Method for evaluating the hardness of virus particles and method for early detection of changes in the properties of antigen particles

Abstract

In the case of microstructures that are observable with an electron microscope, such as vaccine antigen particles, there is a challenge in evaluating their hardness, such as whether they are "hard" or "soft," because their scale is so small. This invention has been made in view of these circumstances and provides a method for evaluating the hardness of microstructures in a relatively simple manner. [Solution] A fixed sample was prepared by immobilizing a predetermined microstructure according to a conventional method, and an unfixed sample was prepared without immobilization treatment. The difference in shape due to the effect of surface tension was observed using a transmission electron microscope, and the greater the degree of structural collapse of the microstructure in the unfixed sample compared to the fixed sample, the softer the microstructure was evaluated to solve the problem.

Description

Technical Field

[0001] The present invention relates to a method for evaluating the hardness of microstructures and a method for early detection of changes in the properties of antigen particles.

Background Art

[0002] Conventionally, microparticles of about 0.1 μm that are difficult to observe with an optical microscope have been observed using an electron microscope.

[0003] Especially when the observation target is a virus, observation with a transmission electron microscope plays an important role in capturing its appearance, knowing its shape, and directly grasping its dynamics (Non-Patent Document 1).

[0004] In observation using an electron microscope, since the sample is placed under vacuum, chemical treatment, that is, fixation treatment, is performed in advance as necessary so that the observation target does not warp due to drying. For example, when performing negative staining and observing a virus with a transmission electron microscope, as a general fixation method, fixation with 2% glutaraldehyde (Patent Document 1) or fixation with 4% formalin is performed (Patent Document 2).

[0005] As an inactivator for preparing a virus as a vaccine antigen, formalin or glutaraldehyde of 0.1% or less is used. This is to prevent excessive structural changes of the virus and maintain its antigenicity. However, when antigen particles of a vaccine such as an inactivated pathogen or a constituent fragment of the pathogen deteriorate over time and the antigenicity decreases, as a sign thereof, the structural hardness (robustness) is gradually impaired.

[0006] Therefore, if the microstructures to be evaluated are used as antigen particles of a vaccine and it is possible to know that a change has occurred in the hardness of these antigen particles at as early a stage as possible in the production of the vaccine, it becomes possible to detect a decrease in antigen activity at an early stage.

[0007] Furthermore, if we can understand the effect of a certain treatment on the morphology of viral particles, it becomes possible to develop a more optimized vaccine by reviewing treatments that cause morphological changes that reduce antigenicity.

[0008] However, in the case of microstructures that are observable with an electron microscope, such as vaccine antigen particles, their scale is so small that it is difficult to evaluate their physical hardness, and there is a need for evaluation methods.

[0009] While it may be possible to visually observe changes in the properties of antigen particles by observing differences in shape through the observation of immobilized samples, detection requires a change significant enough to detect the change in shape. Specifically, the antigen particles must undergo a change in physical properties (hardness) to the point where they cannot withstand the surface tension caused by drying even after immobilization, and this requires a certain period of time. [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] Japanese Patent Publication No. 2015-15954 [Patent Document 2] International Publication No. 00 / 070055 [Non-patent literature]

[0011] [Non-Patent Document 1] RJKuhn, W.Zhang, MGRossmann, SVPletnev, J.Corver, E.Lenches et al., “Structure of dengue virus: implications for flavivirus organization, maturation, and fusion”, Cell, Vol.108, No.5, USA), Cell Press, 2002, p.717-725. [Overview of the project] [Problems that the invention aims to solve]

[0012] As mentioned above, in the case of microstructures that are the target of observation with an electron microscope, such as vaccine antigen particles, there is a problem in that it is difficult to evaluate hardness, such as "hard" or "soft," because their scale is too small. The present invention has been made in view of these circumstances. Virus particles This provides a relatively simple method for evaluating the hardness of a material.

[0013] Furthermore, the present invention also provides a method that can detect changes in the properties of antigen particles accompanied by a decrease in antigenicity earlier than conventional methods that involve immobilizing a sample and then observing it with an electron microscope. [Means for solving the problem]

[0014] In order to solve the above-mentioned conventional problems, the present inventors have [1] predetermined Virus particles Immobilized samples and unimmobilized samples were prepared, and the differences in shape due to the effect of surface tension were observed using a transmission electron microscope. Virus particles The greater the degree of structural collapse compared to the immobilized sample, the more likely it is that the same Virus particles It is considered soft. Virus particles We have discovered a method for evaluating hardness.

