The purpose of this study was to modulate macrophage polarity from the pro-inflammatory M1 to anti-inflammatory M2 phenotype using plasmid DNA (pDNA) expressing interleukin-4 (IL4) or interleukin-10 (IL10)-encapsulated in hyaluronic acid-poly(ethyleneimine) (HA-PEI) nanoparticles (NPs). The HA-PEI/pDNA NPs with spherical shape, average size of 186 nm were efficiently internalized by J774A.1 macrophages. Transfection of HA-PEI/pDNA-IL4 and HA-PEI/pDNA-IL10 NPs increased IL4 and IL10 gene expression in J774 macrophages which could re-program the macrophages from M1 to M2 phenotype as evidenced by a significant increase in the Arg/iNOS level, and upregulation of CD206 and CD163 compared to untreated macrophages. Following intraperitoneal (IP) injection to C57BL/6 mice, HA-PEI NPs effectively targeted peritoneal macrophages over-expressing CD44 receptor. In an in vivo model of stimulated peritoneal macrophages, IP administration of HA-PEI/pDNA-IL4 and HA-PEI/pDNA-IL10 to C57BL/6 mice significantly increased the Arg/iNOS ratio and CD163 expression in the cells. Furthermore, HA-PEI/pDNA-IL10 NPs significantly increased peritoneal and serum IL10 levels which effectively suppressed LPS-induced inflammation by reducing level of TNF-α and IL-1β in peritoneal macrophages and in the peritoneal fluid. The results demonstrated that pDNA-IL10-encapsulate HA-PEI NPs skewed macrophage functional polarity from M1 toward an anti-inflammatory M2 phenotype which may be a promising platform for the treatment of inflammatory diseases.

Viral infectious diseases threaten human health and global stability. Several vaccine platforms, such as DNA, mRNA, recombinant viral vectors, and virus-like particle-based vaccines have been developed to counter these viral infectious diseases. Virus-like particles (VLP) are considered real, present, licensed and successful vaccines against prevalent and emergent diseases due to their non-infectious nature, structural similarity with viruses, and high immunogenicity. However, only a few VLP-based vaccines have been commercialized, and the others are either in the clinical or preclinical phases. Notably, despite success in the preclinical phase, many vaccines are still struggling with small-scale fundamental research owing to technical difficulties. Successful production of VLP-based vaccines on a commercial scale requires a suitable platform and culture mode for large-scale production, optimization of transduction-related parameters, upstream and downstream processing, and monitoring of product quality at each step. In this review article, we focus on the advantages and disadvantages of various VLP-producing platforms, recent advances and technical challenges in VLP production, and the current status of VLP-based vaccine candidates at commercial, preclinical, and clinical levels.

Leishmaniasis is a severe infectious disease. Drugs used for leishmaniasis are very toxic, and no vaccine is available. We found that the hemoglobin receptor (HbR) of Leishmania was conserved across various strains of Leishmania, and anti-HbR antibody could be detected in kala-azar patients' sera. Our results showed that immunization with HbR-DNA induces complete protection against virulent Leishmania donovani infection in both BALB/c mice and hamsters. Moreover, HbR-DNA immunization stimulated the production of protective cytokines like interferon-γ (IFN-γ), interleukin-12 (IL-12), and tumor necrosis factor-α (TNF-α) with concomitant down-regulation of disease-promoting cytokines like IL-10 and IL-4. HbR-DNA vaccination also induced a protective response by generating multifunctional CD4(+) and CD8(+) T cells. All HbR-DNA-vaccinated hamsters showed sterile protection and survived during an experimental period of 8 months. These findings demonstrate the potential of HbR as a vaccine candidate against visceral leishmaniasis.

Several intracellular pathogens arrest the phagosome maturation in the host cells to avoid transport to lysosomes. In contrast, the Leishmania containing parasitophorous vacuole (PV) is shown to recruit lysosomal markers and thus Leishmania is postulated to be residing in the phagolysosomes in macrophages. Here, we report that Leishmania donovani specifically upregulates the expression of Rab5a by degrading c-Jun via their metalloprotease gp63 to downregulate the expression of miR-494 in THP-1 differentiated human macrophages. Our results also show that miR-494 negatively regulates the expression of Rab5a in cells. Subsequently, L. donovani recruits and retains Rab5a and EEA1 on PV to reside in early endosomes and inhibits transport to lysosomes in human macrophages. Similarly, we have also observed that Leishmania PV also recruits Rab5a by upregulating its expression in human PBMC differentiated macrophages. However, the parasite modulates the endosome by recruiting Lamp1 and inactive pro-CathepsinD on PV via the overexpression of Rab5a in infected cells. Furthermore, siRNA knockdown of Rab5a or overexpression of miR-494 in human macrophages significantly inhibits the survival of the parasites. These results provide the first mechanistic insights of parasite-mediated remodeling of endo-lysosomal trafficking to reside in a specialized early endocytic compartment.

Differential functions of Rab5 isoforms in endocytosis are not well characterized. Here, we cloned, expressed, and characterized Rab5a and Rab5b from Leishmania and found that both of them are localized in the early endosome. To understand the role of LdRab5 isoforms in different modes of endocytosis in Leishmania, we generated transgenic parasites overexpressing LdRab5a, LdRab5b, or their dominant-positive (LdRab5a:Q93L and LdRab5b:Q80L) or dominant-negative mutants (LdRab5a: N146I and LdRab5b:N133I). Using LdRab5a or its mutants overexpressing parasites, we found that LdRab5a specifically regulates the fluid-phase endocytosis of horseradish peroxidase and also specifically induced the transport of dextran-Texas Red to the lysosomes. In contrast, cells overexpressing LdRab5b or its mutants showed that LdRab5b explicitly controls receptormediated endocytosis of hemoglobin, and overexpression of LdRab5b:WT enhanced the transport of internalized Hb to the lysosomes in comparison with control cells. To unequivocally demonstrate the role of Rab5 isoforms in endocytosis in Leishmania, we tried to generate null-mutants of LdRab5a and LdRab5b parasites, but both were lethal indicating their essential functions in parasites. Therefore, we used heterozygous LdRab5a ؉/؊ and LdRab5b ؉/؊ cells. LdRab5a ؉/؊ Leishmania showed 50% inhibition of HRP uptake, but hemoglobin endocytosis was uninterrupted. In contrast, about 50% inhibition of Hb endocytosis was observed in LdRab5b ؉/؊ cells without any significant effect on HRP uptake. Finally, we tried to identify putative LdRab5a and LdRab5b effectors. We found that LdRab5b interacts with clathrin heavy chain and hemoglobin receptor. However, LdRab5a failed to interact with the clathrin heavy chain, and interaction with hemoglobin receptor was significantly less. Thus, our results showed that LdRab5a and LdRab5b differentially regulate fluid phase and receptor-mediated endocytosis in Leishmania.