ANTIBODY FORMATION AND DEGRADATION
I. GENERAL CHARACTERISTICS OF THE ANTIBODY RESPONSE
A. Self/non-self discrimination - One characteristic feature of the specific immune system is that it normally distinguishes between self and non-self and only reacts against non-self.
B. Memory - A second feature of the specific immune response is that it demonstrates memory. The immune system "remembers" if it has seen an antigen before and it reacts to secondary exposures to an antigen in a manner different than after a primary exposure. Generally only an exposure to the same antigen will illicit this memory response.
C. Specificity - A third characteristic feature of the specific immune system is that there is a high degree of specificity in its reactions. A response to a particular
antigen is specific for that antigen or a few closely related antigens.
N.B. These are characteristic of all specific immune responses.
II. ANTIBODY FORMATION
A. Fate of the immunogen
1. Clearance after primary injection - The kinetics of Ag clearance from the body after a primary administration is depicted in Figure 1.
a) Equilibrium phase - The first phase is called the equilibrium or equilibration phase. During this time the Ag equilibrates between the vascular and extravascular compartments by diffusion. This is normally a rapid process. Since particulate antigens don't diffuse, they do not show this phase.
b) Catabolic decay phase - In this phase the host's cells and enzymes metabolize
the antigen. Most of the antigen is taken up by macrophages and other phagocytic cells. The duration will depend upon the immunogen and the host.
c) Immune elimination phase - In this phase newly synthesized antibody combines with the antigen producing antigen/antibody complexes which are phagocytosed and degraded. Antibody appears in the serum only after the immune elimination phase is over.
2. Clearance after secondary injection - If there is circulating antibody in the serum injection of the antigen for a second time results in a rapid immune elimination. If the is no circulating antibody then injection of the antigen for a second time results in all three phases but the onset of the immune elimination phase is accelerated.
B. Kinetics of antibody responses to T-dependent Ag
1. Primary (1o) Ab response - The kinetics of a primary antibody response to and
antigen is illustrated in Figure 2.
a) Inductive, latent or lag phase - In this phase the Ag is recognized as foreign and the cells begin to proliferate and differentiate in response to the antigen. The
duration of this phase will vary depending on the antigen but it is usually 5-7 days.
b) Log or Exponential Phase - In this phase the Ab concentration increases exponentially as the B cells that were stimulated by the antigen differentiate into plasma cells which secrete antibody.
c) Plateau or steady-state phase - In this phase Ab synthesis is balanced by Ab
decay so that there in no net increase in Ab concentration.
d) Decline or decay phase - In this phase the rate of Ab degradation exceeds that
of Ab synthesis and the level of Ab falls. Eventually the level of Ab may reach base line levels.. 2. Secondary (2o), memory or anamnestic response (Figure 3)
a) Lag phase - In a secondary response there is a lag phase by it is normally
shorter than that observed in a primary response.
b) Log phase - The log phase in a secondary response is more rapid and higher Ab levels are achieved.
c) Steady state phase
d) Decline phase - The decline phase is not as rapid and Ab may persist for months, years or even a lifetime.
C. Specificity of 1o and 2o responses
Ab elicited in response to an antigen is specific for that antigen although it may also cross react with other antigens which are structurally similar to the eliciting antigen. In general secondary responses are only elicited by the same antigen used in the primary response. However, in some instances a closely related antigen may produce a secondary response, but this is a rare exception.
D. Qualitative changes in Ab during 1o and 2o responses
1. Ig class variation - In the primary response the major class of Ab produced is IgM whereas in the secondary response it is IgG (or IgA or IgE) (Figure 4). The antibodies that persist in the secondary response are the IgG antibodies.
Fig 4
2. Affinity - The affinity of the IgG Ab produced increases progressively during the response, particularly after low doses of antigen (Figure 5). This is referred to as affinity maturation. Affinity maturation is most pronounced after secondary challenge with antigen.
Fig 5
One explanation for affinity maturation is clonal selection as illustrated in Figure 6. A second explanation for affinity maturation is that, after a class switch has ocurred
Fig. 6
in the immune response, somatic mutations occur which fine tune the antibodies to be of higher affinity. There is experimental evidence for this mechanism, although it is not known how the somatic mutation mechanism is activated after exposure to antigen.
3. Avidity - As a consequence of increased affinity, the avidity of the antibodies
increases during the response.
4. Cross-reactivity - As a result of the higher affinity later in the response there is also an increase in detectible cross reactivity. An explanation for why increasing affinity results in an increase in detectible cross reactivity is illustrated by the following example.
Affinity of Ab for Ag
Early Late
_____________________
Immunizing Ag 10-6 10-9
+ ++
Cross reacting Ag 10-3 10-6
- +
-
If a minimum affinity of 10-6 is needed to detect a reaction, early in an immune response the reaction of a cross reacting antigen with an affinity of 10-3 will not be detected. However, late in a response when the affinities increase 1000 fold, the reaction with both the immunizing and cross reacting antigens will be detected.
