疫苗的備製

Influenza virus growth in eggs流感病毒在雞胚上的生長 10 DECEMBER 2009 Before the development of cell culture, many viruses were propagated in embryonated chicken eggs. Today this method is most commonly used for growth of influenza virus. The excellent yield of virus from chicken eggs has led to their widespread use in research laboratories and for vaccine production. In fact the vast majority of influenza vaccines – both inactivated and infectious – are produced in chicken eggs. How is influenza virus propagated in eggs? 由於流感病毒在雞胚上生長的非常好，所以雖有細胞培養，但流感病毒的研究、疫苗備製仍是使用雞胚。尿膜囊液中富含蛋白酶，可將血凝素切開，使從HA0變HA1,HA2,才具感染力！若用細胞培養方式、則必需加蛋白酶，一般是加trypsin胰蛋白酶 The illustration below shows a cutaway view of an embryonated chicken egg. The different routes of inoculation into the egg are shown, as well as the different compartments in which viruses replicate. For propagation of influenza virus, pathogen-free eggs are used 11-12 days after fertilization. The egg is placed in front of a light source to locate a non-veined area of the allantoic cavity just below the air sac. This is marked with a pencil. After all the eggs have been ‘candled’ in this way, a small nick is made in the shell at this position using a jeweler’s scribe. Next, a hole is drilled at the top of the egg with a Dremel motorized tool. If this is not done, when virus is injected, the pressure in the air sac will simply force out the inoculum.使用受精11至12天的雞胚，用光源照射找出氣囊下方，無靜脈區的尿膜囊allantoic腔,用鉛筆標記，氣囊先打小洞洩壓，方便植入病毒。用27號針將病毒種入尿膜囊allantoic腔中，在37度培養2天。雞蛋上之兩個小洞則用石蠟封堵。。 After all the eggs have been nicked and drilled, they are inoculated with virus using a tuberculin syringe – a 1 ml syringe fitted with a 1/2 inch, 27 gauge needle. The needle passes through the hole in the shell, through the chorioallantoic membrane, and the virus is placed in the allantoic cavity, which is filled with allantoic fluid. The two holes in the shell are sealed with melted paraffin, and the eggs are placed at 37 degrees C for 48 hours. During the incubation period, the virus replicates in the cells that make up the chorioallantoic membrane. As new virus particles are produced by budding, they are released into the allantoic fluid. To harvest the virus, the top of the egg shell – the part covering the air sac – is removed. We used to have a special tool to do this, which was placed over the egg. When the handle of this tool is squeezed, it makes a neat crack around the top of the egg. It was then easy to remove the flap of shell with tweezers. The shell membrane and chorioallantoic membrane are pierced with a pipette which is then used to remove the allantoic fluid – about 10 ml per egg. Sufficient virus may be produced in one or two eggs (depending on the viral strain) to produce one 15 microgram dose of vaccine.病毒顆粒釋放入尿囊液中。用小吸管每顆蛋吸取10cc尿囊液，大約每兩顆蛋可製出一劑15microgram的疫苗。 . Influenza hemagglutination inhibition assay 流感的血凝抑制分析 Centers for Disease Control and Prevention have determined that some adults have serum cross-reactive antibodies to the new influenza H1N1 virus. One of the techniques used to reach this conclusion is the hemagglutination inhibition (HI) assay. How does this assay work?將兩兩稀釋之含有病毒之血清和定量之紅血球放入小井中，若有病毒之存在，紅血球會形成Lattice格子狀結構而在小井表面形成有層膜，若無病毒存在，則紅血球會沈澱在小井底部。這種方法30分內會有結果、根據稀釋倍數，可半定量病毒數。這是叫血凝試驗。 To understand the HI assay, we must discuss the hemagglutination assay. Influenza virus particles have an envelope protein called the hemagglutinin, or HA, which binds to sialic acid receptors on cells. The virus will also bind to erythrocytes (red blood cells), causing the formation of a lattice. This property is called hemagglutination, and is the basis of a rapid assay to determine levels of influenza virus present in a sample. To conduct the assay, two-fold serial dilutions of a virus are prepared, mixed with a specific amount of red blood cells, and added to the wells of a plastic tray. The red blood cells that are not bound by influenza virus sink to the bottom of a well and form a button. The red blood cells that are attached to virus particles form a lattice that coats the well. The assay can be performed within 30 minutes, and is therefore a quick indicator of the relative quantities of virus particles. In the figure above, two-fold dilutions of samples of different influenza viruses (A – H) were prepared, mixed with chicken red blood cells, and added to the wells of a 96-well plate. After 30 minutes the wells were photographed. Sample A causes hemagglutination up to the 1:256 dilution; therefore the HA titer of this virus stock is 256. The sample in row B contains no detectable virus, while that in row D has an HA titer of 512. The HA