Methods for detection of anti-cytomegalovirus neutralizing antibodies
The present disclosure provides methods useful for determining levels of HCMV infection in host cells and, by extension, determining levels of neutralizing antibodies present in a sample. The present disclosure encompasses the recognition that HCMV viruses that have a fluorescent moiety permit detection of viral infection (e.g., by assessing fluorescence in cells after contacting the host cell with the virus). In some embodiments, levels of HCMV infection are determined by fluorescence detection where the virus has been preincubated with a test sample (e.g., a serum sample) from a subject. In some embodiments, the subject has been administered a candidate HCMV vaccine.
1. A method comprising steps of:
(i) mixing heat-inactivated serum from a subject that has been immunized with an HCMV candidate vaccine with an HCMV comprising a fluorescent moiety to form a mixture, wherein the HCMV comprising the fluorescent moiety comprises a gH/gL/gO complex;
(ii) adding 10% guinea pig complement to the mixture from (i);
(iii) contacting a host cell that is susceptible to infection by HCMV, wherein said host cell is a human foreskin fibroblast cell of cell line HFF-1, under conditions that allow infection with the mixture of heat-inactivated serum in the presence of rabbit complement of (ii);
(iv) assessing a fluorescence level of the host cell that has been contacted with the mixture by flow cytometry; and
(v) determining a level of infection of the host cell based on the assessed fluorescence level.
2. The method of claim 1 , wherein the subject that has been immunized is a human.
3. The method of claim 1 , wherein the serum comprises anti-HCMV neutralizing antibodies.
4. The method of claim 1 , wherein the HCMV candidate vaccine comprises VLPs.
5. The method of claim 4 , wherein the VLPs comprise one or more of gB, gH, and pp65 from HCMV.
6. The method of claim 1 , wherein the mixture of (ii) is incubated for at least 15 minutes, at least 30 minutes, at least 1 hour, or at least 2 hours before step (iii).
7. The method of claim 1 , wherein the assessing step (iv) is performed in a high throughput manner.
8. The method of claim 1 , wherein the assessing step (iv) comprises comparing the fluorescence level to a reference.
9. The method of claim 8 , wherein the reference is a historical reference.
10. The method of claim 8 , wherein the reference is a side-by-side reference.
11. The method of claim 1 , further comprising determining an anti-HCMV neutralizing antibody titer in the serum based on the determining step.
12. The method of claim 11 , further comprising evaluating the efficacy of the candidate HCMV vaccine based on the determining step.
13. The method of claim 12 , further comprising selecting the candidate HCMV vaccine as a vaccine that induces neutralizing antibodies if the fluorescence level of the host cell that has been contacted with the mixture is lower than a reference fluorescence level.
14. The method of claim 1 , wherein the contacting step (iii) comprises an incubation of at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, or at least 8 hours.
15. The method of claim 1 , wherein the host cell is further monitored for infection by visual cellular morphology assessment or visual fluorescence assessment.
16. The method of claim 15 , wherein the visual cellular morphology assessment comprises monitoring for cellular swelling and rounding.
17. The method of claim 15 , wherein the fluorescence assessment is conducted using a fluorescence microscope.
18. The method of claim 1 , wherein a fluorescence level of the nucleus of the host cell is assessed.
19. The method of claim 1 , wherein a fluorescence level of the whole host cell is assessed.
20. The method of claim 1 , further comprising a step of quantitating the fluorescence level of the host cell.