Dengue virus infection-enhancing activity in serum samples with neutralizing activity as determined by using FcγR-expressing cells

Abstract

Background: Progress in dengue vaccine development has been hampered by limited understanding of protective immunity against dengue virus infection. Conventional neutralizing antibody titration assays that use FcγR-negative cells do not consider possible infection-enhancement activity. We reasoned that as FcγR-expressing cells are the major target cells of dengue virus, neutralizing antibody titration assays using FcγR-expressing cells that determine the sum of neutralizing and infection-enhancing activity, may better reflect the biological properties of antibodies in vivo.

Methods and findings: We evaluated serum samples from 80 residents of a dengue endemic country, Malaysia, for neutralizing activity, and infection-enhancing activity at 1∶10 serum dilution by using FcγR-negative BHK cells and FcγR-expressing BHK cells. The serum samples consisted of a panel of patients with acute DENV infection (31%, 25/80) and a panel of donors without acute DENV infection (69%, 55/80). A high proportion of the tested serum samples (75%, 60/80) demonstrated DENV neutralizing activity (PRNT(50)≥10) and infection-enhancing activity. Eleven of 18 serum samples from patients with acute secondary DENV infection demonstrated neutralizing activity to the infecting serotype determined by using FcγR-negative BHK cells (PRNT(50)≥10), but not when determined by using FcγR-expressing cells.

Conclusion: Human serum samples with low neutralizing activity determined by using FcγR-negative cells showed DENV infection-enhancing activity using FcγR-expressing cells, whereas those with high neutralizing activity determined by using FcγR-negative cells demonstrate low or no infection-enhancing activity using FcγR-expressing cells. The results suggest an inverse relationship between neutralizing antibody titer and infection-enhancing activity, and that neutralizing activity determined by using FcγR-expressing cells, and not the activity determined by using FcγR-negative cells, may better reflect protection to DENV infection in vivo.

Figure 1. Infection-enhancement activity in serum samples with neutralizing activity to each of the four DENV serotypes.

Sixty serum samples exhibiting neutralizing activity to DENV were analyzed for presence of infection-enhancement activity to each of the four DENV serotypes: (A) DENV-1, (B) DENV-2, (C) DENV-3 and, (D) DENV-4. Infection-enhancement activity to each DENV serotype was determined by using FcγR-expressing cells by the formula: (mean plaque count at 1∶10 serum dilution)/(mean plaque count in the absence of human serum samples), and expressed as fold enhancement to each DENV serotype. Closed bars indicate serum samples with neutralizing titer PRNT₅₀≥10 to the indicated DENV serotype and open bars indicate serum samples with neutralizing titer PRNT₅₀<10 as determined using FcγR-negative BHK cells. (*) indicates P<0.05.

Figure 2. Infection-enhancement activity in serum samples with neutralizing activity to DENV-2.

Six serum samples with high neutralizing activity to DENV-2 at 1∶10 serum dilutions were tested for presence of infection-enhancement activity to DENV-2 at serum dilutions of 1∶10 to 1∶10⁶. (A) serum sample #40, (B) #42, (C) #44 (D) #49, (E) #54 and, (F) #58. Infection-enhancement activity to each DENV serotype was determined by using FcγR-expressing BHK cells and BHK cells by the formula: (mean plaque count at each serum dilution)/(mean plaque count in the absence of human serum samples), and expressed as fold enhancement to DENV-2. Closed bars indicate FcγR-expressing BHK cells and open bars indicate FcγR-negative BHK cells.