[0015] Furthermore, according to the present invention Virus particles The method for evaluating hardness also has the following characteristics: [2] Virus particles A method for evaluating the hardness, which is the physical robustness of, prescribed Virus particles A sample that has undergone the prescribed processing, and Virus particles A sample that has not undergone the aforementioned predetermined treatment is prepared, and an unfixed sample is made from each sample without immobilization treatment. The difference in shape due to the effect of surface tension is observed using a transmission electron microscope, and the difference in shape of one unfixed sample compared to the other unfixed sample. Virus particlesThe greater the degree of structural collapse of Virus particles is evaluated to have become soft due to the implementation or non - implementation of the predetermined treatment Virus particles is a method for evaluating the hardness of [3] The Virus particles is a method for evaluating the physical robustness of Virus particles as described in [1] or [2], characterized in that it is an antigen particle of a vaccine. [4] A method for early detection of the decrease in immunogenicity and titer of whole - particle virus antigen as an antigen particle contained in a vaccine, comprising preparing a sample of the antigen particle without performing an immobilization treatment and observing the structural collapse due to the influence of surface tension with a transmission electron microscope. It is a method for early detection of changes in the properties of antigen particles, characterized by this.

Effect of the Invention

[0016] According to the method for evaluating the hardness of Virus particles according to the present invention, an immobilized sample obtained by subjecting a predetermined Virus particles to an immobilization treatment and an un - immobilized sample without the immobilization treatment are prepared, and the difference in shape due to the influence of surface tension is observed with a transmission electron microscope. The greater the degree of structural collapse of Virus particles in the un - immobilized sample compared to the immobilized sample, the more Virus particles is evaluated to be soft. Therefore, Virus particles it is possible to provide a method for evaluating the hardness of Virus particles comparatively simply.

[0017] Also, Virus particles it is a method for evaluating the hardness, which is the physical robustness of A sample in which a predetermined Virus particles has been subjected to a predetermined treatment and a sample in which the Virus particles has not been subjected to the predetermined treatment are prepared, and for each sample, an un - immobilized sample without the immobilization treatment is prepared, and the difference in shape due to the influence of surface tension is observed with a transmission electron microscope. For one un - immobilized sample, Virus particlesThe greater the degree of structural collapse, the greater the amount of material in the sample used to prepare the other unfixed sample. Virus particles Since it was decided that the softening was due to the implementation or non-implementation of the prescribed treatment, Virus particles We can learn about the effect on hardness, and furthermore, Virus particles The cause of the change in physical properties that occurred can be easily estimated.

[0018] Also, the above Virus particles If we assume that these are vaccine antigen particles, then we can evaluate the hardness of the vaccine antigen particles.

[0019] Furthermore, according to the method for early detection of changes in the properties of antigen particles according to the present invention, since the sample is prepared without immobilizing the antigen particles and structural collapse due to the effect of surface tension is observed using a transmission electron microscope, it is possible to provide a method that can detect changes in the properties of antigen particles accompanied by a decrease in antigenicity, in particular the decrease in immunogenicity and titer of whole-particle viral antigens, earlier than conventional methods that involve immobilizing the sample and then observing it with an electron microscope. [Brief explanation of the drawing]

[0020] [Figure 1] This is an explanatory diagram showing the antigen particle image of B / MA SK_1 according to the first embodiment. [Figure 2] This is an explanatory diagram showing the antigen particle image of B / MA SK_2 according to the first embodiment. [Figure 3] This is an explanatory diagram showing the antigen particle image of the hepatitis A vaccine according to the second embodiment. [Modes for carrying out the invention]

[0021] The present invention relates to a method for evaluating the hardness of microstructures, and more particularly to a method for evaluating the hardness of microstructures that allows for relatively simple evaluation of the hardness of microstructures.

[0022] In this evaluation method, immobilized samples are prepared by applying an immobilization treatment to a specified microstructure according to a conventional method, and unimmobilized samples are prepared without the immobilization treatment.

