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Effect of formalin treatment on antigenicity of herpes simplex virus type 1 glycoprotein D
Abstracts
Effect of formalin treatment on antigenicity of herpes simplex virus type 1 glycoprotein D P.H. Ridgeway and R.J. Phillpotts Centre for Applied Microbiology and Research, Porton Down, Wiltshire SP4 0JG, UK Formalin has traditionally been used to inactivate virus in vaccine preparations. In addition to the problems which may arise due to inadequate inactivation of virus, formalin treatment may also affect the epitopic structure of antigens. Not only is there the possibility that biologically important epitopes may be destroyed, but new irrelevant epitopes may be created. In an attempt to define the changes in epitopic structure which take
place when a virus glycoprotein is treated with formalin, and to examine their biological significance, we have raised monoclonal antibodies to native and formalin-treated herpes simplex virus type 1 (HSV-1) glycoprotein D. These have been examined for their reactivity with native and formalin-treated HSV-1 antigen in ELISA, and for their biological properties.
Preparation of immune complex herpes simplex vaccine using human sera D. Sharitf Department of Medical Microbiology, The Medical School, University of Birmingham, Birmingham B15 2TJ, UK The preparation of an immune complex is described which may be suitable for immunization of human subjects. Human serum was reacted with virus particle-free DNAse-treated HSV antigen and the precipitate formed was then washed at least six times in the appropriate buffers. The polypeptide profile of the complex vaccine indicated a number of virus-specific polypeptides which were found to be antigenic by immunoblotting against a number of different human sera. Immunization of a rabbit with this vaccine stimulated neutralizing and lmmunoprecipitating antibodies, which also reacted in ELISA, Western blot and
inhibited release of herpes virus from infected cells. As anticipated there was also evidence of reactivity against human immunoglobulin and a very low level of activity against host cell antigen. There was less than 50 pg D N A as determined by blot hybridization in one putative vaccine dose ( ~ 2 x 10 7 cell equivalents). The protective efficacy of vaccination is now under investigation in a mouse model system. This approach may permit employment of other than human cell lines for human vaccine production and pre-selection of virus antigens depending on the immunoprecipitating antiserum.
Glycosylated and non-glycosylated proteins of HSV-1: relevance to vaccine development G.R.B. Skinner and S. Hawkins Department of Medical Microbiology, University of Birmingham, Edgbaston, Birmingham B15 2TJ, UK While glycoproteins of HSV-1 play a major role in protective immunity, other viral components from virus particles and virus-infected cells are antigenic and immunogenic; it was important, therefore, to examine the non-glycosylated proteins in more detail with particular attention to their role in the immuno-response. Glycoproteins were removed from virusinfected cell extracts and from the HSV-1 vaccine preparation by immobilized lectins or antisera which have been raised against lectin-purified HSV glycoproteins. Eluate fractions, obtained as described, reacted against hyperimmune serum prepared against HSV-1 infected cell extracts, and on immunoblotting there was reactivity against a number of polypeptides,
including the 'glycoprotein region' in the lectin eluant fractions. In addition, both eluant fractions removed neutralizing antibody from hyperimmune and anti-glycoprotein sera. Preparation of antisera against these fractions resulted in development of antibody directed against glycoprotein regions with a greater breadth of type-specific reactivity than was obtained from antisera against glycoprotein-rich eluate fractions. Preparation of a vaccine by elution on lectin columns will, therefore, lose components which are capable of stimulating neutralizing antibody reactivity against both non-glycosylated and glycosylated polypeptides.
Preparation of a subunit vaccine against herpes simplex G.R. Talei Department of Medical Microbiology, The Medical School, University of Birm&gham, Birmingham B15 2TJ, UK A subunit vaccine was prepared in serum-starved BHK-21 cells infected with type 2 herpes simplex virus. The infected cells were treated with detergent and cell nuclei removed by low-speed centrifugation; this also served to strip envelope glycoproteins and other proteins from the virus particles. Virus particles were removed by ultracentrifugation over 20% sucrose and the proteins extracted by acetone precipitation. The vaccine contained at least two immunoprecipitating antigens against hyperimmune sera (one was glycoprotein D) and reacted to high titre by ELISA tests lmmunoprecipitating and
neutralizing antibody were stimulated in vaccinated rabbits and there was protective efficacy against challenge by type 2 herpes simplex virus in mice. The vaccine was found to contain <0.05% of original infected cell D N A and residual D N A was < 100 base pairs in size as analysed by agarose gels. As 50-95 % of genital herpes infections are now caused by the type 2 herpes simplex virus, it is intended to develop a subunit type 2 herpes simplex vaccine prepared in MRC-5 or Vero cells for immunization of human subjects.
