Assays for the evaluation of seasonal covid-influenza combination vaccines

A method for determining if a biological sample contains antibodies that neutralize a SARS-COV-2 virus, comprising:

• a. obtaining the biological sample, wherein the biological sample is from a subject that has been simultaneously administered an immunogenic composition directed to a seasonal influenza virus and an immunogenic composition directed to a SARS-COV-2 virus;
• b. contacting the biological sample with a replication-deficient Maloney murine leukemia virus (MLV) pseudovirus to form a sample-pseudovirus mixture, the MLV pseudovirus comprising a recombinant SARS-COV-2 S (rS) glycoprotein and a gene of interest (GOI) encoding a reporter protein;
• c. adding cells expressing angiotensin converting enzyme 2 (ACE2) to the sample-pseudovirus mixture, wherein the cells are A549 or HEK293T cells;
• d. incubating the sample-pseudovirus mixture with the cells expressing ACE2 at 37° C., and quantifying expression of the reporter protein in the cells expressing ACE2 at 48 hours of incubation;
• e. contacting control A549 or HEK293T cells expressing ACE2 with the MLV pseudovirus to form a virus-only control, wherein the MLV pseudovirus has not been contacted with the biological sample;
• and
• f. incubating the virus-only control with the control cells at 37° C., and quantifying expression of the reporter protein in the control cells expressing ACE2 at 48 hours of incubation;
• wherein if the expression of the reporter protein in the cells of step d is less than expression of the reporter protein in the control cells of step f, then the biological sample contains neutralizing antibodies against the SARS-COV-2 virus.

A method for simultaneously determining if a biological sample contains neutralizing antibodies against two or more SARS-COV-2 virus strains, comprising:

• a. obtaining the biological sample, wherein the biological sample is from a subject that has been simultaneously administered an immunogenic composition directed to a seasonal influenza virus and an immunogenic composition directed to a SARS-COV-2 virus;
• b contacting the biological sample with a first replication-deficient MLV pseudovirus and a second replication-deficient MLV pseudovirus to form a sample-pseudovirus mixture, the first MLV pseudovirus comprising an rS glycoprotein from a first SARS-COV-2 virus strain and a GOI encoding a first reporter protein, and the second MLV pseudovirus comprising an rS glycoprotein from a second SARS-COV-2 virus strain and a GOI encoding a second reporter protein;
• c. adding cells expressing ACE2 to the sample-pseudovirus mixture, wherein the cells are A549 or HEK293T cells;
• d. incubating the sample-pseudovirus mixture with the cells expressing ACE2 at 37° C., and quantifying expression of the first reporter protein and the second reporter protein in the cells expressing ACE2 at 48 hours of incubation;
• e. contacting control A549 or HEK293T cells expressing ACE2 with the first MLV pseudovirus and the second MLV pseudovirus to form a virus-only control, wherein the first MLV pseudovirus and the second MLV pseudovirus have not been contacted with the biological sample;
• and
• f. incubating the virus-only control with the control cells at 37° C., and quantifying expression of the first reporter protein and the second reporter protein in the control cells expressing ACE2 at 48 hours of incubation;
• wherein if the expression of the first reporter protein in the cells of step d is less than expression of the first reporter protein in the control cells of step f, then the biological sample contains neutralizing antibodies against the first SARS-COV-2 virus strain, and
• wherein if the expression of the second reporter protein in the cells of step d is less than expression of the second reporter protein in the control cells of step f, then the biological sample contains neutralizing antibodies against the second SARS-COV-2 virus strain.

A method for determining if a biological sample contains antibodies that bind to a SARS-COV-2 Spike(S) glycoprotein, comprising:

• a. obtaining the biological sample, wherein the biological sample is from a subject that has been simultaneously administered an immunogenic composition directed to a seasonal influenza virus and an immunogenic composition directed to a SARS-COV-2 virus;
• b providing a surface coated with a recombinant SARS-COV-2 S (rS) glycoprotein;
• c. exposing the surface to the biological sample;
• d. exposing the surface to a secondary antibody;
• and
• e. detecting the secondary antibody that is bound to the surface;
• wherein the biological sample contains antibodies that bind to the rS glycoprotein if the secondary antibody is detected.