Biotecnologia Aplicada Elfos Scientiae ISSN: 0684-4551 Vol. 15, Num. 1, 1998, pp. 35-42

Trabajos selecionados del Congreso Biotecnologia Habana'97. diciembre 1-6, 1997

Selected papers from Congreso Biotecnologia Habana'97. December 1-6, 1997

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NOVEL PATHWAYS FOR NEGATIVE REGULATION OF INFLAMMATORY CYTOKINES

Alberto Mantovani

Instituto di Ricerche Farmacologiche "Mario Negri", Via Eritrea 62-20157 Milan, Italy and Dept. of Biotechnology, Section of General Pathology, University of Brescia, Italy.

Introduction

Inflammatory cytokines act in cascades. One can schematically recognize primary inflammatory cytokines, the prototype of which is IL-1, and secondary effector molecules, among which chemokines play an important role in recruitment. Pro and anti-inflammatory signals regulate the production of primary and secondary inflammatory cytokines, sometimes in unexpected ways (1). The possibility that microenvironmental signals may regulate the action of proinflammtory cytokines by acting at the receptor level has been less extensively studied. Here we will review recent results on the action of pro and anti-inflammatory signals on receptors for IL-1, which in fact is a complex system and for the chemokine monocyte chemotactic protein-1 (MCP-1), a prototypic inflammatory cytokine.

Given the existence of two distinct IL-1R, a number of studies have investigated the actual role played by each of them in IL-1 signaling. As summarized briefly hereafter, all available evidences indicate the IL-1- induced activities are mediated exclusively via the IL-1RI, whereas IL- 1RII has no signaling activity and inhibits IL-1 activities by acting as a decoy for IL-1 (1).

MCP-1 is a prototypic C-C chemokine active on mononuclear phagocytes, basophils, T cells and NK cells (2-4). Other CC chemokines, including the recently identified mocrophage derived chemokine (MDC), are also active on dendritic cells. We examined the effect of LPS on chemokine receptor expression and we found that LPS causes a drastic and rapid downregulation of the expression of CCR2, a receptor for MCP-1 and -3. The ED50 of LPS was 1 ng/mL and half maximal effect was reached with an optimal dose in 45 min. Inhibition of MCP-1 receptor expression was functionally relevant since LPS-treated monocytes showed a reduced capacity to bind and to respond to MCP-1 chemotactically. The action of LPS on C-C chemokine receptors was specific in that CXCR2 was unaffected. In neutrophils, LPS and TNF-alpha were reported to inhibit the expression of IL-8 receptors, while G-CSF increased it. IL-2 was shown to induce CCR2 in T lymphocytes and NK cells, and observation confirmed here for monocytes. Interestingly, CCR2 induction in T cells was a slow process, requiring four days of exposure to cytokine. The results reported here show a dramatic, rapid and differential downregulation of chemokine receptors by LPS in monocytes.

LPS did not inhibit the rate of nuclear transcription of CCR2, but did reduce the mRNA half life from 1.5 h to 45 min. regulation of CCR chemokine receptor expression, in addition to agonist production, is likely a crucial point for regulation of the chemokine system. We speculate that the divergent effect of certain proinflammatory signals on agonist versus receptor expression may serve to retain mononuclear phagocytes at sites of inflammation, to prevent their reverse transmigration, and, possibly, to limit excessive recruitment.

Primary and secondary inflammatory cytokines are highly regulated by diverse signals. Emphasis has largely been on how pro- and anti- inflammatory molecules affect cytokine production. The results summarized here obtained with IL-1 receptors and with the MCP-1 receptor indicate that receptor expression may represent a crucial regulatory element for the tuning of the action of primary and secondary inflammatory cytokines.

References

1. Colotta F, Dower SK, Sims JE, Mantovani A. The type II 'decoy' receptor: novel regulatory pathway for interleukin-1. Immunol Today 1994;15:562- 566.

