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مقدمهای بر واکسن های ژنی
Abstract
این کتاب که در سال 1382 پیرو آغاز پروژه واکسن های ژنی در ایران در سال 1380 توسط دکتر علی کرمی شروع شد منتشر گردید و به توضیح نسل چهارم واکسن ها یعنی استفاده از مواد ژنتیکی شامل
DNA یا RNA بجای میکرب یا پروتئین نوترکیب بعنوان واکسن برای القا سیستم ایمنی در اثر بیان ژن در داخل سلول ها می پردازد
تا کنون واکسن ها شامل میکرب های کشته یا ضعیف شده یا ساب یونیتی یعنی بخشی از میکرب یا سم بودند ولی در این روش از ماده ژنتیکی رمز کننده پروتئین مورد نظر استفاده می شود در این فناوری که با استفاده از بیوتکنولوژی و مهندسی ژنتیک ژن رمز کننده پروتئین ایمنی زا به یک حامل یا پلاسمنید خاص کلون می شود این پلاسمید دارای ویژگی بیان ژن در داخل سلول است بنابر این پروتئین نوترکیب تولید شده در داخل سلول سبب القا سیستم ایمنی هومورال و سلولار شده و هم آنتی بادی ضد آنتی ژن تولید می شود و هم ایمنی دراز مدت سلولی علیه میکرب یا سم ایجاد شده و مزایای بسیاری نسبت به واکسن های نسل اوب تا سوم را دارد. دکتر کرمی در این پروژه که بنیان آن را در ایران نهادند این فرضیه را مطرح کردند که از ژن رمز کننده پروتئین های حفاظت دهنده یا ایمنی زا می توان بجای میکرب کشته یا ضعیف شده یا پروتئین نوترکیب استفاده کرد. دکتر کرمی این گون واکسن ها را تهیه و آزمایشات بسیاری بروی حیوانات آزمایشگاهی انجام دادند و مقالات بسیاری نیز منتشر کردند ولی بنا به احتیاط های ایمنی هر گز این واکسن ها در انسان ها آزمایش نشدند چون نیاز به بررسی های بیشتری برای اطمینان کامل از ایمنی آنها در تزریق به جمعیت انسانی وجود دارد. به هر حال بنیان گزار واکسن های ژنی در ایران دکتر کرمی بودند. برای اطلاع دانشمندان بخش هایی از این کتاب به شکا رایگان در اینجا منتشر می شود. اصل کتاب منتشر شده توسط انتشارات دانشگاه دانشگاه امام حسین ع می باشد.
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DNA vaccine have many potential advantage over traditional vaccine. There is no risk of infection , its superior to protein or polysaccharides and recombinant vaccine because DNA is very stable, its production is easy and most suitable for the use in developing countries. But most exciting finding about advantage of DNA vaccine is its ability to induce both cellular and humoral response, it works like inactivated live viral vaccines. Several experiment have shown immunogenicity of Nucleic Acid immunization with HBs-Ag encoding plasmid.
We have studied the expression of HBs antigen and anti HBs response in mice by intramuscular and intradermal injection of recombinant eukaryotic vector encoding hepatitis B surface antigen sequence next to CMV promoter. ELISA analysis of serum samples from mice injected with 50 or 100 ug of endotoxin free DNA with and without special new polymer synthetized in IRAN (dendrosome) revealed that : small amount of HBsAg expressed in mice tissue can strongly stimulate immune response and high level of anti-HBs was detected in both endotoxin freeDNA alone and with polymer but Dendrosome mediated DNA vaccination has elevetated antibody production comparing to non adjuvant DNA. Enhanced immune response was detected as 10 fold comparing to four times administration of human recombinant (protein) HBs vaccine. PCR analysis of blood and muscle tissue injection with DNA vaccine for presence of HBs sequence in different time intervals revealed that after 72hr we were not able to detect from HBs-Ag encoding sequence.
