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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.

Epitope exposure strategies often fail due to high conformational instability, resulting in loss of native conformation and fast degradation. Protein scaffolding, where a structural element of an antigen protein is transplanted into a scaffold acceptor protein, has been employed as an alternative solution. However, while this approach aims to preserve the epitope structure, it frequently results in unstable chimeric scaffolds. To overcome this issue, we employed a novel computational approach to rationally engineer conformational antigens into a highly stable scaffold protein. This strategy is showcased to display conformational HIV-1 gp41-based epitopes in their native structure. From HIV-1 antigen sequence databases, we have identified short sequences with the most probable antibody-recognizable regions. These sequences were inserted into or replaced regions of the original Top7 protein with analogous secondary structure assignment. Molecular dynamics simulations were used to characterize the protein stability and structural dynamic of the chimeric proteins, leading to a selection of promising candidates whose immunogenic epitopes are suitably exposed for antibody recognition. These computer-designed recombinant proteins were produced in bacteria using codon optimized DNA sequences and their diagnostic performance was assessed by liquid microarray against a human cohort of 47 sera samples. Our results show that the Top7 protein is a suitable scaffold to provide the required structural stability to predetermined target shapes and sequences, allowing the potential use of the chimeric proteins as antigens for specific antibody recognition and/or stimulation of immune response.