2. Mantovani A, Sozzani S, Proost P, Van Damme J. The monocyte chemoattractant protein family. In: Chemoattractant Ligands and Their Receptors, Horuk R ed., CRC Press, Inc. Boca Raton 1996;pp.169-192.

3. Godiska R, Chantry D, Raport CJ, Sozzani S, Allavena P, Leviten D, Mantovani A, Gray PW. Human macrophage derived chemokine (MDC) a novel chemoattractant for monocytes, monocyte derived dendritic cells, and natural killer cells. J Exp Med 1997;185:1595-1604.

4. Sica A, Saccani A, Borsatti A, Power CA, Wells TNC, Luini W, Polentarutti N, Sozzani S, Mantovani A. Bacterial lipopolysaccharide rapidly inhibits expression of C-C chemokine receptors in human monocytes. J Exp Med 1997; 185:969-974.

THERAPEUTIC APPLICATLONS OF THE INTERLEUKINS

Kendall A Smith

The New York Hospital-Cornell Medical Center, 525 East 68th Street, New York, New York, 10021. USA

Introduction

The use of cytokines (interleukins) in the clinic to augment host defenses has been problematic because of toxic side effects. The symptoms produced by cytokines that stimulate lymphocytes and monocytes are those usually associated with inflammation caused by infecttous microbes, including fever, rigor, fatigue, myalgia, malaise and vascular collapse.

Thus, the classic signs of inflammation, rubor, calor, tumor and dolor are now understood to result from the actions of cytokines produced during the host defense reaction, and not due to the direct effects of microbial products per se. Accorcfingly, the therapeutic chatlenge in attempting to stimulate or enhance host defenses is to establish a therapeutic index, i.e. a beneficial therapeutic: toxic ratio.

Interleukin 2 (IL-2), the first cytokine molecule to be identified and characterized is prototypic, in that when first used in the clinic very high doses were administered as intravenous bolus injections. Thus, 50 million IU (3.3 mg) were injected every 8 hours for a total dose of 15 million IU (10 mg) per day. This regimen results in severe toxicity (WHO Grade III-IV), and consists of the Systemic Inflammatory Response Syndrome (SIRS). Consequently, individuals treated in this manner can only tolerate such toxicity for a few days, and must be cared for in an intensive care unit. The basis for the toxicity of high dose lL-2 can be traced to the type and distribution of IL- receptors (IL-2R). There are 3 classes of lL- 2Rs, which are distinguishabie by the affinity with which they bind IL-2. High affinity IL- 2Rs (Kd=10-11 M) are onty expressed by antigen-activated T cells and B cells, and 10% of Natural Killers (NK) cells. By comparison, intermediate affinity IL-2Rs (Kd=10-9 M) are expressed by the majority of NK cells. Because NK cells comprise 10% of circulating mononuclear cells, there are (approximately) 109 NK cells that are capable of responding to IL-2, when high doses are administered. NK cells produce secondary cytokines when activated by IL-2, in particular cytokines that target monocytes and macrophages, including IFN-gamma, TNF-alpha, and GM-CSF. These cytokines stimulate their target cells to release pro-inflammatory cytokines, such as IL-1, li-6, IL-12 and TNFalpha. The result is the SIRS.

It is possible to circumvent the activation of this cascade of cytokine release by lowering the IL-2 dose 400-fold, to 0.375 million IU (25 ug). At this dose injected subcutaneously, a peak plasma IL-2 concentration of only 25 pM is attained. Since 25 pM will occupy (approximately) 70% of high affinity IL-2Rs, but <2% of the intermediate affinity IL-2Rs expressed by most NK cells, the large amounts of cytokines produced by the NK cells can be avoided. In this manner, daily subcutaneous injections of IL-2 in ultralow doses can be given continuously without toxicity. Thus far, we have administered IL-2 for more than 2 years. This therapy boosts host defenses, as indicated by increased circulating NK cells, eosinophils, monocytes, and T cells. Moreover, delayed-type hypersensitivity is markedly augmented. Therefore, there are now many indications whereby lL-2 immunostimulatory therapy may be beneficial, including infectious diseases, immunodeficiencies and cancer.