[0023] The following describes in detail the conventional method for preparing the sample, but any procedure commonly used for electron microscopy observation of suspension protein solutions is acceptable, and the invention is not limited to the embodiments described below.

[0024] (Immobilized sample) (1) Fixing the sample The method used to fix microstructures is not particularly limited, as long as it is a commonly used method. For example, fixation with glutaraldehyde can be employed. More specifically, the sample can be fixed by adding 2% glutaraldehyde to a suspension of proteins.

[0025] (2) Stirring of the sample solution First, thoroughly mix the sample solution. Cool the sample on ice, mix it by inverting it to avoid creating bubbles, and if necessary, mix it carefully using a vortex mixer.

[0026] (3) Hydrophilization treatment of the grid The grid with the support film to be used should be treated to make it hydrophilic. The process from hydrophilic treatment to placing the sample should be carried out as quickly as possible. If it takes too long, it is best to repeat the hydrophilic treatment.

[0027] (4) Securing the grid to the grid holder The hydrophilic treated grid with a support membrane is fixed to the grid holder.

[0028] (5) Collection of sample solution Gently mix the sample to be applied to the grid again, and collect 5-7 μL of the sample solution using a micropipette. Any volume between 5 and 7 μL is acceptable as long as the volume is consistent across all samples.

[0029] (6) Apply When a droplet of sample solution is formed at the tip of a micropipette and brought close to a grid with a support film, the droplet is drawn in and adheres to the grid.

[0030] (7)Stand still Next, let the grid, after applying the sample solution, stand for about 5 minutes.

[0031] (8) Absorption of the sample solution Next, use a piece of filter paper cut to an appropriate size to absorb the sample liquid from the grid. After absorbing the sample liquid, press the filter paper against the part of the grid holder that is holding the grid for 30 seconds.

[0032] (9) Applying the staining solution After drawing up the sample solution and letting it stand for 5 minutes, place an appropriate amount (7-10 μL) of staining solution onto the grid using a micropipette. Ensure that the amount of staining solution is consistent across all samples.

[0033] (10) Dyeing Let it stand for about 5 minutes before staining.

[0034] (11) Absorption of staining solution Next, use filter paper to absorb the staining solution from the grid. After absorbing the staining solution, press the filter paper against the part of the grid holder that is holding the grid for 30 seconds.

[0035] (12) Left to dry Leave it for at least 20 minutes to air dry.

[0036] Furthermore, the preparation of unfixed samples without immobilization treatment can be achieved by preparing the sample without performing the "(1) Sample Fixation" step.

[0037] The microstructures described here are not particularly limited and may be biologically derived structures or other structures. However, the method for evaluating the hardness of microstructures according to the present invention is highly useful for biologically derived structures, particularly structures composed of proteins, and examples of such microstructures include inactivated pathogens, constituent fragments of pathogens, and vaccine antigen particles.

[0038] The immobilized and unimmobilized samples prepared in this manner are subjected to a transmission electron microscope to observe the differences in shape resulting from the influence of surface tension.

[0039] Furthermore, a distinctive feature of this invention is that the greater the degree of structural collapse of the microstructure in an unfixed sample compared to a fixed sample, the softer the microstructure is considered to be, thereby allowing for a relatively simple evaluation of the hardness of the microstructure.

[0040] To explain in more detail, when antigen particles treated with different concentrations of drugs during the vaccine manufacturing process are prepared as immobilized and unimmobilized samples and observed, the greater the degree of structural collapse of the microstructure in the unimmobilized sample compared to the immobilized sample, the softer the microstructure can be considered to be.

[0041] Furthermore, in this invention, a sample in which the microstructure has been subjected to a predetermined treatment and a sample in which the microstructure has not been subjected to the predetermined treatment are prepared, and an unfixed sample is made from each sample. The difference in shape due to the effect of surface tension is observed using a transmission electron microscope, and the greater the degree of structural collapse of the microstructure in the other unfixed sample compared to one unfixed sample, the more it can be evaluated that the microstructure in the sample used to prepare the other unfixed sample is relatively softer. In addition, two unfixed samples are prepared with or without the predetermined treatment, and the greater the degree of structural collapse between the unfixed samples, the more it can be evaluated that the microstructure in the sample used to prepare the other unfixed sample is relatively softer.