E. Cellular events during 1o and 2o responses to T-dependent Ag
1. Primary response (Figure 7)
a) Lag phase - Clones of T and B cells with the appropriate antigen receptors bind antigen, become activated and begin to proliferate. The expanded clones of B cells differentiate into plasma cells which begin to secrete antibody.
b) Log phase - The plasma cells initially secrete IgM antibody since the C: heavy
chain gene is closest to the rearranged VDJ gene. Eventually some B cells switch from making IgM to IgG, IgA or IgE. As more B cells proliferate and differentiate into antibody secreting cells the antibody concentration increases exponentially.
c) Stationary phase - As antigen is depleted, T and B cells are no longer activated. In addition, mechanisms which down regulate the immune response come into play. Furthermore, plasma cells begin to die. When the rate of antibody synthesis equals the rate of antibody decay the stationary phase is reached.
d) Decline phase - When no new antibody is produced because the antigen is no
longer present to activate T and B cells and the residual antibody slowly is degraded, the decay phase is reached.
2. Secondary response (Figure 8)
Not all of the T and B cells that are stimulated by antigen during primary challenge
with antigen die. Some of them are long lived cells and constitute what is refer to as the memory cell pool. Both memory T cells and memory B cells are produced and memory T cells survive longer than memory B cells. Upon secondary challenge with antigen not only are virgin T and B cells activated, the memory cells are also activated and thus there is a shorter lag time in the secondary response. Since there is an expanded clone of cells being stimulated the rate of antibody production is also increased during the log phase of antibody production and higher levels are achieved. Also, since many if not all of the memory B cells will have switched to IgG (IgA or IgE) production, IgG is produced earlier in a secondary response. Furthermore since there is an expanded clone of memory T cells which can help B cells to switch to IgG (IgA or IgE) production, the predominant class of Ig produced after secondary challenge is IgG (IgA or IgE).
F. Ab response to T-independent Ag - Responses to T-independent Ag are characterized by the production of almost exclusively IgM Ab and no secondary response. Secondary exposure to the Ag results in another primary response to the Ag as illustrated in Figure 9.
G. Class switching
During an antibody response to a T-dependent antigen a switch occurs in the class of Ig produced from IgM to some other class (except IgD). Our understanding of the structure of the immunoglobulin genes, helps explain how class switching occurs (Figure 10).
During class switching another DNA rearrangement occurs between a switch site
(S:) in the intron between the rearranged VDJ regions and the C: gene and another
Fig 10
switch site before one of the other heavy chain constant region genes. The result of this recombination event is to bring the VDJ region close to one of the other constant region genes, thereby allowing expression of a new class of heavy chain. Since the same VDJ gene is brought near to a different C gene and since the antibody specificity is determined by the hypervariable regions within the V region, the antibody produced after the switch occurs will have the same specificity as before.
Cytokines secreted by T helper cells can cause the switch to certain isotypes.
H. Membrane and secreted immunoglobulin
The specificity of membrane immunoglobulin on a B cell and the Ig secreted by the plasma cell progeny of a B cell is the same. An understanding of how the specificity of membrane and secreted Ig from an individual B cell can be the same comes from an understanding of immunoglobulin genes (Figure 11). There are two potential polyA sites in the immunoglobulin gene. One after the exon for the last heavy chain domain and the other after the exons that code for the trans- membrane domains. If the first polyA site is used, the pre-mRNA is processed to produce a secreted protein. If the second polyA site is used, the premRNA is processed to produce a membrane form of the immunoglobulin. However, in all cases the same VDJ region is used and thus the specificity of the antibody remains the same. All C regions genes have these additional membrane pieces associated with them and thus after class switching other classes of immunoglobulins can be secreted or expressed on the surface of B cells.
Degradation of antibodies:
The carbohydrate content of immunoglobulin varies from 3 to 12% of the molecular weight of immunoglobulin. The carbohydrate content of immunoglobulin is mostly associated with heavy chain and rarely to light chains. The oligosaccharide units are attached to the heay cahain via N-glycoside linkage as N-acetile glucosamine residuesto aspergine in asequence of Asn-X-Ser, Asn-X-Thr of the immunoglobulin. X is any amino acid except proline. The oligosaccharide branch terminateswith Galactose to which sialic acid (NGNA). NGNA bound to galactose units are removed the enzyme nuraminidase and the immunoglobulins are bound to the receptors on hepatocytes with high affinity. The bound Ig are taken into the hepatocyte by receptor mediated endoctosis and are degraded.
Friday, January 23, 2009
Wednesday, January 21, 2009
Immunology
DNA VACCINES
Methods
The methods described next outline (1) selection of a vaccine gene, (2) construction of a DNA vaccine, (3) demonstration of vaccine protein expression.