MOLECULAR MECHANISMS OF APOPTOSIS

James W Larrick and Susan C Wright

Palo Alto Institute of Molecular Medicine; 2462 Wyandotte Street; Mountain View, CA 94043 USA email; jwlarrick@aol.com

Introduction

Apoptosis is the normal physiological process of cell death essential for the maintence of homeostasis (reviewed in 1-3). Because of its important role in development and recent evidence implicating dysregulation of this process underiying various pathological conditions (3), there has been intense interest in the biochemical mechanism of apoptosis. Work in this laboratory has analyzed the apoptotic pathway using the human monocyte-like cell line U037 asa model system. Our studies indicate that both TNF and UV light initiate similar biochemicai processes culminating in internucleosomal DNA fragmentation. A key even in this pathway is the activation of a serine protease termed AP24 (apoptotic protease of 24 KD). This enzyme was purified from apoptotic U937 cells following exposure to UV light and was shown to initiate internucleosomal DNA fragmentation in nuclei isolated from normal U937 cells (4). Since protein synthesis is not required for apoptosis in this system (5), AP24 must be expressed in an inactive form or else in sequestered from its substrate in normal U937 cel! s. The upstream signaling events leading to activation of AP24 have been the subject of recent work. Recent work demonstrating a role for the caspase cascade (6) AOP-ribosylation (7) and calcium calmodulin kinase II (8) in the upstream activation of AP24 witl be presented.

References

1. Walker Nl, Harmon BV, Goge GC, Kerr JFK. Patterns of celi death. Meth Achiev Exp Pathol 1988;13:18-54.

2. Gerchenson LE, Rotello RJ. Apoptosis: a different type of cell death. FASEB J 1995; 6:2450.

3. Thompson CB. Apoptosis in the pathogenesis and treatment of disease. Science 1992;267:1456.

4. Wright SC, Wei QS, Zhong J, Zheng H, Kinder DH, Larrick JW. Purification of a 24 kD protease from apoptotic tumor cells that activates DNA fragmentation. J Exp Med 1994;180:2113.

5. Wright SC, Kumar P, Tam AW, Shen N, Varma M, Larrick JW. 1992. Apoptosis and DNA fragmentation preceed TNA-induced cytolysis in U937 cells. J Cell Biochem 1994;48:344.

6. Wright SC, Schellenberger U, Wnat H, Kinder DH, Larrick JW. Activation of C((32-like proteases is NOT sufficient to lead to apoptosis: Inhibition of apoptosis by agents that suppress activation of AP24 but not DEVD- cleaving activity. 1997(submitted).

7. Wright SC, Wei QS, Kinder Ok, Larrick JW. Biochemical pathways of apoptosis: NAD deficient cells are resistant to TNK or UV light activation of the 24kD apoptotic protease and DNA fragmentation. J Exp Med 1996; 183:463.

8. Wright SC, Ji L, Wang H, Larrick JW. 1997. Biochemical pathways of apoptosis: Calmodulin dependent protein kinase II mediates signal transduction in apoptosis. FASEB J (in press).

DNA VACCINES: NEW APPLICATIONS OF BIOTECHNOLOGY TO INFECTIOUS DISEASES

Robert Whalen

Director of Research, French National Center of Scientific Research, Paris, France, E-mail: whalen@pasteur.fr The ONA Vaccine Web: http://www.genweb.com/Dnavax/

Introduction

Although direct gene transfer into tissues is a relatively inefficient process, it is nonethetess possible to synthesize enough of a foreign protein after injection of purified plasmid DNA expression vector to induce an immune response, The expression of several types of antigens from a large number of pathogens has shown that the immune responses induced are broad-based, These responses include induction of antibodies as well as the production of cytotoxic and helper T lymphocytes, and they result in long- lasting T-cell memory in the animal.