Experiments were designed to test the effect of introns on gene expression in transgenic mice. Four different pairs of gene constructs, which were identical except that one member of each pair lacked all introns, were compared for expression of mRNA after introduction into the murine germ line by microinjection of fertilized eggs. The expression of two chimeric genes, made by fusing either the mouse metallothionein I or the rat elastase 1 promoter/enhancer to the rat growth hormone gene, was assayed in fetal liver or pancreas, respectively, while two natural genes, an oligonucleotide-marked mouse metallothionein I gene and the human beta-globin gene, were assayed in fetal liver. In each case there was, on average, 10- to 100-fold more mRNA produced from the intron-containing construct. Moreover, mRNA levels were proportional to the relative rates of transcription that were measured in isolated nuclei. However, when the expression of the two mouse metallothionein I gene-based constructs was tested after transfection into cultured cells, little difference was observed. These observations suggest that introns play a role in facilitating transcription of microinjected genes and that this effect may be manifest only on genes exposed to developmental influences.
Genetic immunization is a simple method for producing polyclonal antibodies in mice. To test if this approach could be used for monoclonal antibody production, biolistic transfection was used to immunize a mouse. High levels of polyclonal antibodies against human growth hormone (hGH) were elicited following three inoculations with the gene for hGH. When hybridoma cells were created from the mouse's splenocytes, approximately 17% secreted antibodies vs. hGH. Of these, some recognized only native or denatured hGH, while most recognized both forms of the protein. These findings demonstrate the utility of genetic immunization as a method to produce monoclonal antibodies.
A live attenuated AroA− auxotrophic mutant ofSalmonella typhimurium (SL7207) has been used as carrier for the pCMVβ vector that contains the β-galactosidase (β-gal) gene under the control of the immediate early promoter ofCytomegalovirus (CMV). We tested whether orally administered bacterial carrier could enter and deliver the transgene to antigen-presenting cells (APCs) through the natural enteric route of infection and whether β-gal expression could generate a protective response against an aggressive murine fibrosarcoma transduced with the β-gal gene (F1.A11) that behaves operationally as a tumor-associated antigen. After three courses, at 15-day intervals, mice developed both cell-mediated and systemic humoral responses to β-gal. Mice vaccinated with the Salmonella harboring pCMVβ, but not with plasmid-less carrier, showed resistance to a challenge with F1.A11 cells. These experiments suggest that Salmonella-based DNA immunization allows us to specifically target antigen expression in vivo to APCs. To prove that the transgene is actually expressed by APCs as a function of an eukaryotic promoter, the green fluorescent protein (GFP) was placed under the control of either the eukariotic CMV or a prokaryotic promoter. Using cytofluorometric analysis, GFP was detected only in splenocytes of mice receiving a Salmonella carrier harboring GFP under the CMV promoter. These results indicate that transgene expression occurs because of a Salmonella-mediated gene transfer to eukaryotic cells. Finally, approximately 19% of the splenocytes expressed GFP. Among them, F4/80+ macrophages and CD11cbright dendritic cells (DCs) were scored as positive for GFP expression. Extensive work has been performed trying to optimize the way to transfect DCs, ex vivo, with genes coding for relevant antigens. We show here, for the first time, that DCs can be directly and specifically transduced in vivo such to induce DNA vaccination against tumors.
© 1998 by The American Society of Hematology.
In contrast to adult mice immunized with influenza A virus strain WSN and plasmid expressing WSN hemagglutinin (HA) gene which developed primary and secondary anti-HA antibody responses, mice immunized as neonates with virus failed to produce anti-HA antibodies while those immunized with plasmid developed weak primary but strong secondary responses. Analysis of the frequency of HA-specific B clonotypes as well as their reactivity pattern (RP) showed that viral or genetic immunization of adults increased the frequency of clonotypes which exhibit broad RP. The most striking observation of our study is that immunization of neonates with plasmid leads to increased synthesis of anti-HA antibodies as well as to an increased frequency of clonotypes exhibiting an adult-like RP. In contrast, neonatal immunization with virus caused a longlasting unresponsiveness and the few clonotypes stimulated in vitro exhibited only a monoreactive pattern. Isotype patterns of mAb are also diversified in the case of mice immunized with plasmid as neonates. Rapid replacement of neonatal with adult clonotypes may explain the significant survival of the mice immunized with plasmid and challenged 1 or 3 months later with lethal doses of virus.