Selection of a Vaccine Gene
The first aspect of designing a DNA vaccine is to decide whether the vaccine is intended to induce antibodies and protect against infection or generate cell-mediated responses for the treatment of an established infection or a tumor. Antibodies can neutralize an incoming pathogen and prevent infection, and they can inhibit the spread of newly replicated progeny to other cells. DNA vaccines designed to prevent infection most commonly aim to induce the neutralizing antibody. To produce neutralizing antibody the most appropriate target is usually a protein on the surface of the pathogen in its extracellular state. For example, DNA vaccination against the papillomavirus (PV) major capsid protein L1 induced high-titered PV-specific antibody responses and virtually completes protection in an animal model. Clinical trials of a human papillomavirus (HPV) L1-based virus-like particle vaccine have shown complete protection of vaccinated women against infection after a median follow-up time of 17 mo.
Certain antibodies may be useful for treating human tumors. For example, trastuzumab (Herceptin™), a monoclonal IgG1 antibody found on the transmembrane protein HER2/neu that is overexpressed in a subset of breast cancers, has shown significant therapeutic efficacy in patients whose tumors overexpress HER2/neu. Several mechanisms of action for trastuzumab have been proposed, including antibody-dependent cellular cytotoxicity (ADCC). Although trastuzumab is monoclonal, it is nevertheless possible that an effective anti-tumor response could be induced by vaccination to induce antibody responses to other tumor-associated cell transmembrane proteins.
DNA vaccines designed to eliminate an established infection or a tumor aim to produce cell-mediated immune responses. Suitable antigens for this purpose are expressed intracellularly during infection and/or after malignant progression. Ideally, the target protein is expressed at high levels in all infected or tumor cells. Viruses and other infectious agents evolve with their hosts in order to be able to persist in the face of an intact immune system. Several viruses express proteins that evade immune recognition. For example, the Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) contains a glycine-alanine repeat (GAr) domain that protects the protein from proteasomal degradation and major histochemical (MHC) class I presentation to cytotoxic T cells. Deletion of this domain, however, allows an EBNA1-specific immune response to be generated through vaccination. Furthermore, cytotoxic T cells that recognize epitopes upstream or downstream of the GAr domain also can kill EBVtransfected lymphobastoid cell lines that express wild-type EBNA1.
A safety consideration in designing anticancer vaccines for humans is the use of tumor-associated proteins and oncoproteins. For example, the HPV E6 and E7 oncoproteins are excellent targets for vaccination but also transform primary human epithelial cells in vitro, raising the possibility that they could do so in vivo. Because bacterially derived plasmids do not replicate in mammalian cells, transformation would require the persistence of the E6/E7 genes through integration into host chromatin. However, recent studies have shown that a DNA vaccine inoculated multiple times into rat muscle or reproductive organs was not integrated into the host genome at any time up to 45 d after the final inoculation. Nevertheless, the potential for in vivo oncogenicity can be overcome by constructing a DNA vaccine composed of multiple, short overlapping sequences (encoding approx 30 amino acids) ordered in a scrambled order relative to the wild-type protein. The scrambled gene encodes a dysfunctional oncogene that maintains all of its immunogenic epitopes.
Construction of DNA Vaccine
The construction of a DNA vaccine requires a plasmid backbone, such as pcDNA3 (Invitrogen Co., Carlsbad, CA). pcDNA3 contains the cytomegalovirus (CMV) promoter, a multiple cloning sequence and a downstream polyadenylation
signal from bovine growth hormone (BGH). The strong CMV promoter is most commonly used because it drives high-level expression of the vaccine gene, a known benefit for inducing a strong immune response. The gene to be targeted is often available in another construct but rarely with useful cloning sites. Therefore, PCR primers are designed to contain appropriate restriction sites upstream of the ATG and downstream of the stop codon for directional cloning into the plasmid backbone. Sometimes the target gene is not available but its sequence is known. In this situation, the first step is to generate cDNA from an organ or tissue that expresses relatively high levels of the gene and then proceed with PCR as described. By using different restriction sites upstream and downstream of the vaccine gene (directional cloning), the gene is cloned in the proper orientation relative to the regulatory elements for its expression.
The choice of restriction sites for cloning requires knowledge of whether the vaccine gene contains any internal site(s) for a restriction enzyme in the multiple cloning sequence. If no internal sites are present, the upstream primer is designed to contain about 20 nucleotides homologous to the 5'-end of the vaccine gene (including the ATG), the appropriate upstream restriction site, and a “tail” of several nucleotides that facilitates restriction endonuclease cleavage of the PCR product. The downstream primer is designed in an analogous fashion. PCR is performed, and the PCR product and plasmid vector are then cleaved with the same (or compatible) restriction enzymes. The PCR product may then be purified and ligated to the vector. Bacteria are transformed and colonies with the desired insert are identified.
Demonstration of Vaccine Protein Expression
To demonstrate that the newly constructed vector expresses the vaccine gene, expression is analyzed in transiently transfected mammalian cells. The pcDNA3 vector offers an advantage in this regard because it contains an SV40 origin of replication and therefore replicates to high-copy number in COS and other cell lines containing the SV40 T antigen. Control cells are transfected with the pcDNA3 vector containing no insert or an irrelevant gene. After 48–72 h, the transfected cells are harvested and either lysed for Western blotting or fixed for cellular immunostaining. Western blotting has the advantage of confirming the size of the expressed protein as well as showing its identity (by antibody staining). Cellular immunostaining has the advantage of revealing intracellular location, which is of particular importance when designing vaccine genes for intracellular trafficking.