In several cases, the immunity resulting from such DNA-based immunization is able to protect the animal from challenge by the pathogenic organism. Thus, such DNA vaccines have been clearly demonstrated to be a realistic approach to vaccine development, and further work in this field can concentrate on the practical maters of formulation of such novel vaccines so that they can be used safely and reliably for human and veterinary vaccination purposes, Several phase l/ll clinical trials are currently underway to evaluate the safety and immunogenicity of DNA vaccines in humans.

Such a methodology has many advantages with respect to vaccine developmment, The ability to immunize with a simple ONA plasmid a1lows researchers to test many ideas using a very simp1e methodology. The plasmids can be introduced by intramuscular or intradermal needle injection, or the DNA can be delivered after absorption to gold particle and then bombarded into the epidermis. The use of the latter technology, often referred to as a "gene gun", appears to give very efficient DNA uptake, and strong immune responses can be achieved with a few micrograms or less of DNA. This technology has also allowed the sirnu1taneous introduction of thousands of clones of potentially antigenic genes, in an approach called "expression-library immunization".

Results and Discussion

In our experimental work, plasmids encoding the envelope protein of the hepatitis B virus give strong immune responses, and these results wilt serve to illustrate the potential of DNA vaccines. Intramuscular irnrnunization induces a particular effective T cell priming which can protect chimpanzees from disease after challenge by the hepatitis B virus. Other results indicate that a DNA vaccine encoding HBV proteins could be effective in treating chronic hepatitis B, which is a great public health problem in Asia, Africa and many other countries. It is clear that plasmid DNA itself functions as an immunostimulatory molecule, and in particular accounts for the induction of a T-helper type 1 phsnotype characterized by the secretion of the cytokines interferon-gamma and interleukin-2.

Conclusion

High quality plasmid DNA is relatively easy to prepare, using straightforward fermentation and chromatography methods. The DNA itself is very stable, and this aspect, combined with the inexpensive production process, could result in DNA vaccines which are affordable in most aceas of the world, lndeed, the manufacturing expertise required should be implemented in as many countries as possible, to provide foc "vaccine independence" and the ability to move quickly to produce vaccines relevant to each country's specific infectious diseases problems.

References

1. Donnelly JJ, Ulrner JB, Shiver JW, Liu MA. DNA vaccines. Annu Rev Immunol 1997;15:617-648.

2. Pisetsky DS. Immune activation by bacterial DNA: a new genetic code. Immunity 1996;5:303-310.

3. Whalen RG. DNA vaccines for emerging infectious diseases. Emerging lnfectious Oiseases 1996;2:168-175. Available online at:

NEW APPROACHES FOR THE DESIGN OF AIDS VACCINES

Jose V Torres and Maria P Carlos

Medical Microbiology and Immunology, School of Medicine, University of California, Davis, CA 95B16, USA.

Introduction

Although several AIDS vaccine candidates in clinical trials such as synthetic peptides, recombinant subunit, live vector and combination vaccines have been shown to induce HlV specific antibodies and CTL responses in non-human primate models and humans, major obstacles facing the development of AIDS vaccine still remain, These include the induction of long-lasting protective immunity, identification of the correlates of immune protection, cross-protection against diverse natural isolates, protection against infection with cell-associated virus end efficacy against mucosal transmission, Recently, plasmid DNA vaccines for genetic Immunization have induced high titars of neutralizing antibodies and strong cytotoxic T lymphocyte responses. Immunization with multiple doses of an HIV-1 env DNA vaccine and HIV-1 env protein preparation have protected monkeys from intravenous infection with an SIV virus expressing the HIV-1 envelope glycoprotein.

