Detection of IgG3 antibodies specific to the human immunodeficiency virus type 1 (HIV-1) p24 protein as marker for recently acquired infection

Abstract

Reducing the risk of human immunodeficiency virus type 1 (HIV-1) transmission is still a public health priority. The development of effective control strategies relies on the quantification of the effects of prophylactic and therapeutic measures in disease incidence. Although several assays can be used to estimate HIV incidence, these estimates are limited by the poor performance of these assays in distinguishing recent from long-standing infections. To address such limitation, we have developed an assay to titrate p24-specific IgG3 antibodies as a marker of recent infection. The assay is based on a recombinant p24 protein capable to detect total IgG antibodies in sera using a liquid micro array and enzyme-linked immunosorbent assay. Subsequently, the assay was optimised to detect and titrate anti-p24 IgG3 responses in a panel of sequential specimens from seroconverters over 24 months. The kinetics of p24-specific IgG3 titres revealed a transient peak in the 4 to 5-month period after seroconversion. It was followed by a sharp decline, allowing infections with less than 6 months to be distinguished from older ones. The developed assay exhibited a mean duration of recent infection of 144 days and a false-recent rate of ca. 14%. Our findings show that HIV-1 p24-specific IgG3 titres can be used as a tool to evaluate HIV incidence in serosurveys and to monitor the efficacy of vaccines and other transmission control strategies.

Keywords: ELISA; Early infection marker; HIV/AIDS; recent infection.

Fig. 1.

Immunoreactivity and diagnostic performance assessment of the designed HIV-1 p24 protein. (a) – Median fluorescence intensity (MFI) for the p24 protein against HIV-1 positive and negative (control) human serum samples and against Hepatitis B and C, Chagas Disease, Syphilis and HTLV-1/2 positive serum samples. The results are presented as the median values over a 100 events for individual samples (dots) along with the corresponding standard deviation (P < 0.001). Mean values and correspondent standard deviations are shown. The P-values of two-tailed t-test and one-way ANOVA are indicated above each analysis. (b) – ROC curve analysis of the HIV-1 p24 protein. The paired results for sensitivity and specificity were plotted as points in a ROC space and the trade-off between these measures for different discrimination cut-offs are graphically represented.

Fig. 2.

Detection and titration of p24-specific total IgG antibodies in clinical samples by ELISA assay. (a) – Scatter plot representation of p24-specific IgG antibodies detection in serum samples from 48 healthy participants and 48 HIV-1 chronically infected individuals. The results are shown as mean values over all measurements and corresponding standard error of the mean (s.e.m.). Dots represent individual measurements. A dashed line indicates the cut-off value and the P-value of two-tailed t-test is indicated above. (b) – Titration of p24-specific total IgG antibodies in paired samples from 11 HIV-1 positive individuals comprising the last negative sample and first positive sample within 6–12 months after seroconversion. Each sample was tested in duplicate and the mean values are shown. Dotted lines connect paired samples and the P-value of two-tailed t test is indicated above the graph.

Fig. 3.

p24-specific total IgG and IgG3 antibody titres as measured by our ELISA assay in 150 sequential serum samples from 30 HIV-1 positive and recently seroconverted individuals. The total IgG (a) and IgG3 (c) response towards p24 was determined by ELISA in 150 serum samples from 30 HIV-1 positive individuals who recently seroconverted. The samples were divided into five groups based on the time post-eroconversion and over a total period of 24 months. The antibody titres are plotted vs. the intervals after seroconversion in months. Individual measurements are shown (grey symbols) along with the median (black lines) and correspondent 95% confidence interval. The black dotted line in (c) represents the cut-off value (titre: 190) selected based on the final end of the 95% confidence interval of the mean of first time-point. (b) – Titration of p24-specific total IgG antibodies in relation to days since seroconversion. Each line represents a single individual with sequential specimens collected over time.

Fig. 4.

Kinetics of p24-specific IgG3 antibodies as measured by our ELISA assay in 150 sequential serum samples from 30 HIV-1 positive and recently seroconverted individuals in relation to time since seroconversion (days). (a) – Each line represents a single individual with sequential specimens collected over time post-seroconversion (in days). Cut-off (titre = 190) is shown as a red dashed line, whereas the mean duration of recent infection (144 days) and corresponding confidence interval (95% CI 132-156) are shown as a vertical black line and shaded grey area, respectively. (b) – Confidence levels of the developed assay are shown as the frequency of individuals correctly classified as recent (1–4 months) and non-recent (5–24 months) according to the assay cut-off described in (a).