A new and unusual approach for evoking an immune response has recently been introduced—that of DNA-based immunization. Purified plasmid DNA, containing protein coding sequences and the necessary regulatory elements to express them, can be introduced into tissues of the organism by means of a parenteral injection or by particle bombardment. The number of cells transfected and the amount of protein produced is sufficient to produce a remarkably strong and broad-based immune response to a wide variety of foreign proteins. The absence of an exogenous infectious agent or immunogen results in the abrupt appearance of a foreign protein within the normal cells of an immunologically mature and healthy animal and provokes an energetic and efficient reaction to this form of antigen presentation. This review summarizes the results obtained with the various experimental models that have been described to date and considers in greater depth the immune response to the surface antigen of the human hepatitis B virus that has been achieved using DNA-based immunization. Several issues are addressed in a prospective manner in order to anticipate some future developments and to point out topics likely to be pertinent to this field. DNA-mediated induction of immune responses may soon be applied as a form of therapeutic treatment. Although this method may constitute a revolution for vaccination, many issues must first be dealt with, especially concerning the safety of using DNA as an immunizing molecule.
This chapter explores and compares different possible mechanisms by which hepadnaviruses maytrigger liver cell proliferation and transformation, and considers the factors that may influence the primacy of some oncogenic pathways over others in tumors induced by different viruses of the same family. Primary hepatocellular carcinoma (HCC) is one of the most common cancers in many parts of the world and is also one of the rare human cancers showing seroepidemiologic association with a viral infection. The role of hepatitis B virus (HBV) as a causal agent of HCC is established and the increased risks of developing HCC are estimated. Productive HBV infections potentiate the action of exogenous carcinogenic factors like aflatoxins and alcohol. The clinical and immunological aspects of HBV infections and their modes of transmission are also discussed as are genetic organization of the HBV genome, genome structure and replication, and regulated expression of viral genes. HBV DNA integration patterns into host-cell DNA have led to a different hypothesis on the contribution of HBV to hepatocarcinogenesis. Hepatitis B is rendered highly contagious by the unusual stability of infectious HBV virions present in the blood and other body fluids like saliva, urine, and semen.
Myoblast transfer has emerged as a promising treatment for inherited myopathies such as Duchenne muscular dystrophy (DMD). Further development of the technique's therapeutic potential requires an experimental system in which issues of graft rejection can be clearly discriminated from those related to myoblast biology. Here we report the development and initial application of a quantitative assay for myogenic cells bearing a wild-type dystrophin gene following transfer into the mdx mouse. The technique relies upon the ability of a mutagenizing polymerase chain reaction (PCR) primer to create a new restriction site in the amplification production of the wild-type, but not the mdx dystrophin gene. The ratio of host to donor cells can be determined from muscle biopsies as small as 1 mg, regardless of donor H-2 background. This simple technique should allow a number of basic questions related to myoblast and direct gene transfer to be addressed using the mdx mouse model.
This paper describes in vitro experiments with two types of intramolecular duplex structures that inhibit translation in cis
by preventing the formation of an initiation complex or by causing the complex to be abortive. One stem-loop structure (delta
G = -30 kcal/mol) prevented mRNA from engaging 40S subunits when the hairpin occurred 12 nucleotides (nt) from the cap but
had no deleterious effect when it was repositioned 52 nt from the cap. This result confirms prior in vivo evidence that the
40S subunit-factor complex, once bound to mRNA, has considerable ability to penetrate secondary structure. Consequently, translation
is most sensitive to secondary structure at the entry site for ribosomes, i.e., the 5' end of the mRNA. The second stem-loop
structure (hp7; delta G = -61 kcal/mol, located 72 nt from the cap) was too stable to be unwound by 40S ribosomes, hp7 did
not prevent a 40S ribosomal subunit from binding but caused the 40S subunit to stall on the 5' side of the hairpin, exactly
as the scanning model predicts. Control experiments revealed that 80S elongating ribosomes could disrupt duplex structures,
such as hp7, that were too stable to be penetrated by the scanning 40S ribosome-factor complex. A third type of base-paired
structure shown to inhibit translation in vivo involves a long-range interaction between the 5' and 3' noncoding sequences.
Peptides from endogenous proteins are presented by major histocompatibility complex class I molecules, but antigens (Ags)
in the extracellular fluids are generally not. However, pathogens or particulate Ags that are internalized into phagosomes
of macrophages (M phi s) stimulate CD8 T cells. The presentation of these Ags is resistant to chloroquine but is blocked by
inhibitors of the proteasome, a mutation in the TAP1-TAP2 transporter, and brefeldin A. Moreover, phagocytosis of a ribosomal-inactivating
protein inhibited M phi protein synthesis. These results demonstrate that M phi s transfer Ags from phagosomes into the cytosol
and that endogenous and exogenous Ags use a final common pathway for class I presentation.