assay can be easily modified to determine the level of antibodies to influenza virus present in serum samples. In the CDC study cited below, the authors wished to determine whether stored serum samples contained antibodies to the new influenza H1N1 strain. First they obtained a preparation of one of the new influenza viruses, specifically A/California/04/2009 and determined its HA titer by the method described above. They added a fixed amount of virus to every well of a 96-well plate, equivalent to 32 – 64 HA units. Then they prepared two-fold dilutions of each serum to be tested, and added each dilution series along a row of wells. Finally, they added red blood cells and incubated for 30 minutes.但若將血凝試驗加以modified,則可用來半定量病人血清中的抗體量！方法叫血凝抑制試驗！小井中放的是某特定病毒，約相當32至64HA單位，其餘都和血凝試驗同，病人血清中若有特定抗體存在，則會先和病毒結合，那紅血球就會沈澱至底部！CDC説只要血凝試驗，稀釋到1：40仍能和病毒作用，（即紅血球沈澱），就可降低感染率一半！讓血凝抑制試驗成為流行病學上一有力工具！ The basis of the HI assay is that antibodies to influenza virus will prevent attachment of the virus to red blood cells. Therefore hemagglutination is inhibited when antibodies are present. The highest dilution of serum that prevents hemagglutination is called the HI titer of the serum. If the serum contains no antibodies that react with the new H1N1 strain, then hemagglutination will be observed in all wells. Likewise, if antibodies to the virus are present, hemagglutination will not be observed until the antibodies are sufficiently diluted. The CDC report contains the statement “…serum HI antibody titers of 40 are associated with at least a 50% reduction in risk for influenza infection or disease in populations”. A serum HI antibody titer of 40 means that at a dilution of 1:40, the serum blocked hemagglutination. By determining HI titers and comparing them with influenza attack rates in populations, it is possible to calculate the significance of the HI antibody titer with respect to susceptibility to influenza virus infection. When used in this manner, the HI assay is a powerful epidemiological tool. J Katz, PhD, K Hancock, PhD, V Veguilla, MPH, W Zhong, PhD, XH Lu, MD, H Sun, MD, E Butler, MPH, L Dong, MD, PhD, F Liu, MD, PhD, ZN Li, MD, PhD, J DeVos, MPH, P Gargiullo, PhD, N Cox, PhD (2009). Serum Cross-Reactive Antibody Response to a Novel Influenza A (H1N1) Virus After Vaccination with Seasonal Influenza Vaccine Morbid. Mortal. Weekly Rep., 58 (19), 521-524 Influenza microneutralization assay 28 MAY 2009另一個CDC用來測定成人血清是否存在和新H1N1病毒有交叉反應抗體的試驗：流感微中和試驗。病毒感染常造成細胞病變效應：細胞變圓，從培養皿上脫落，利用血清中是否存在抗體、中和掉病毒，而不造成細胞病變效應。從血清稀釋的量來半定量存在的抗體。 The microneutralization assay is another technique used by the Centers for Disease Control and Prevention to determine that some adults have serum cross-reactive antibodies to the new influenza H1N1 virus. Let’s explore how this assay works. Viral replication is often studied in the laboratory by infecting cells that are grown in plastic dishes or flasks, commonly called cell cultures. Many viruses kill such cells. Here is an example of HeLa cells being killed by poliovirus: The upper left panel shows uninfected cells, and the other panels show the cells at the indicated times after infection. As the virus replicates, infected cells round up and detach from the cell culture plate. These visible changes are called cytopathic effects. There is another way to visualize viral cell killing without using a microscope: by staining the cells with a dye. In the example shown below, cells have been plated in the small wells of a 96 well plate. One well was infected with virus, the other was not. After a period of incubation, the cells were stained with the dye crystal violet, which stains only living cells. It is obvious which cells were infected with virus and which were not. We can use this visual assay to determine whether a serum sample contains antibodies that block virus infection. A serum sample is mixed with virus before infecting the cells. If the serum contains antibodies that block viral infection, then the cells will survive, as determined by staining with crystal violet. If no antiviral antibodies are present in the serum, the cells will die. In its present form, this assay tells us only whether or not there are antiviral antibodies in a serum sample. To make the assay quantitative, two-fold dilutions of the serum are prepared, and each is mixed with virus and used to infect cells. At the lower dilutions, antibodies will block infection, but at higher dilutions, there will be too few antibodies to have an effect. The simple process of dilution provides a way to compare the virus-neutralizing abilities of different sera. The neutralization titer is expressed as