Our research goal is the development of a therapeutic AIDS vaccine through the SIV in rhesus macaques animal model system, We believe that both humoral (strong neutralizing antibodies) and cellular (cytotoxic T Cell lymphocyte) responses are necessary for an effective therapeutic or preventive vaccine. One of the major challenges to the development of an HIV vaccine is the antigenic variability which results in overwhelming diversity. The main obstacle to achieve this goal continues to be the lack of a viable approach to account for epitope glycoprotein variability by immunization. Most of the antigenic variability is found at specific regions of the envelope glycoprotein (gp120), This protein is important for attachment and entry into the target cells and is critical for eliciting broad humoral responses, including neutralizing antibodies. Therefore, we have designed and prepared a vaccine component consisting of a cocktail of peptides representing the accumulation of the in vivo variability seen in the envelope glycoprotein. This construct is also designed to overcome MHC restriction limitations in vaccines against variable pathogens which are intended for outbred populations such as humans. This Hypervariable Epitope Construct (HEC) induced a broad humoral immune response in rabbits and rhesus macaques. As a direct result of immunization with this construct we also demonstrated clear capacity to overcome MHC restriction in mice. Following initial proof of concept in the SIV model, we are pursuing this vaccination concept in HIV-1. A previous study involving rhesua macaques immunized with HIV-1 envelope peptide miniproteins from three HIV-1 strains was determined to be safe and immunogenic at the dose of vaccine to be used in humans. Following demonstration of lack of toxicity in monkeys, this HIV-1 synthetic vaccine candidate was approved for clinical trials.

A second generation, more complex synthetic HIV-1 immunogen has been designed and prepared in our laboratory. This new vaccine construct is recognized by antibodies from HIV-1 positive individuals representing HlV-1 subtypes A, B, C, D, E, F as well as HIV-2 strains. Immunization of rabbits and rhesus macaques resulted in the induction of high titers of HIV-1 antibodies, T Cell proliferative response and delayed-type hypersensitivity (DTH) reactions. Our collective data suggest that this preparation is a promising component of an AlDS vaccine. In addition, our concept constitutes a new class of custom-made vaccine that can be prepared to be effective against all antigenically variable pathogens. In addition to the Induction of broad humoral responses with our peptide constructs, a strong CTL response eight also be required for an effective vaccine. Therefore, we have designed a combination vaccine using a recombinant vector to express SIV proteins (gag, pol, env, and nef) and synthetic peptide constructs in rhesus macaques.

Preliminary studies have shown strong CTL, secreted IgA/IgG, high antibody titers, neutralizing antibody responses and broad, cross-reactive overa0 immunogenicity. This combination vaccine is currently one of the vaccines under a Multicenter Preclinical Trial (NIH Protocol 33) being tested as a potential vaccine for use in humans.

DNA vaccination is being developed as a very promising addition to the battle against infectious diseases, There is data that supports the notion that plasmids expressing viral genes are capable of inducing protective antiviral lmmune responses in some animal models, it is clear that genetic vaccines directed against lentiviruses which cause fatal immunodeficiency in primates scan now be developed and tested in a comprehensive manner. We have prepared an SiV plasmid with a deletion encompassing the coding regions for integrase, vif, vpx and vpr. These deletions were introduced into the SIVmac background where the 5' U3 region of the LTR was replaced with a CMV promoter. Several rabbits received injections of 300 ug of the plasmid at 0, 13 and 43 weeks using a needleless injection system and were boosted IM at week 60. Antibody responses to gp130 and disrupted SIV were detected in all subjects by ELlSA. High levels of T-cell proliferative responses to gag and env peptldes, disrupted SIV, and gp130 were also demonstrated. Our tab has also produced whole defective particles for genetic immunization and have tested the lmmune response to HIV-1 in rabbits and SIVmac in macaques and rabbits. DNA and RNA constructs are designed to express HIV and SIV proteins.

We are now monitoring long term humoral and cellular immune responses in macaques. At the end of this study the government expects the development of a construct suitable for clinical trials, Our new projects also include development of a safe retroviral vector to be specifically directed for expression in antigen specific cells and a complex improved large plasmid to be used for immunization though direct targeting of lymphoid tissues.

Copyright 1998 Elfos Scientiae