cell-attached membrane patches. In excised patches of IEC-CF7 cells, low-conductance Cl- channels could be activated by addition of the catalytic subunit of the adenosine 3',5'-cyclic monophosphate-dependent protein kinase A (PKA) plus ATP. During bath fluid replacement studies, the activated low-conductance channel remained active in the absence of ATP at room temperature and showed saturation kinetics. Rectifying (32 pS) Cl- channels were not observed in either IEC-6 cells or IEC-CF7 cells, indicating that there is no relation between CFTR expression and the incidence of this channel. Our data strongly support the conclusion that CFTR can act as a low-conductance Cl- channel, gated by PKA. The IEC-6-derived cell line IEC-CF7 may prove to be a useful model in the study of CFTR function because of the absence of 32-pS Cl- channel activity and its potential for differentiation.

Differential localization of the cystic fibrosis transmembrane conductance regulator in normal and cystic fibrosis airway epithelium.

Deletion of the amino acid residue Phe 508 of the cystic fibrosis transmembrane conductance regulator (CFTR) protein represents the most common mutation identified in cystic fibrosis (CF) patients. A monoclonal and a polyclonal antibody directed against different regions of CFTR were used to localize the CFTR protein in normal and CF airway epithelium derived from polyps of non-CF and CF subjects homozygous for the delta Phe 508 CFTR mutation. To identify the cellular and subcellular localization of CFTR, immunofluorescent light microscopy, confocal scanning microscopy, and immunogold transmission electron microscopy were performed on cryofixed tissue. A markedly different subcellular distribution was identified between normal and CF airway epithelial cells. In normal epithelium, labeling was restricted to the surface apical compartment of the ciliated cells. In contrast, in the epithelium from homozygous delta Phe 508 CF patients, CFTR markedly accumulated in the cytosol of all the epithelial cells. These findings are consistent with the concept that the CFTR delta Phe 508 mutation modifies the intracellular maturation and trafficking of the protein, leading to an altered subcellular distribution of the delta Phe 508 mutant CFTR.

Expression of the cystic fibrosis transmembrane conductance regulator gene can be regulated by protein kinase C.

Epithelial cells utilize at least two types of apical Cl- channels, the cAMP-activated cystic fibrosis transmembrane conductance regulator (CFTR) and the Ca2+/calmodulin-dependent Cl- channel. While phorbal ester (PMA) activates only CFTR-dependent Cl- secretion and the Ca2+ ionophore A23187 only the Ca2+/calmodulin-dependent Cl- secretion, PMA and A23187 share the ability to down-regulate expression of the CFTR gene at the transcriptional level. Since both PMA and A23187 can activate protein kinases, we hypothesized that protein kinase pathways may be involved in the regulation of CFTR gene expression. Exposure of HT-29 human colon carcinoma cells to the protein kinase C activator SC9 down-regulated CFTR mRNA levels in a dose-dependent fashion, similar to that seen with PMA. The reduction in CFTR transcript levels by SC9 and PMA was blocked by H7, an inhibitor of protein kinases. In a similar fashion, the down-regulation of CFTR transcript levels by A23187 was blocked by H7 as well as staurosporine, another protein kinase inhibitor. Interestingly, both H7 and staurosporine themselves increased CFTR mRNA levels. Quantification of CFTR gene transcription rate showed a reduction by SC9 (similar to that with PMA and A23187) that was prevented by H7 and that H7 by itself increased CFTR transcription. Together, these observations suggest that protein kinase pathways, likely including protein kinase C, are involved in the regulation of CFTR gene expression, with activation or inhibition of protein kinase activity down-regulating or up-regulating CFTR gene expression, respectively.

A 17.6 kbp region located upstream of the rabbit WAP gene directs high level expression of a functional human protein variant in transgenic mouse milk.

We have investigated whether DNA regions present in the rabbit whey acidic protein (WAP) promoter/5' flanking sequence could potentially confer, in vivo, high level expression of reporter genes. Transgenic mice were generated expressing a variant of human alpha 1-antitrypsin, which has inhibitory activity against plasma kallikrein under the control of a 17.6 kbp DNA fragment located upstream of the rabbit WAP gene. Up to 10 mg/ml of active and correctly processed recombinant protein were detected in mouse milk, thus suggesting that the far upstream DNA sequences from the rabbit WAP gene might be useful for engineering efficient protein production in the mammary glands of transgenic animals.

