Attempts to Influence the Incomplete Reproductive Cycle of Influenza Virus in HeLa Cells by Antibodies
Authors: Löffler Hans , Henle Gertrude , Henle Werner 
Published: December 2022

Summary
The question whether antibodies are truly incapable of entering cells was studied in HeLa cultures infected with the PR8 strain of influenza A virus and in uninfected populations treated with antiviral or normal rabbit sera. The results were evaluated by staining of cells vitally or after acetone fixation with fluorescein isothiocyanate-coupled antibodies to PR8 virus or rabbit γ-globulin, as well as by hemagglutination and complement fixation tests.
Replication of influenza virus in HeLa cells is restricted to one cycle because solely noninfectious hemagglutinins (NIHA) are produced. The ultimate yields of NIHA and the maximal percentage of cells containing influenza antigens were strictly proportional to the dose of seed virus employed. Few, if any, cells reacted with fluorescent antibodies in the second passage, and none after further passages.
Differential staining of acetone-fixed cells from infected, untreated cultures showed that S antigen developed first and remained restricted to the nucleus. V antigen appeared initially in the region of the Golgi apparatus, from where it spread throughout the cytoplasm and, in time, reached the cell surface, as evident from vital staining with labeled anti-PR8 γ-globulin.
Exposure of infected or uninfected cultures to antiviral or normal rabbit serum provided the following data: Vital staining of infected antiserum-treated cells with fluorescent anti-PR8 yielded essentially negative results, whereas labeled anti-rabbit γ-globulin produced brilliant fluorescence, restricted to the cell surface. The reverse was noted with infected cells maintained in the presence of normal serum; i.e., surface fluorescence with labeled antibodies to PR8 virus but not to rabbit γ-globulin. Uninfected cells, treated or not, failed to stain with either of the fluorescent antibodies.Acetone-fixed cells from antiviral or normal serum-treated cultures revealed the same degrees of virus-specific nuclear and cytoplasmic fluorescence. With labeled anti-rabbit γ-globulin, only the former became stained, but again apparently only at the cell surface in that no characteristic nuclear or perinuclear fluorescence could be elicited. Uninfected, treated or untreated cultures gave negative results with both types of fluorescent antibodies.Disintegrated cell suspensions from infected, antiserum-treated cultures fixed specifically some plement per se, but not those derived from populations exposed to normal rabbit serum. After addition of excess anti-V or anti-S, both preparations fixed the same numbers of complement units, indicating that the presence of antiviral serum in the medium did not affect the production of the two antigens.Exposure of infected cultures to anti-PR8 serum prevented the demonstration of NIHA formation as long as antibodies were present throughout or were added late in the incubation period, but not when they were removed at the 4th hr after infection. This secondary effect of antiserum may be accounted for by combination of viral antigen at the cellular surface with antibodies which on disruption of the cells may interact with liberated NIHA by means of free determinant groups or after dissociation in the course of reestablishing antigen-antibody equilibrium. All attempts to remove the attached antibodies were unsuccessful.
As the data stand, no evidence was obtained to indicate entry of antibodies into normal or infected cells in sufficient amounts to be detectable by the immunofluorescence techniques employed or by reduction in production of viral antigens.