METHOD OF PREPARING A VACCINE

Abstract: A vaccine against an infectious agent is prepared by a method in which infectious cells, which may be virus-infected cells, bacteria or fungi, are reacted with an antibody to the antigen required for the vaccine, to provide a solution containing antigen-antibody complexes, separating the precipitable macroscopic fraction of the complexes from the solution, and separating soluble complexes from the solution by means of centrifugation, the separated soluble complexes comprising at least one active constituent of the required vaccine. The vaccine may also include the precipitable fraction of the complexes.

Description: METHOD OF PREPARING A VACCINE There is need for vaccines which are substantially free of nucleic acid which may have oncogenic potential in immunised subjects. A known method of achieving this objective is to extract the required antigenic material from infectious cells by complexing cell material with antibody to the required antigen and subsequent washing the immune complexes to remove unco plexed antigens, excess antibody, host cell components, serum components and nucleic acid. Known methods of preparation of immune complex vaccines have depended on separation of precipitable antigen- antibody complexes which can be identified by the naked- eye or following low speed centrifugation where a pellet of antigen-antibody complexes may be visible to the naked-eye. Physical precipitation of antigen by antibody occurs at optimal concentrations of both reagents and if there is excess antigen or excess antibody there may be formation either of soluble antigen-antibody complexes or redissolution of antigen. Soluble immune complexes are discarded with solution from which the precipitable complexes have been separated. The soluble complexes contain valuable antigenic properties and can therefore make a substantial contribution to the effectiveness of a vaccine. The present invention relates to a method of vaccine preparation by means of which the soluble complexes form at least one active component of the vaccine. In this connection soluble complexes are to be understood as those which can be induced to form a deposit under appropriately high gravitational force. References to infectious cells herein are to be understood as including bacteria and fungi, as well as host cells infected with a virus. According to the invention there is provided a method of preparing a vaccine against an infectious agent, in which an infectious cell starting material capable of acting as said agent is reacted with an antibody to the antigen required for the vaccine, to form antigen-antibody complexes which are suspended in a solution, and separating the precipitable fraction of said complex from said solution, characterised in that soluble immune complexes in said solution are separated therefrom by ultracentrifugation, said soluble immune complexes providing an active constituent of the vaccine. The following example describes preparation of a vaccine against cytomegalovirus, using ultracentrifuged soluble immune-complexes. A cytomegalovirus antigen was prepared by infecting human embryo lung cells with cytomegalovirus at a multiplicity of 0.1 pfu per cell. The infected cells ere incubated in a container for 7 days at 37°C, and harvested by removal from the container by shaking, washing the cells in phosphate buffered saline and finally resuspending the cells the cells in phosphate buffered saline at a concentration of 10 7 to 4 x 10 7 cells per ml to give an infectious cell starting material. The cytomegalovirus-infected cells forming the starting material were disrupted by freezing and thawing or by ultrasonic vibration. The preparation was then inactivated by addition of Nonidet, to a concentration of 1%, which also serves to strip important antigenic material from the envelope of the virus particles. Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) were digested by addition of DNase at a concentration of 80μg/ml and RNase at a concentration of 800μg\ml respectively, and incubated for 2 hours at 37°C. Virus particles were removed by preliminary ultracentrifugation at 140,000g for 20 minutes to provide a purified extract. In some experiments where it is considered essential to completely remove virus particles from the preparation the ultracentrifugation procedure can be carried out over 20% sucrose as previously described in Skinner et al, Medical Microbiology and Immunology (1980) 169, pp 39-51. The extract obtained following the ultracentrifugation procedure is added to an appropriate volume, most usually an equal volume, of a serum which contains antibody against cytomegalovirus antigens and left at 25°C for _-hr and overnight at 4°C. The serum may be animal or human immune serum, or a monoclonal antibody serum. Prior to use the serum was clarified by ultracentrifugation and to inactivate complement was heated at 60°C for 10 minutes. Most usually the antisera will contain immunoprecipitating antibody to cytomegalovirus antigen(s) but this is not an essential feature of the preparation process. The resulting antigen-antibody preparation is now clarified of any macroscopic precipitate by centrifugation at 13,000 rpm for 5 minutes in an Eppendorf centrifuge (ll,600g). The supernatant is then ultracentrifuged at 45,000 rpm for 20 minutes on an MSE superspeed 65 centrifuge (145,000g) this procedure normally obtains a visible tightly packed pellet of the soluble antigen-antibody complexes. The pellet is then washed at least 5 times in phosphate buffered saline by repeated resuspension, agitation with a Vortex mixer, and ultracentrifugation as previously described. The washed pellet is finally resuspended in phosphate buffered saline and stored at -70°C. The resulting vaccine preparation was shown to contain cytomegalovirus polypeptides by polyacrylamide gel electrophoresis and was antigenic as evidenced by ELISA solid phase assay and immunoblotting against a human serum previously shown to react to high titre against cytomegalovirus antigen. There was evidence of immunogenicity following inoculation of this complex into a hamster. The vaccine preparation was safe and did not induce evidence of pathogenic effect on inoculation into mice or rabbits. There was no evidence of DNA in the vaccine preparation of a size greater than 200 bases. In another example the infectious cell starting material is treated with a detergent which will disrupt both the nuclei of the host cells and the virus particles therein. The disrupted cell material is then treated with a neutralizing agent for the detergent, for example a potassium salt. The material may be further treated with nucleases (RNase and DNase) and subjected to as preliminary centrifugation step to provide a purified extract which is free from cell and virus material. From that purified extract both the macroscopic precipitate and the soluble complexes may be recovered by the same ultracentrifugation step, after addition of antibody to the required antigen. A particular detergent which will disrupt both the cell nuclei and the virus particles is sodium dodecyl sulphate (SDS). If SDS is used the preliminary centrifugation step may be dispensed with. A further example is particularly appropriate to preparation of vaccines against bacteria or fungi, which by themselves constitute an infectious cell starting material, those cells are disrupted with a known detergent, or an organic solvent or an appropriate enzyme. The disrupting agent is then removed or neutralized to leave a non-infectious extract to which antibody to the antigen is added. Both the macroscopic and soluble complexes are recovered, either by successive respective centrifugation steps, or by the same ultracentrifugation step, as described above. The methods described above provide the following advantages: 1 A greater yield of material is obtained from a given quantity of antigen in that antigen in the form of soluble antigen-antibody complexes is obtained by the ultracentrifugation procedure. This material would normally be discarded. In addition, it is possible to add more antibody to the supernatant following ultracentrifugation and obtain a further deposit of soluble immune-complexes by further ultracentrifugation. 2 Antigenic or immunogenic material can be obtained under conditions where it is not possible to achieve macroscopic or microscopic immunopreciptitation prior to ultracentrifugation. 3 In the example described there is evidence that the material obtained following ultracentrifugation has a higher level of antigenic reactivity on testing by solid phase ELISA against hyperimmune antisera prepared against the antigen than the material obtained by first precipitation and removed by low speed centrifugation. This may relate to a lesser degree of degradation of antigenic sites than occurs when antigens are precipitated-out on complexing with antisera. 4 The process of ultracentrifugation results in a smaller more compact pellet which permits more efficient washing of the pellet from other unwanted materials. 5 Exclusion of the first precipitate obtained by Eppendorf centrifugation may be used to remove unwanted residual large molecular weight material which has been precipitated at this stage, for example, virus particles which have remained following ultracentrifugation without employment of the sucrose cushion.