A potential problem associated with Western blotting and cellular immunostaining is the lack of an antibody against the target protein. One solution is to add an epitope tag to the vaccine gene and use a commercially available antibody to the epitope tag. Another solution is to generate an antibody to the target protein. This can be achieved using a procedure that does not require a polypeptide immunogen. The vaccine gene is fused to a secretory signal (if it does not have one), and intracellular localization signals (e.g., nuclear localization if it has one) are deleted. The new construct is then used as a DNA vaccine in rabbits, which produce high titered polyclonal antisera. Expression of some proteins can be determined indirectly without the need for antibody.
Design of Plasmid DNA Constructs for Vaccines
An ideal vector for DNA vaccines should be safe in humans and easily produced at commercial scale. The most obvious safety issue for a DNA vaccine vector is the possibility of the plasmid integrating into the human chromosome. To minimize the risk of integration, the vector should not replicate in mammalian cells. Therefore, a vector should be chosen that does not contain a mammalian origin of replication (ORI). To further minimize the possibility of the vector integrating into the human genome, the vector sequence should be blasted into the human genomic sequence to make sure there are few, if any, strong homologies to human genes. Large numbers of molecules per dose are required for an effective DNA vaccine; therefore, a commercially viable vaccine must give high yields of plasmid, preferably through a simple production process. For this reason, bacterial plasmids propagated in the well-studied Gram-negative bacterium, Escherichia coli, is the most widely used production system. The bacterial replicon of a DNA vaccine vector has to allow high yield of plasmid molecules to meet the commercial needs, given that the issues of potential integration into the host genome have been addressed. Because pUC-based vectors yield between 500 and 700 copies per bacterial cell and are readily available as a result of their widespread use for recombinant protein expression, they have been the basis of many DNA vaccine vectors. Unlike their ancestral ColE1-type vectors, pUC plasmids do not require amplification to achieve high yields from the fermentation process, although the copy number can be increased by manipulating the growth rate of the host cells. From a process development perspective, so-called “runaway replication” vectors also seem to be an attractive choice for DNA vaccines. Such plasmids are initially low-copy but loss of replication control can be induced to cause accumulation of plasmid DNA to high levels, up to copy numbers near 1000. Induced amplification, usually through a temperature shift, would result in a slightly more complicated fermentation process to achieve the desired high DNA yields. These authors found no examples of such vectors being exploited for DNA vaccine production. In addition to carrying the sequences necessary for replication in bacteria, the plasmid must also contain a selectable marker for growth in E. coli, which is usually a drug resistance gene. This gene cannot confer resistance to penicillin or other β-lactam antibiotics as these can cause severe allergic reactions in humans, and the use of such antibiotics in the manufacture of products for humans is not permitted by the Food and Drug Administration (FDA). The selectable marker should be the only gene that is expressed in E. coli, because bacterial growth and plasmid production can be adversely affected by the expression of multiple genes, especially if the gene products are toxic. Taking all of these issues into consideration, a good DNA vaccine vector should be designed with minimal functions such that the only gene expressed in E. coli is the selectable marker and the only gene expressed in mammalian cells is the antigen. Any additional plasmid functions, such as an f1 (+) origin or lacZ gene, should be removed.
The mammalian promoter and polyA termination signal also need to be addressed. The amount of plasmid that is internalized in vivo has been estimated
to be in the picogram range after injection into mouse muscle and in the picogram to femtogram range in tissues from 1 to 7 d after intravenous delivery of DNA. Because the plasmid will not replicate in the cells, the amount of plasmid available for expression is very low. For this reason, a strong mammalian promoter/terminator should be chosen to drive expression of the antigen gene. Attention should be paid to the transcription terminator used in conjunction with the promoter. We have found that the choice of the transcription terminator/polyA signal can have a dramatic effect on the strength of the promoter (unpublished data). The combination of the cytomegalovirus (CMV) promoter and bovine growth hormone (BGH) terminator provides a high level of transcription. The vector that we designed consists of a pUC backbone, the CMV promoter with intron A, the BGH terminator, and a kanamycin resistance gene and has been described in previous papers. This vector can be obtained from Vical (www.vical.com). There are two forms of the vector: one requires that all translation signals be cloned in with the gene of interest, and the other is a fusion vector where the gene can be fused in-frame to the signal sequence of the human tissue-specific plasminogen activator (tPA) gene for secretion. There are also commercially available vectors that have been designed for DNA vaccines. For example, Invitrogen sells pVAX1, which is similar to our vector except that it contains the CMV promoter without intron A. It is a nonfusion vector and requires that the inserted gene contains Kozak translation initiation sequence (Kozak), an initiation codon (ATG), and a termination codon (TAA, TGA, or TAG). Both the vector from Invitrogen and the vectors from Vical are designed to stimulate cellular as well as humoral immune responses.