the reciprocal of the highest dilution at which virus infection is blocked. In the example shown here, the serum blocks virus infection at the 1:2 and 1:4 dilutions, but less at 1:8 and not at all at 1:16. Each serum dilution was tested in triplicate, which allows for more accuracy. In this sample, the neutralization titer would be 4, the reciprocal of the last dilution at which infection was completely blocked. This explanation should clarify how the neutralization titers were obtained that are reported in the CDC study cited below. By the way, microneutralization simply means that the neutralization assay is done in a small format, such as a 96 well plate, instead of larger cell culture dishes. The authors of the CDC study note that “although serum hemagglutination inhibition (HI) antibody titers of 40 are associated with at least a 50% reduction in risk for influenza infection or disease in populations, no such correlate of protection exists for microneutralization antibody titers”. They used mathematical analysis to determine the relationship between HI and microneutralization titers. They found that in sera from children, an HI titer of 40 corresponded to a microneutralization titer of 40. However, in adults, an HI titer of 40 corresponded to a microneutralization titer of 160 or more. I don’t know the reason for this difference, but one possibility is that not all neutralizing antibodies in adult sera are able to inhibit hemagglutination. Understanding why this situation might occur will require a discussion of how antibodies block viral infection. J Katz, PhD, K Hancock, PhD, V Veguilla, MPH, W Zhong, PhD, XH Lu, MD, H Sun, MD, E Butler, MPH, L Dong, MD, PhD, F Liu, MD, PhD, ZN Li, MD, PhD, J DeVos, MPH, P Gargiullo, PhD, N Cox, PhD (2009). Serum Cross-Reactive Antibody Response to a Novel Influenza A (H1N1) Virus After Vaccination with Seasonal Influenza Vaccine Morbid. Mortal. Weekly Rep., 58 (19), 521-524 Detecting viruses: the plaque assay 6 JULY 2009用plaque斑塊分析來測定病毒量。 One of the most important procedures in virology is measuring the virus titer – the concentration of viruses in a sample. A widely used approach for determining the quantity of infectious virus is the plaque assay. This technique was first developed to calculate the titers of bacteriophage stocks. Renato Dulbecco modified this procedure in 1952 for use in animal virology, and it has since been used for reliable determination of the titers of many different viruses.斑塊測定最早是用於噬菌體測試，1952年Renato Dulbecco將它修改用於動物病毒量之測試，沿用至今。 To perform a plaque assay, 10-fold dilutions of a virus stock are prepared, and 0.1 ml aliquots are inoculated onto susceptible cell monolayers. After an incubation period, to allow virus to attach to cells, the monolayers are covered with a nutrient medium containing a substance, usually agar, that causes the formation of a gel. When the plates are incubated, the original infected cells release viral progeny. The spread of the new viruses is restricted to neighboring cells by the gel. Consequently, each infectious particle produces a circular zone of infected cells called a plaque. Eventually the plaque becomes large enough to be visible to the naked eye. Dyes that stain living cells are often used to enhance the contrast between the living cells and the plaques. Only viruses that cause visible damage of cells can be assayed in this way. An example of plaques formed by poliovirus on a monolayer of HeLa cells is shown at left. In this image, the cells have been stained with crystal violet, and the plaques are readily visible where the cells have been destroyed by viral infection. The titer of a virus stock can be calculated in plaque-forming units (PFU) per milliliter. To determine the virus titer, the plaques are counted. To minimize error, only plates containing between 10 and 100 plaques are counted, depending on the size of the cell culture plate that is used. Statistical principles dictate that when 100 plaques are counted, the sample titer will vary by plus or minus 10%. Each dilution is plated in duplicate to enhance accuracy. In the example shown below, there are 17 plaques on the plate made from the 10-6 dilution. The titer of the virus stock is therefore 1.7 x 108 PFU/ml. Next we’ll consider how the plaque assay can be used to prepare clonal virus stocks, a step that is essential for studying viral genetics. Dulbecco, R., & Vogt, M. (1953). Some problems of animal virology as studied by the plaque technique. Cold Spring Harbor Symp. Quant. Biol., 18, 273-279 How many viruses are needed to form a plaque? 8 JULY 2009動物病毒，一個病毒顆粒就足以造成一個斑塊。 