Claims: . CLAIMS 1 A method of preparing vaccine against an infectious agent, in which an infectious cell starting material capable of acting as said agent is reacted with an antibody to the antigen required for the vaccine, to form antigen-antibody complexes which are suspended in a solution, and separating the precipitable fraction of said complex from said solution, characterised in that soluble immune complexes in said solution are separated therefrom by ultracentrifugation, said soluble immune complexes providing an active constituent of the vaccine. 2 A method as claimed in claim 1 in which the complexes in said precipitable fraction also form an active constituent of the vaccine. 3 A method as claimed in claim 2 in which said precipitable fraction and said soluble complexes are separated by the same ultracentrifugation step. 4 A method as claimed in any preceding claim in which said ultracentrifugation is carried out at more than 50,000g. 5 A method as claimed in claim 4 in which said ultracentrifugation is carried out at more than 140,000g. 6 A method as claimed in any preceding claim in which the infectious cells in the starting material are disrupted. 7 A method as claimed in claim 6 in which DNA and RNA in the disrupted cells are digested by- addition of nucleases. 8 A method as claimed in claim 6 or claim 7 in which virus particles are removed from the disrupted cells by means of a preliminary ultracentrifugation step. 9 A method as claimed in claim 8 in which said preliminary ultracentrifugation step is carried out over sucrose. 10 A method as claimed in any preceding claim in which the separated soluble immune complexes are resuspended. 11 A method as claimed in claim 10 in which the steps of separating said soluble immune complexes by ultracentrifugation, and subsequent resuspension of the separated complexes is performed a plurality of times.

Inventors: BUCHAN ALEXANDER, SKINNER GORDON ROBERT BRUCE, BUCHAN, ALEXANDER, SKINNER, GORDON, ROBERT, BRUCE
Applicants: UNIV BIRMINGHAM, THE UNIVERSITY OF BIRMINGHAM
Application year: 1990
Main classification: A61K39
IPC codes: A61K39