Although DNA immunization remains a very attractive method to induce immunity to a variety of pathogens, the transfection efficiency is still relatively low. This is especially true in species other than mice. One way of improving this efficiency is to temporarily permeabilize the cells to allow cellular uptake of DNA plasmids. One way to permeabilize cells is by electroporation. The principle behind electroporation is to temporarily permeabilize cell membranes to allow for increased uptake of large molecules such as plasmid DNA. Because electroporation permeabilizes membranes it can work in a wide variety of tissues including skin and muscle, the most commonly used tissues for administering DNA vaccines. In addition, electroporation has also been demonstrated to work effectively in several animal species including rabbits, pigs, sheep, and mice. Although electroporation has been used to deliver plasmids to different tissues it seems to be more effective in enhancing the level of expression in muscle tissue compared to skin tissue.
The development of needle-free injection originally stemmed from a general apprehension of needle injections, disease transmission by accidental needle-sticks, and the need for effective mass immunization. Naked DNA vaccines, as attractive and universal as they appear, have not produced robust immune responses in test systems. However, proof of principle for DNA vaccines has been validated with a number of vaccine candidates in a variety of test systems, and the concept of DNA vaccines as a generic platform for vaccines still remains viable and attractive. Many avenues are being explored to enhance the immunogenicity of DNA vaccines. The easiest and most straightforward approach that can be quickly transitioned to a clinical trial setting is vaccine delivery by a needle-free jet injector. This approach has shown much potential in a number of cases and should become the lead method for enhancing DNA vaccines. This approach requires no additional development, and with an expanding market and willingness from jet injector manufacturers to produce prefilled syringes, the technique should become feasible for larger phase II/phase III trials.
As an alternative to DNA-based vaccines, messenger RNA (mRNA)-based ccines present additional safety features: no persistence, no integration in the genome, no induction of autoantibodies. Moreover, mRNA which are generated by in vitro transcription, are easy to produce in large amounts and very high purity. This feature facilitates the good manufacturing practices process and guaranties batch-to-batch reproducibility. Vaccination can be achieved by several
delivery methods including direct injection of naked mRNA, injection of mRNA encapsulated in liposomes Gene Gun delivery of mRNA loaded on gold beads or in vitro transfection of the mRNA in cells followed by re-injection of the cells into the patients. Two of these technologies are being evaluated in human clinical trials: (1) in vitro mRNA-transfection of dendritic cells to be adoptively transferred and (2) direct injection of globin-stabilized mRNA..
Applications:
In some parts of sub-Saharan Africa, it is believed that most of the deaths attributed to malaria occur in infants. For this and other logistical reasons, if a malaria vaccine is developed and licensed, it will have to be administered to neonates or young infants, when they have maternally acquired antibodies against malaria parasite proteins. Pre-erythrocytic malaria vaccines in development rely on CD8+ T cells as immune effectors, yet some studies indicate that neonates do not mount optimal CD8+ T-cell responses. We report that BALB/c mice first immunized as neonates (7 d) with a Plasmodium yoelii circumsporozoite protein (PyCSP) DNA vaccine mixed with a plasmid expressing murine granulocyte macrophage-colony stimulating factor (DG) and boosted at 28 d with pox virus expressing PyCSP were protected (93%) as well as mice immunized entirely as adults (70%). Like adults, protection was dependent on CD8+ T cells and accompanied by excellent anti-PyCSP interferon-γ and cytotoxic T-lymphocyte responses. Mice born of immune mothers (previously exposed to P. yoelii parasites or immunized with the same vaccine given to the neonates) were also protected and had excellent T-cell responses. These data support assessment of this immunization strategy in neonates/young infants in areas where malaria exacts the greatest toll.
The DNA vaccine revolution has opened a vast scope of novel approaches for protective and therapeutic treatments of type I allergy. The ability of DNA vaccines to stimulate Th1 type reactions has rendered them a promising tool for immunotherapy of type I allergy.
T-lymphocytes are essential participants of adaptive immunity, essential for cellular and humoral recognition of foreign antigens. In pathogenic situations T cells may, however, also recognize self-antigens, causing detrimental autoimmune responses that ultimately lead to autoimmune disease. Experimental autoimmune encephalomyelitis (EAE) is a murine model for the autoimmune disease multiple sclerosis, in which T cells invade the central nervous system and destroy the myelin sheath around neuronal axon fibers. In some EAE systems, the sequence of the α- or β-chains of the pathogenic T-cell receptor is known and makes it possible to induce an immune response that eliminates these self-specific T cells. DNA vaccination allows the induction of an immune response to protect mice from the development of EAE.
DNA vaccines hold promise for generating protective immunity against a wide variety of pathogens.
Methods
The methods described next outline (1) selection of a vaccine gene, (2) construction of a DNA vaccine, (3) demonstration of vaccine protein expression.