[0042] When performing such an evaluation, first prepare a sample in which a predetermined microstructure has been subjected to a predetermined treatment, and a sample in which the same microstructure has not been subjected to the predetermined treatment.

[0043] The prescribed treatment is not particularly limited; any treatment that can prepare both a treated sample and an untreated sample is acceptable. Examples of such treatments include treatment over time, treatment related to storage conditions, and treatment involving the addition of chemicals or other substances.

[0044] To explain in more detail, examples of treated and untreated samples that have undergone specific processing regarding time-dependent treatment and storage conditions include antigen particles contained in a vaccine stored at 37°C for one month and antigen particles contained in a vaccine stored at 37°C for three months.

[0045] Thus, the method for evaluating the hardness of microstructures according to the present invention, having the above-described configuration, provides a method for evaluating the hardness of microstructures that allows for relatively simple evaluation of the hardness of microstructures.

[0046] Furthermore, according to the method for evaluating the hardness of microstructures according to the present invention, if the only substantial difference between a treated sample and an untreated sample is the presence or absence of a predetermined treatment, the softening of the microstructure's hardness may be evaluated as being due to the predetermined treatment.

[0047] Furthermore, in the method for evaluating the hardness of microstructures according to the present invention, in addition to unfixed treated or untreated samples, fixed treated or untreated samples can also be observed in the same manner, and the hardness can be evaluated by taking into account the observation results of these four samples.

[0048] In this way, by comparing immobilized samples with unimmobilized samples, comparing unimmobilized treated samples with unimmobilized untreated samples, and performing evaluations through a combination of these comparisons, the causes of changes in physical properties that occur in microstructures can be easily estimated.

[0049] Furthermore, the present invention also provides a method for detecting changes in the properties of antigen particles early on, by interpreting changes in the hardness of antigen particles, which are microstructures, as a sign of decreased antigen activity.

[0050] The method for early detection of changes in the properties of antigen particles according to the present invention is characterized in that a sample is prepared with the antigen particles unfixed without any immobilization treatment, and structural collapse due to the effect of surface tension is observed using a transmission electron microscope.

[0051] As mentioned earlier, when vaccine antigen particles, such as inactivated pathogens or fragments of pathogens, deteriorate and their antigenicity decreases, a gradual loss of structural rigidity (robustness) is a warning sign of this deterioration.

[0052] Therefore, if we can identify the changes in the hardness of the antigen particles in the vaccine, using the microstructures being evaluated as vaccine antigen particles, we can detect any subsequent decrease in antigen activity as early as possible.

[0053] The present invention will be described in detail below with reference to examples, but the present invention is not limited in any way to these examples.

[0054] Example 1: Egg-derived precipitated influenza whole particle vaccine Whole-particle influenza vaccine, obtained by inoculating chicken eggs with a seed influenza virus, culturing it, and purifying it, was used as a sample.

[0055] In the vaccine manufacturing process, B / MA SK_1 treated with 0.02% formalin and B / MA SK_2 treated with 0.08% formalin were prepared as immobilized samples according to standard methods, and unimmobilized samples were prepared. The differences in shape due to the effect of surface tension were observed using a transmission electron microscope. It has been found that B / MA SK_1 has lower immunogenicity in mice compared to B / MA SK_2.

[0056] Figure 1(a) shows an image of antigen particles from a sample immobilized with B / MA SK_1, and Figure 1(b) shows an image of antigen particles from an unimmobilized sample that was not immobilized with B / MA SK_1. Similarly, Figure 2(a) shows an image of antigen particles from a sample immobilized with B / MA SK_2, and Figure 2(b) shows an image of antigen particles from an unimmobilized sample that was not immobilized with B / MA SK_2. All of these Figures 1(a), 1(b), 2(a), and 2(b) are negative staining images obtained using the method according to the present invention.

[0057] As shown in Figures 1(a) and 1(b), when comparing B / MA SK_1 immobilized samples treated according to conventional methods with unimmobilized samples, it was confirmed that the unimmobilized samples were clearly more fragmented.

