Kinetics of neutralizing antibodies in patients naturally infected by H5N1 virus

Abstract

Background: Little is known about the kinetics of anti-H5 neutralizing antibodies in naturally H5N1-infected patients with severe clinical illness or asymptomatic infection.

Methods: Using H5N1 microneutralisation (MN) and H5-pseudotype particle-based microneutralisation assays (H5pp) we analyzed sera sequentially obtained from 11 severely ill patients diagnosed by RT-PCR (follow-up range 1-139 weeks of disease onset) and 31 asymptomatically infected individuals detected in a sero-epidemiological study after exposure to H5N1 virus (follow-up range: 1-2 month-11 months after exposure).

Results: Of 44 sera from 11 patients with H5N1 disease, 70% tested positive by MN (antibody titre > or = 80) after 2 weeks and 100% were positive by 3 weeks after disease onset. The geometric mean MN titers in severely ill patients were 540 at 1-2 months and 173 at 10-12 months and thus were higher than the titers from asymptomatic individuals (149 at 1-2 months, 62.2 at 10-12 months). Fractional polynomial regression analysis demonstrated that in all severely ill patients, positive titers persisted beyond 2 years of disease onset, while 10 of 23 sera collected 10-11 months after exposure in asymptomatically infected individuals tested negative.

Conclusions: Our results indicate that people with asymptomatic H5N1 infection have lower H5N1 antibody titres compared to those with severe illness and that in many asymptomatically infected patients the antibody titer decreased to levels below the threshold of positivity within one year. These data are essential for the design and interpretation of sero-epidemiological studies.

Figure 1. H5 neutralizing antibody titers by H5pp (1a) and MN (1b) tests in 31 asymptomatic/mild individuals.

Orange lines: Fig. 1a: Linear regression line (slope of –0.06, p value  = 0.172). Fig1b: Linear regression line (slope of –0.31, p value  = 0.024). Cross points: Seropositive cases' neutralizing antibody titers at Weeks 7, 9, 48 and 51 after exposure (n = 54). Green line: Threshold titer at 80.

Figure 2. H5 neutralizing antibody titers by H5pp (2a) and MN (2b) tests in 11 severely ill H5N1 patients.

Orange dots: Geometric means of titers by week (n = 47). Blue lines: Fractional polynomial regression line. Grey zones: 95% confidence interval around fractional polynomial regression line. Green line: Threshold titer at 80.

Figure 3. Comparison of H5pp and MN tests by Bland & Altman method.

On the x axis, the means of the H5 titers observed with the two methods are shown for individual samples. On the y axis, the difference between the methods divided by the means of the titers presented in percent. The limits of agreements are depicted. A total of 101 sera were included in the analysis. Bland and Altman plot, N  = 101. Bias: 31.9% [95% Confidence Interval  = +17.1% to +46.7%]. Limits of agreement  = −115.6% and +179.5%.