Selection of a Vaccine Gene
The first aspect of designing a DNA vaccine is to decide whether the vaccine is intended to induce antibodies and protect against infection or generate cell-mediated responses for the treatment of an established infection or a tumor. Antibodies can neutralize an incoming pathogen and prevent infection, and they can inhibit the spread of newly replicated progeny to other cells. DNA vaccines designed to prevent infection most commonly aim to induce the neutralizing antibody. To produce neutralizing antibody the most appropriate target is usually a protein on the surface of the pathogen in its extracellular state. For example, DNA vaccination against the papillomavirus (PV) major capsid protein L1 induced high-titered PV-specific antibody responses and virtually completes protection in an animal model. Clinical trials of a human papillomavirus (HPV) L1-based virus-like particle vaccine have shown complete protection of vaccinated women against infection after a median follow-up time of 17 mo.
Certain antibodies may be useful for treating human tumors. For example, trastuzumab (Herceptin™), a monoclonal IgG1 antibody found on the transmembrane protein HER2/neu that is overexpressed in a subset of breast cancers, has shown significant therapeutic efficacy in patients whose tumors overexpress HER2/neu. Several mechanisms of action for trastuzumab have been proposed, including antibody-dependent cellular cytotoxicity (ADCC). Although trastuzumab is monoclonal, it is nevertheless possible that an effective anti-tumor response could be induced by vaccination to induce antibody responses to other tumor-associated cell transmembrane proteins.
DNA vaccines designed to eliminate an established infection or a tumor aim to produce cell-mediated immune responses. Suitable antigens for this purpose are expressed intracellularly during infection and/or after malignant progression. Ideally, the target protein is expressed at high levels in all infected or tumor cells. Viruses and other infectious agents evolve with their hosts in order to be able to persist in the face of an intact immune system. Several viruses express proteins that evade immune recognition. For example, the Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) contains a glycine-alanine repeat (GAr) domain that protects the protein from proteasomal degradation and major histochemical (MHC) class I presentation to cytotoxic T cells. Deletion of this domain, however, allows an EBNA1-specific immune response to be generated through vaccination. Furthermore, cytotoxic T cells that recognize epitopes upstream or downstream of the GAr domain also can kill EBVtransfected lymphobastoid cell lines that express wild-type EBNA1.
A safety consideration in designing anticancer vaccines for humans is the use of tumor-associated proteins and oncoproteins. For example, the HPV E6 and E7 oncoproteins are excellent targets for vaccination but also transform primary human epithelial cells in vitro, raising the possibility that they could do so in vivo. Because bacterially derived plasmids do not replicate in mammalian cells, transformation would require the persistence of the E6/E7 genes through integration into host chromatin. However, recent studies have shown that a DNA vaccine inoculated multiple times into rat muscle or reproductive organs was not integrated into the host genome at any time up to 45 d after the final inoculation. Nevertheless, the potential for in vivo oncogenicity can be overcome by constructing a DNA vaccine composed of multiple, short overlapping sequences (encoding approx 30 amino acids) ordered in a scrambled order relative to the wild-type protein. The scrambled gene encodes a dysfunctional oncogene that maintains all of its immunogenic epitopes.
Construction of DNA Vaccine
The construction of a DNA vaccine requires a plasmid backbone, such as pcDNA3 (Invitrogen Co., Carlsbad, CA). pcDNA3 contains the cytomegalovirus (CMV) promoter, a multiple cloning sequence and a downstream polyadenylation
signal from bovine growth hormone (BGH). The strong CMV promoter is most commonly used because it drives high-level expression of the vaccine gene, a known benefit for inducing a strong immune response. The gene to be targeted is often available in another construct but rarely with useful cloning sites. Therefore, PCR primers are designed to contain appropriate restriction sites upstream of the ATG and downstream of the stop codon for directional cloning into the plasmid backbone. Sometimes the target gene is not available but its sequence is known. In this situation, the first step is to generate cDNA from an organ or tissue that expresses relatively high levels of the gene and then proceed with PCR as described. By using different restriction sites upstream and downstream of the vaccine gene (directional cloning), the gene is cloned in the proper orientation relative to the regulatory elements for its expression.
The choice of restriction sites for cloning requires knowledge of whether the vaccine gene contains any internal site(s) for a restriction enzyme in the multiple cloning sequence. If no internal sites are present, the upstream primer is designed to contain about 20 nucleotides homologous to the 5'-end of the vaccine gene (including the ATG), the appropriate upstream restriction site, and a “tail” of several nucleotides that facilitates restriction endonuclease cleavage of the PCR product. The downstream primer is designed in an analogous fashion. PCR is performed, and the PCR product and plasmid vector are then cleaved with the same (or compatible) restriction enzymes. The PCR product may then be purified and ligated to the vector. Bacteria are transformed and colonies with the desired insert are identified.
Demonstration of Vaccine Protein Expression
To demonstrate that the newly constructed vector expresses the vaccine gene, expression is analyzed in transiently transfected mammalian cells. The pcDNA3 vector offers an advantage in this regard because it contains an SV40 origin of replication and therefore replicates to high-copy number in COS and other cell lines containing the SV40 T antigen. Control cells are transfected with the pcDNA3 vector containing no insert or an irrelevant gene. After 48–72 h, the transfected cells are harvested and either lysed for Western blotting or fixed for cellular immunostaining. Western blotting has the advantage of confirming the size of the expressed protein as well as showing its identity (by antibody staining). Cellular immunostaining has the advantage of revealing intracellular location, which is of particular importance when designing vaccine genes for intracellular trafficking.