The plaque assay is an essential tool for determining virus titers. The concept is simple: virus infection is restricted to neighboring cells by a semisolid overlay. By counting the number of plaques, the virus titer can be calculated in PFU per ml. A key question is: how many viruses are needed to form a single plaque? For most animal viruses, one infectious particle is sufficient to initiate infection. This conclusion can be reached by studying the relationship between the number of infectious virus particles and the plaque count. A linear relationship means that one infectious particle can form a plaque. In this case the virus is said to infect cells with one-hit kinetics. This concept is illustrated below. In this figure, the number of plaques produced by a virus with one-hit kinetics or two-hit kinetics is plotted versus the relative concentration of the virus. There are some examples of viruses with two-hit kinetics: in other words, two different types of viral particles must infect a cell to initiate the infectious cycle. Examples include the genomes of some (+) strand RNA viruses of plants, which consists of two RNA molecules that are packaged in different particles. The dose-response curve of such viruses is parabolic rather than linear. When a single virus particle can form a plaque, the viral progeny within the plaque are clones. Virus stocks prepared from a single plaque are called plaque purified virus stocks. To prepare such virus stocks, the tip of a small pipette is inserted into the agar overlay above the plaque. The plug of agar is removed and placed in buffer. The viruses within the agar plug move into the buffer, which can then be used to infect cultured cells. To ensure purity, this process is usually repeated at least one more time. Plaque purification is used extensively in virology to establish clonal virus stocks. The ability to prepare clonal virus stocks was an essential development that permitted genetic analysis of viruses. Measurement of viruses by end-point dilution assay 13 JULY 2009 The plaque assay is a terrific method for determining virus titers, but it doesn’t work for all viruses. Fortunately there are several alternative methods available, including the end-point dilution assay. The end-point dilution assay was used to measure virus titer before the development of the plaque assay, and is still used for viruses that do not form plaques. Serial dilutions of a virus stock are prepared and inoculated onto replicate cell cultures, often in multi-well formats (e.g. 96 well plastic plates). The number of cell cultures that are infected is then determined for each virus dilution, usually by looking for cytopathic effect. In this example of an end-point dilution assay, 10 monolayer cell cultures were infected with each virus dilution. After an incubation period, plates that displayed cytopathic effects were scored with a +. At high dilutions, none of the cell cultures are infected because no particles are present. At low dilutions, every cell culture is infected. Half of the cell cultures showed cytopathic effects at the 10-5 dilution. This is the end point: the dilution of virus at which 50% of the cell cultures are infected. This number can be calculated from the data and expressed as 50% infectious dose (ID50) per milliliter. The virus stock in this example contains 105 ID50 per ml. In real life, the 50% end point does not usually fall exactly on a dilution as shown in the example. Therefore statistical procedures are used to calculate the end point of the titration. End-point dilution methods can also be used to determine the virulence of a virus in animals. The same approach is used: serial dilutions of viruses are made and inoculated into multiple test animals. Infection of the animal can be determined by death or clinical symptoms such as fever, weight loss, or paralysis. The results are expressed as 50% lethal dose (LD50) per ml or 50% paralytic dose (PD50) per ml when lethality or paralysis are used as end points. The following example illustrates the use of end point dilution to measure the lethality of poliovirus in mice. Eight mice were inoculated per virus dilution, and the end point was death. The statistical method of Reed and Muench was used to determine the 50% end point. In this method, the results are pooled, and the mortality at each dilution is calculated. The 50% end point, which falls between the fifth and sixth dilutions, is calculated to be 10-6.5. Therefore the virus sample contains 106.5 LD50 units. Reed, L.J., & Muench, H. (1938). A simple method of estimating fifty percent endpoints. Am. J. Hygiene, 27, 493-497