Covalent coupling of HIV-1 glycoprotein trimers to biodegradable calcium phosphate nanoparticles via genetically encoded aldehyde-tags.

The usage of antigen-functionalized nanoparticles has become a major focus in the field of experimental HIV-1 vaccine research during the last decade. Various molecular mechanisms to couple native-like trimers of the HIV-1 envelope protein (Env) onto nanoparticle surfaces have been reported, but many come with disadvantages regarding the coupling efficiency and stability. In this study, a short amino acid sequence ("aldehyde-tag") was introduced at the C-terminus of a conformationally stabilized native-like Env. The post-translational conversion of a tag-associated cysteine to formylglycine creates a site-specific aldehyde group without alteration of the Env antigenicity. This aldehyde group was further utilized for bioconjugation of Env trimers. We demonstrated that the low acidic environment necessary for this bioconjugation is not affecting the trimer conformation. Furthermore, we developed a two-step coupling method for pH-sensitive nanoparticles. To this end, we conjugated aldehyde-tagged Env with Propargyl-PEG3-aminooxy linker (oxime ligation; Step-one) and coupled these conjugates by copper-catalyzed azide-alkyne cycloaddition (Click reaction; Step-two) to calcium phosphate nanoparticles (CaPs) functionalized with terminal azide groups. CaPs displaying orthogonally arranged Env trimers on their surface (o-CaPs) were superior in activation of Env-specific B-cells (in vitro) and induction of Env-specific antibody responses (in vivo) compared to CaPs with Env trimers coupled in a randomly oriented manner. Taken together, we present a reliable method for the site-specific, covalent coupling of HIV-1 Env native-like trimers to the surface of nanoparticle delivery systems. This method can be broadly applied for functionalization of nanoparticle platforms with conformationally stabilized candidate antigens for both vaccination and diagnostic approaches. STATEMENT OF SIGNIFICANCE: During the last decade antigen-functionalized nanoparticles have become a major focus in the field of experimental HIV-1 vaccines. Rational design led to the production of conformationally stabilized HIV-1 envelope protein (Env) trimers - the only target for the humoral immune system. Various molecular mechanisms to couple Env trimers onto nanoparticle surfaces have been reported, but many come with disadvantages regarding the coupling efficiency and stability. In this paper, we describe a highly selective bio-conjugation of Env trimers to the surface of medically relevant calcium phosphate nanoparticles. This method maintains the native-like protein conformation and has a broad potential application in functionalization of nanoparticle platforms with stabilized candidate antigens (including stabilized spike proteins of coronaviruses) for both vaccination and diagnostic approaches.

Adjuvant properties of AcF1, an immunostimulant fraction of Alchornea cordifolia extract.

As a result of strong experimental data supporting effectiveness and safety, herb-based immunomodulators are paving way as alternative sources of potent adjuvants for vaccines. In this study, the immunostimulatory and adjuvant properties of AcF1, a flavonoids-rich fraction of Alchornea cordifolia extract, was evaluated. In vitro, AcF1 was shown to activate total splenocytes, CD4+ T cells, and B cells, inducing remarkable increases in CD69 expression, profound proliferation, and increased IL-4 and IFN-gamma expression by the naïve splenic cells in a concentration-dependent manner. Lympho-activation and proliferation induced by AcF1 was partially inhibited by U0126, a selective mitogen activated protein kinase kinase (MKK) inhibitor. Additionally, AcF1 was shown to induce structural and functional maturation of bone marrow-derived dendritic cells (BM-DCs) and their specific-antigen presentation functions. Used as an adjuvant in a homologous prime-boost OVA immunisation in C57BL/6 mice, AcF1 significantly (P<0.05) increased the level of OVA-specific antibody titres in the sera of immunised mice, compared to the control group immunised with OVA alone. The results of this study show AcF1 as a potent immunostimulant and a potential adjuvant for further study in combination with other vaccine antigens.

Effects of granulocyte-macrophage colony-stimulating factor produced by intestinal epithelial cells on functional activity of hemopoietic stem cells.

The population of epithelial cells in the small intestine includes precursor cells of hemopoiesis possessing the ability to form splenic colonies on day 8. Intestinal epithelial cells stimulates proliferative and colony-forming activity of hemopoietic stem cells by producing granulocyte-macrophage colony-stimulating factor.