A potential problem associated with Western blotting and cellular immunostaining is the lack of an antibody against the target protein. One solution is to add an epitope tag to the vaccine gene and use a commercially available antibody to the epitope tag. Another solution is to generate an antibody to the target protein. This can be achieved using a procedure that does not require a polypeptide immunogen. The vaccine gene is fused to a secretory signal (if it does not have one), and intracellular localization signals (e.g., nuclear localization if it has one) are deleted. The new construct is then used as a DNA vaccine in rabbits, which produce high titered polyclonal antisera. Expression of some proteins can be determined indirectly without the need for antibody.
Design of Plasmid DNA Constructs for Vaccines
An ideal vector for DNA vaccines should be safe in humans and easily produced at commercial scale. The most obvious safety issue for a DNA vaccine vector is the possibility of the plasmid integrating into the human chromosome. To minimize the risk of integration, the vector should not replicate in mammalian cells. Therefore, a vector should be chosen that does not contain a mammalian origin of replication (ORI). To further minimize the possibility of the vector integrating into the human genome, the vector sequence should be blasted into the human genomic sequence to make sure there are few, if any, strong homologies to human genes. Large numbers of molecules per dose are required for an effective DNA vaccine; therefore, a commercially viable vaccine must give high yields of plasmid, preferably through a simple production process. For this reason, bacterial plasmids propagated in the well-studied Gram-negative bacterium, Escherichia coli, is the most widely used production system. The bacterial replicon of a DNA vaccine vector has to allow high yield of plasmid molecules to meet the commercial needs, given that the issues of potential integration into the host genome have been addressed. Because pUC-based vectors yield between 500 and 700 copies per bacterial cell and are readily available as a result of their widespread use for recombinant protein expression, they have been the basis of many DNA vaccine vectors. Unlike their ancestral ColE1-type vectors, pUC plasmids do not require amplification to achieve high yields from the fermentation process, although the copy number can be increased by manipulating the growth rate of the host cells. From a process development perspective, so-called “runaway replication” vectors also seem to be an attractive choice for DNA vaccines. Such plasmids are initially low-copy but loss of replication control can be induced to cause accumulation of plasmid DNA to high levels, up to copy numbers near 1000. Induced amplification, usually through a temperature shift, would result in a slightly more complicated fermentation process to achieve the desired high DNA yields. These authors found no examples of such vectors being exploited for DNA vaccine production. In addition to carrying the sequences necessary for replication in bacteria, the plasmid must also contain a selectable marker for growth in E. coli, which is usually a drug resistance gene. This gene cannot confer resistance to penicillin or other β-lactam antibiotics as these can cause severe allergic reactions in humans, and the use of such antibiotics in the manufacture of products for humans is not permitted by the Food and Drug Administration (FDA). The selectable marker should be the only gene that is expressed in E. coli, because bacterial growth and plasmid production can be adversely affected by the expression of multiple genes, especially if the gene products are toxic. Taking all of these issues into consideration, a good DNA vaccine vector should be designed with minimal functions such that the only gene expressed in E. coli is the selectable marker and the only gene expressed in mammalian cells is the antigen. Any additional plasmid functions, such as an f1 (+) origin or lacZ gene, should be removed.
The mammalian promoter and polyA termination signal also need to be addressed. The amount of plasmid that is internalized in vivo has been estimated
to be in the picogram range after injection into mouse muscle and in the picogram to femtogram range in tissues from 1 to 7 d after intravenous delivery of DNA. Because the plasmid will not replicate in the cells, the amount of plasmid available for expression is very low. For this reason, a strong mammalian promoter/terminator should be chosen to drive expression of the antigen gene. Attention should be paid to the transcription terminator used in conjunction with the promoter. We have found that the choice of the transcription terminator/polyA signal can have a dramatic effect on the strength of the promoter (unpublished data). The combination of the cytomegalovirus (CMV) promoter and bovine growth hormone (BGH) terminator provides a high level of transcription. The vector that we designed consists of a pUC backbone, the CMV promoter with intron A, the BGH terminator, and a kanamycin resistance gene and has been described in previous papers. This vector can be obtained from Vical (www.vical.com). There are two forms of the vector: one requires that all translation signals be cloned in with the gene of interest, and the other is a fusion vector where the gene can be fused in-frame to the signal sequence of the human tissue-specific plasminogen activator (tPA) gene for secretion. There are also commercially available vectors that have been designed for DNA vaccines. For example, Invitrogen sells pVAX1, which is similar to our vector except that it contains the CMV promoter without intron A. It is a nonfusion vector and requires that the inserted gene contains Kozak translation initiation sequence (Kozak), an initiation codon (ATG), and a termination codon (TAA, TGA, or TAG). Both the vector from Invitrogen and the vectors from Vical are designed to stimulate cellular as well as humoral immune responses.
