# A temperature-responsive PLA-based nanosponge as a novel nanoadjuvant and efficient delivery carrier of Ag85B for effective vaccine against Mycobacterium tuberculosis

## TL;DR

A new temperature-sensitive nanoparticle system delivers a TB vaccine protein more effectively, offering better protection and immune response than the current BCG vaccine.

## Contribution

A novel temperature-responsive nanosponge system is developed as a delivery carrier and adjuvant for Ag85B, a TB vaccine protein.

## Key findings

- Ag85B@aPNS showed superior protective efficacy against M. tuberculosis in mice compared to BCG.
- The nanosponge system enhanced cellular uptake of antigens by macrophages without cytotoxicity.
- Ag85B@aPNS improved immunogenicity in BCG-primed mice when used as a booster.

## Abstract

Tuberculosis (TB) is a contagious disease and the second leading cause of death worldwide. The Bacille Calmette–Guérin (BCG) vaccine, the only licensed TB vaccine, has insufficient protective efficacy in adults, necessitating the development of new TB vaccines. Ag85B, a protein-subunit TB vaccine, is a promising candidate due to its high immunogenicity. However, its hydrophobicity presents challenges in manufacturing, expression, and purification, and Ag85B alone does not elicit sufficient immune stimulation. To overcome these limitations, this study aimed to design a temperature-responsive amine-terminated polylactic acid (PLA)-based nanosponge (aPNS) as both a nanoadjuvant and an efficient delivery carrier for Ag85B.

Ag85B was produced using an EZtag fusion tag vector, achieving high product yield and purity. It was then loaded into aPNS, a nanoparticle system with a PLA core and Pluronic shell, through a temperature-responsive process at 4 °C that preserved protein bioactivity. The stability and sustained-release profile of Ag85B@aPNS were evaluated. In vitro cytotoxicity and cellular uptake studies were conducted using macrophages. Protective efficacy and immunogenicity were assessed in M. tuberculosis-challenged mice and BCG-primed mice.

The Ag85B protein was successfully produced and loaded into aPNS, which exhibited good colloidal stability and a sustained-release profile. Neither the synthesized Ag85B nor the aPNS showed significant cytotoxicity. aPNS enhanced the cellular uptake of antigens by macrophages. Compared to BCG, Ag85B@aPNS demonstrated superior protective efficacy against M. tuberculosis in mice and improved immunogenicity in BCG-primed mice.

Ag85B@aPNS is a viable candidate for TB vaccination, showing potential as both a standalone vaccine and a BCG-booster. Its ability to enhance immunogenicity and provide protection highlights its promise in addressing the limitations of current TB vaccines.

The online version contains supplementary material available at 10.1186/s12964-025-02105-2.

Tuberculosis (TB) remains a major global health challenge, and while the current BCG vaccine provides some protection, its effectiveness varies and it does not fully prevent the disease in adults. To address this, researchers are developing new vaccines and boosters that enhance the immune system’s ability to fight TB. This study introduces a novel vaccine candidate called Ag85B@aPNS, which combines a TB-specific protein (Ag85B) with a specialized nanoparticle delivery system (aPNS). The researchers designed the aPNS to carry and protect the Ag85B protein, ensuring it remains stable and active for a longer time. These nanoparticles also respond to temperature changes, making them more effective in releasing the protein under human body conditions. Experiments showed that Ag85B@aPNS was safe for cells and enhanced the ability of immune cells to recognize and process the protein. This means the vaccine can better stimulate the immune system without causing harm. In animal studies, the vaccine was tested in mice infected with TB bacteria. Mice that received Ag85B@aPNS had fewer bacteria in their lungs and less lung damage compared to those given the standard BCG vaccine. Furthermore, when used as a booster in BCG-primed mice, the vaccine strengthened the immune response significantly. These findings suggest that Ag85B@aPNS is a promising candidate for improving TB vaccination strategies, either as a replacement for BCG or as a booster to enhance its effects. This could lead to better protection against TB in the future.

The online version contains supplementary material available at 10.1186/s12964-025-02105-2.