Immunocytochemical analysis reveals differences between the subcellular localization of normal and delta Phe508 recombinant cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis (CF) is caused by mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). The most common mutation responsible for CF is the deletion of amino acid residue Phe508, with an average allelic frequency of 70%. We have isolated an anti-CFTR monoclonal antibody which specifically recognizes recombinant normal and delta Phe508-CFTR produced by a vaccinia virus expression system. Immunocytochemical analysis of L cells expressing either normal or delta Phe508-CFTR showed a marked difference in subcellular distribution. Normal CFTR had a distinct localization in the perinuclear area and was also associated with the plasma membrane. delta Phe508-CFTR essentially lacked the membrane-associated distribution and was present throughout the cytoplasm. This heterologous expression system thus provides a model system for studying the subcellular localization of different mutant forms of CFTR.

The promoter of the human cystic fibrosis transmembrane conductance regulator gene directing SV40 T antigen expression induces malignant proliferation of ependymal cells in transgenic mice.

Transgenic mice bearing a human cystic fibrosis transmembrane conductance regulator (CFTR) promoter-SV40 T antigen fusion transgene were generated in order to localize in vivo the potential oncogenesis linked to the tissue-specific activity of the promoter for the CFTR gene. Surprisingly, the only site of tumors resulting from expression of the reporter onc gene was ependymal cells lining the brain ventricles. SV40 T antigen expression in these cells led to a consistent pathology in the first weeks of age: ependymoma and consequent hydrocephaly. Tumor-derived cell lines were established, characterized and shown to originate from SV40 T antigen-induced ependymoma. No pathological alterations were found in other organs, such as lungs and pancreas, in which cystic fibrosis is pathologically manifest in humans. Such transgenic mice and derived cell lines may represent valid models for analysing (1) the role of SV40 T antigen in ependymoma formation and (2) CFTR function in ependymal cells.

Down-regulation of cystic fibrosis transmembrane conductance regulator gene expression by agents that modulate intracellular divalent cations.

In cystic fibrosis (CF), epithelial cells are unable to normally up-regulate apical membrane Cl- secretion in response to agents which increase cyclic AMP, but they do increase Cl- secretion in response to increases in intracellular Ca2+. Since intracellular divalent cations regulate the expression of many genes, we hypothesized that mobilization of intracellular Ca2+ and/or other divalent cations might modulate not only Ca(2+)-dependent Cl- channels but also cystic fibrosis transmembrane conductance regulator (CFTR) gene expression. To evaluate this concept, HT-29 human colon carcinoma cells were cultured under various conditions designed to manipulate intracellular divalent cation concentrations and CFTR gene expression was quantified at the levels of transcription, mRNA accumulation, mRNA half-life, and protein. Exposure to the divalent cation ionophores A23187 and ionomycin (agents which increase intracellular divalent cation concentrations) caused dose- and time-dependent reductions of CFTR mRNA levels, which could be blocked by the use of Ca(2+)- and Mg(2+)-free media. Ionophore-induced CFTR gene modulation was also observed with T84 human colon carcinoma cells and freshly isolated normal human bronchial epithelial cells. Incubation of HT-29 cells with thapsigargin, an agent that releases Ca2+ from intracellular stores, or in medium containing increased extracellular concentrations of Ca2+ or Mg2+ also caused down-regulation of CFTR mRNA levels. Transcription run-on analysis showed that, parallel with the decrease in CFTR mRNA levels, A23187 reduced the rate of transcription of the CFTR gene, while CFTR mRNA transcript half-life was unaffected. Consistent with the down-regulation of CFTR gene expression, CFTR protein levels also decreased after exposure to A23187. Thus, despite the independence of Ca(2+)-dependent Cl- channels and cyclic AMP-dependent CFTR-related Cl- channels in epithelial cells, increases in intracellular divalent cation concentrations down-regulate the expression of the CFTR gene at the transcriptional level, with consequent decreases in CFTR mRNA and protein.

In vivo transfer of the human cystic fibrosis transmembrane conductance regulator gene to the airway epithelium.

Direct transfer of the normal cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene to airway epithelium was evaluated using a replication-deficient recombinant adenovirus (Ad) vector containing normal human CFTR cDNA (Ad-CFTR). In vitro Ad-CFTR-infected CFPAC-1 CF epithelial cells expressed human CFTR mRNA and protein and demonstrated correction of defective cAMP-mediated Cl- permeability. Two days after in vivo intratracheal introduction of Ad-CFTR in cotton rats, in situ analysis demonstrated human CFTR gene expression in lung epithelium. PCR amplification of reverse transcribed lung RNA demonstrated human CFTR transcripts derived from Ad-CFTR, and Northern analysis of lung RNA revealed human CFTR transcripts for up to 6 weeks. Human CFTR protein was detected in epithelial cells using anti-human CFTR antibody 11-14 days after infection. While the safety and effectiveness remain to be demonstrated, these observations suggest the feasibility of in vivo CFTR gene transfer as therapy for the pulmonary manifestations of CF.