Vaccine, Vol. 7, April 1989 187
Effect of formalin treatment on antigenicity of herpes simplex virus type 1 glycoprotein D P.H. Ridgeway and R.J. Phillpotts Centre for Applied Microbiology and Research, Porton Down, Wiltshire SP4 0JG, UK Formalin has traditionally been used to inactivate virus in vaccine preparations. In addition to the problems which may arise due to inadequate inactivation of virus, formalin treatment may also affect the epitopic structure of antigens. Not only is there the possibility that biologically important epitopes may be destroyed, but new irrelevant epitopes may be created. In an attempt to define the changes in epitopic structure which take
place when a virus glycoprotein is treated with formalin, and to examine their biological significance, we have raised monoclonal antibodies to native and formalin-treated herpes simplex virus type 1 (HSV-1) glycoprotein D. These have been examined for their reactivity with native and formalin-treated HSV-1 antigen in ELISA, and for their biological properties.
Preparation of immune complex herpes simplex vaccine using human sera D. Sharitf Department of Medical Microbiology, The Medical School, University of Birmingham, Birmingham B15 2TJ, UK The preparation of an immune complex is described which may be suitable for immunization of human subjects. Human serum was reacted with virus particle-free DNAse-treated HSV antigen and the precipitate formed was then washed at least six times in the appropriate buffers. The polypeptide profile of the complex vaccine indicated a number of virus-specific polypeptides which were found to be antigenic by immunoblotting against a number of different human sera. Immunization of a rabbit with this vaccine stimulated neutralizing and lmmunoprecipitating antibodies, which also reacted in ELISA, Western blot and
inhibited release of herpes virus from infected cells. As anticipated there was also evidence of reactivity against human immunoglobulin and a very low level of activity against host cell antigen. There was less than 50 pg D N A as determined by blot hybridization in one putative vaccine dose ( ~ 2 x 10 7 cell equivalents). The protective efficacy of vaccination is now under investigation in a mouse model system. This approach may permit employment of other than human cell lines for human vaccine production and pre-selection of virus antigens depending on the immunoprecipitating antiserum.
Glycosylated and non-glycosylated proteins of HSV-1: relevance to vaccine development G.R.B. Skinner and S. Hawkins Department of Medical Microbiology, University of Birmingham, Edgbaston, Birmingham B15 2TJ, UK While glycoproteins of HSV-1 play a major role in protective immunity, other viral components from virus particles and virus-infected cells are antigenic and immunogenic; it was important, therefore, to examine the non-glycosylated proteins in more detail with particular attention to their role in the immuno-response. Glycoproteins were removed from virusinfected cell extracts and from the HSV-1 vaccine preparation by immobilized lectins or antisera which have been raised against lectin-purified HSV glycoproteins. Eluate fractions, obtained as described, reacted against hyperimmune serum prepared against HSV-1 infected cell extracts, and on immunoblotting there was reactivity against a number of polypeptides,
including the 'glycoprotein region' in the lectin eluant fractions. In addition, both eluant fractions removed neutralizing antibody from hyperimmune and anti-glycoprotein sera. Preparation of antisera against these fractions resulted in development of antibody directed against glycoprotein regions with a greater breadth of type-specific reactivity than was obtained from antisera against glycoprotein-rich eluate fractions. Preparation of a vaccine by elution on lectin columns will, therefore, lose components which are capable of stimulating neutralizing antibody reactivity against both non-glycosylated and glycosylated polypeptides.
Preparation of a subunit vaccine against herpes simplex G.R. Talei Department of Medical Microbiology, The Medical School, University of Birm&gham, Birmingham B15 2TJ, UK A subunit vaccine was prepared in serum-starved BHK-21 cells infected with type 2 herpes simplex virus. The infected cells were treated with detergent and cell nuclei removed by low-speed centrifugation; this also served to strip envelope glycoproteins and other proteins from the virus particles. Virus particles were removed by ultracentrifugation over 20% sucrose and the proteins extracted by acetone precipitation. The vaccine contained at least two immunoprecipitating antigens against hyperimmune sera (one was glycoprotein D) and reacted to high titre by ELISA tests lmmunoprecipitating and
neutralizing antibody were stimulated in vaccinated rabbits and there was protective efficacy against challenge by type 2 herpes simplex virus in mice. The vaccine was found to contain <0.05% of original infected cell D N A and residual D N A was < 100 base pairs in size as analysed by agarose gels. As 50-95 % of genital herpes infections are now caused by the type 2 herpes simplex virus, it is intended to develop a subunit type 2 herpes simplex vaccine prepared in MRC-5 or Vero cells for immunization of human subjects.
Vaccine, Vol. 7, April 1989 187