[0058] On the other hand, as shown in Figures 2(a) and 2(b), for B / MA SK_2, no clear difference in shape due to the influence of surface tension was observed when comparing immobilized samples treated according to conventional methods with unimmobilized samples that had not undergone immobilization treatment.

[0059] Furthermore, as shown in Figures 1(a) and 2(a), no difference in shape can be observed when comparing the immobilized samples of B / MA SK_1 and B / MA SK_2. However, as shown in Figures 1(b) and 2(b), a difference in shape can be observed by comparing the unimmobilized samples.

[0060] In other words, structural hardness (robustness), which cannot be evaluated by conventional methods, can be evaluated by observing the unfixed sample in this invention.

[0061] Furthermore, B / MA SK_1 is thought to have lower structural rigidity (robustness) compared to B / MA SK_2, and therefore cannot maintain its epitope, resulting in lower immunogenicity in mice.

[0062] Example 2: Hepatitis A vaccine Whole-particle hepatitis A vaccine, obtained by inoculating cultured cells with hepatitis A virus as a seed, culturing, and purifying the cells, was used as a sample.

[0063] A comparison was made between a vaccine stored at 37°C for one month (as a sample without the prescribed treatment) and a vaccine stored at 37°C for an additional two months (a total of three months) as part of the prescribed treatment.

[0064] Figure 3(a) shows an image of antigen particles in an unfixed vaccine sample stored at 37°C for one month without the prescribed treatment, and Figure 3(b) shows an image of antigen particles in an unfixed vaccine sample stored at 37°C for a further two months with the prescribed treatment. Both Figures 3(a) and 3(b) are negative staining images obtained using the method according to the present invention.

[0065] As shown in Figure 3(a), vaccine antigen particles stored at 37°C for one month without the prescribed treatment showed almost no structural degradation and retained their shape. However, as shown in Figure 3(b), vaccine antigen particles stored at 37°C for an additional two months as part of the prescribed treatment changed to a flattened shape, and some particles showed a significant degree of structural degradation and did not retain their shape.

[0066] Based on these findings, it was determined that the vaccine antigen particles became unstable (plastically deformed) after being stored at 37°C for an additional two months (a total of three months).

[0067] Finally, the above-described embodiments represent only one aspect of the present invention, and the present invention is not limited to the embodiments described above. Therefore, it goes without saying that various modifications are possible depending on the design, etc., even in embodiments other than those described above, as long as they do not depart from the technical spirit of the present invention. [Industrial applicability]

[0068] According to this disclosure, a fixed sample is prepared by immobilizing a predetermined microstructure according to a conventional method, and an unfixed sample is prepared without immobilization treatment. The difference in shape due to the effect of surface tension is observed using a transmission electron microscope, and the greater the degree of structural collapse of the microstructure in the unfixed sample compared to the fixed sample, the softer the microstructure is evaluated to be. Thus, a method for evaluating the hardness of a microstructure that allows for relatively simple evaluation of the hardness of the microstructure can be provided.

Claims

1. A method for evaluating the hardness of virus particles involves preparing immobilized samples of a given virus particle and unimmobilized samples without immobilization treatment, observing the difference in shape due to surface tension using a transmission electron microscope, and evaluating the softness of the virus particle if the degree of structural collapse of the virus particle in the unimmobilized sample is greater than that of the immobilized sample.

2. A method for evaluating the hardness, which is the physical robustness of a virus particle, A method for evaluating the hardness of virus particles, comprising preparing a sample in which a predetermined treatment has been applied to a predetermined virus particle and a sample in which the same virus particle has not been applied to the predetermined treatment, preparing an unfixed sample from each sample without immobilization treatment, observing the difference in shape due to the effect of surface tension using a transmission electron microscope, and evaluating that the greater the degree of structural collapse of the virus particles in the other unfixed sample compared to one unfixed sample, the softer the virus particles in the sample used to prepare the other unfixed sample have become due to the application or non-application of the predetermined treatment.

3. The method for evaluating the physical robustness of a virus particle according to claim 1 or 2, characterized in that the aforementioned virus particle is an antigen particle of a vaccine.

4. A method for early detection of a decrease in the antigenicity of antigen particles contained in a vaccine, characterized by preparing a sample of the antigen particles without immobilization treatment and observing structural collapse due to the effect of surface tension using a transmission electron microscope.

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