Although DNA immunization remains a very attractive method to induce immunity to a variety of pathogens, the transfection efficiency is still relatively low. This is especially true in species other than mice. One way of improving this efficiency is to temporarily permeabilize the cells to allow cellular uptake of DNA plasmids. One way to permeabilize cells is by electroporation. The principle behind electroporation is to temporarily permeabilize cell membranes to allow for increased uptake of large molecules such as plasmid DNA. Because electroporation permeabilizes membranes it can work in a wide variety of tissues including skin and muscle, the most commonly used tissues for administering DNA vaccines. In addition, electroporation has also been demonstrated to work effectively in several animal species including rabbits, pigs, sheep, and mice. Although electroporation has been used to deliver plasmids to different tissues it seems to be more effective in enhancing the level of expression in muscle tissue compared to skin tissue.
The development of needle-free injection originally stemmed from a general apprehension of needle injections, disease transmission by accidental needle-sticks, and the need for effective mass immunization. Naked DNA vaccines, as attractive and universal as they appear, have not produced robust immune responses in test systems. However, proof of principle for DNA vaccines has been validated with a number of vaccine candidates in a variety of test systems, and the concept of DNA vaccines as a generic platform for vaccines still remains viable and attractive. Many avenues are being explored to enhance the immunogenicity of DNA vaccines. The easiest and most straightforward approach that can be quickly transitioned to a clinical trial setting is vaccine delivery by a needle-free jet injector. This approach has shown much potential in a number of cases and should become the lead method for enhancing DNA vaccines. This approach requires no additional development, and with an expanding market and willingness from jet injector manufacturers to produce prefilled syringes, the technique should become feasible for larger phase II/phase III trials.
As an alternative to DNA-based vaccines, messenger RNA (mRNA)-based ccines present additional safety features: no persistence, no integration in the genome, no induction of autoantibodies. Moreover, mRNA which are generated by in vitro transcription, are easy to produce in large amounts and very high purity. This feature facilitates the good manufacturing practices process and guaranties batch-to-batch reproducibility. Vaccination can be achieved by several
delivery methods including direct injection of naked mRNA, injection of mRNA encapsulated in liposomes Gene Gun delivery of mRNA loaded on gold beads or in vitro transfection of the mRNA in cells followed by re-injection of the cells into the patients. Two of these technologies are being evaluated in human clinical trials: (1) in vitro mRNA-transfection of dendritic cells to be adoptively transferred and (2) direct injection of globin-stabilized mRNA..
Applications:
In some parts of sub-Saharan Africa, it is believed that most of the deaths attributed to malaria occur in infants. For this and other logistical reasons, if a malaria vaccine is developed and licensed, it will have to be administered to neonates or young infants, when they have maternally acquired antibodies against malaria parasite proteins. Pre-erythrocytic malaria vaccines in development rely on CD8+ T cells as immune effectors, yet some studies indicate that neonates do not mount optimal CD8+ T-cell responses. We report that BALB/c mice first immunized as neonates (7 d) with a Plasmodium yoelii circumsporozoite protein (PyCSP) DNA vaccine mixed with a plasmid expressing murine granulocyte macrophage-colony stimulating factor (DG) and boosted at 28 d with pox virus expressing PyCSP were protected (93%) as well as mice immunized entirely as adults (70%). Like adults, protection was dependent on CD8+ T cells and accompanied by excellent anti-PyCSP interferon-γ and cytotoxic T-lymphocyte responses. Mice born of immune mothers (previously exposed to P. yoelii parasites or immunized with the same vaccine given to the neonates) were also protected and had excellent T-cell responses. These data support assessment of this immunization strategy in neonates/young infants in areas where malaria exacts the greatest toll.
The DNA vaccine revolution has opened a vast scope of novel approaches for protective and therapeutic treatments of type I allergy. The ability of DNA vaccines to stimulate Th1 type reactions has rendered them a promising tool for immunotherapy of type I allergy.
T-lymphocytes are essential participants of adaptive immunity, essential for cellular and humoral recognition of foreign antigens. In pathogenic situations T cells may, however, also recognize self-antigens, causing detrimental autoimmune responses that ultimately lead to autoimmune disease. Experimental autoimmune encephalomyelitis (EAE) is a murine model for the autoimmune disease multiple sclerosis, in which T cells invade the central nervous system and destroy the myelin sheath around neuronal axon fibers. In some EAE systems, the sequence of the α- or β-chains of the pathogenic T-cell receptor is known and makes it possible to induce an immune response that eliminates these self-specific T cells. DNA vaccination allows the induction of an immune response to protect mice from the development of EAE.
DNA vaccines hold promise for generating protective immunity against a wide variety of pathogens.
Biochemistry
msc biochemistry is a two year program deals with the basics of biochemistry, biotechnology,molecular biology, immunology, microbiology & other related subjects. Any information regarding the above- u can contact me at amaras.vsl@gmail.com Or amaras_vsl@yahoo.co.in
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