Background: Bacillus Calmette–Guérin, the attenuated strain of Mycobacterium bovis, remains the only available vaccine against tuberculosis (TB). However, its ineffectiveness in adults against pulmonary TB and varied protective efficacy (0–80%) speak to an urgent need for the development of an improved and efficient TB vaccine. In this milieu, poly(lactic-co-glycolic acid) (PLGA), is a preferential candidate, due to such properties as biocompatibility, targeted delivery, sustained antigen release, and atoxic by-products. Methods: In this study, we formulated PLGA nanoparticles (NPs) encapsulating the bivalent H1 antigen, a fusion of Mycobacterium tuberculosis (Mtb) Ag85B and ESAT6 proteins, and investigated its role in immunomodulation and protection against Mtb challenge. Using the classical water–oil–water solvent-evaporation method, H1-NPs were prepared, with encapsulation efficiency of 86.1%±3.2%. These spherical NPs were ~244.4±32.6 nm in diameter, with a negatively charged surface (ζ-potential -4±0.6 mV). Results: Under physiological conditions, NPs degraded slowly and the encapsulated H1 antigen was released over a period of weeks. As a proof-of-concept vaccine candidate, H1 NPs were efficiently internalized by the THP-1 human macrophages. Six weeks after a single-dose vaccination, H1 NP–immunized C57BL/6J mice showed significant increase in the production of total serum IgG (P<0.0001) and its isotypes compared to H1 alone, IgG_2a being the predominant one, followed by IgG₁. Further, the cytokine-release profile of antigen-stimulated splenocyte-culture supernatant indicated a strong T_H1-biased immunoresponse in H1 NP–vaccinated mice, with ~6.03- and ~2.8-fold increase in IFNγ and TNFα cytokine levels, and ~twofold and 1.6 fold increase in IL4 and IL10 cytokines, respectively, compared to H1 alone–immunized mice. In protection studies, H1 NP–vaccinated mice displayed significant reductions in lung and spleen bacillary load (P<0.05) at 5-week post–Mtb H37Rv challenge and prolonged survival, with a mean survival time of 177 days, compared to H1 alone–vaccinated mice (mean survival time 80 days). Conclusion: Altogether, our findings highlight the significance of the H1-PLGA nanoformulation in terms of providing long-term protection in mice with a single dose.

Introduction: Aluminum salts, although they have been used as adjuvants in many vaccine formulations since 1926, exclusively induce a Th2-biased immune response, thereby limiting their use against intracellular pathogens like Mycobacterium tuberculosis. Methods and Results: Herein, we synthesized amorphous and crystalline forms of aluminum hydroxide nanoparticles (AH nps) of 150– 200 nm size range. Using Bacillus anthracis protective antigen domain 4 (D4) as a model antigen, we demonstrated that both amorphous and crystalline forms of AH nps displayed enhanced antigen D4 uptake by THP1 cells as compared to commercial adjuvant aluminum hydroxide gel (AH gel). In a mouse model, both amorphous and crystalline AH nps triggered an enhanced D4-specific Th2- and Th1-type immune response and conferred superior protection against anthrax spore challenge as compared to AH gel. Physicochemical characterization of crystalline and amorphous AH nps revealed stronger antigen D4 binding and release than AH gel. Conclusion: These results demonstrate that size and crystallinity of AH nps play important roles in mediating enhanced antigen presenting cells (APCs) activation and potentiating a strong antigen-specific immune response, and are critical parameters for the rational design of alum-based Th1-type adjuvant to induce a more balanced antigen-specific immune response.

Introduction: In this study, we have investigated the immunogenicity and protective efficacy of a niosomal formulation encapsulating protective antigen (PA) and PA domain 4 (D4) of Bacillus anthracis. Methods: Nonionic surfactant–based vesicles (NISV) + PA and NISV + D4 were prepared from span-60 and cholesterol by reverse-phase evaporation method and were evaluated for in vitro characteristics and immunological studies. Results: Particle characterization using transmission electron microscopy and atomic force microscopy analysis showed that the niosomal formulation was spherical in shape. The entrapment efficiency values were calculated to be 58.5% and 44.75% for PA and D4, respectively. Confocal microscopy and flow cytometry studies showed an enhanced uptake of antigen in THP1 macrophages by niosome as compared to antigen only. An in vitro release assay showed a burst release of antigen from niosome within 24 hours followed by a gradual release for 144 hours. Immunological studies showed that both PA- and D4-encapsulated niosome elicited a robust IgG titer. Antibody isotyping and cytokine profile showed that NISV + PA and NISV + D4 enhanced both Th1 and Th2 responses in mice, suggesting a mixed Th1/Th2 response. Both NISV + PA and NISV + D4 elicited high levels of anti-inflammatory cytokine interleukin-10 with low levels of pro-inflammatory cytokine tumor necrosis factor-α, suggesting the anti-inflammatory property of niosome. Both the niosomal formulations were also able to confer protection against BA infection as compared to only PA and D4. Conclusion: PA and D4 encapsulated NISV formulation could modulate both the Th1 and Th2 adaptive immune system and was found to be a better prophylactic against anthrax.

Cancer-specific CD8⁺ cytotoxic T cells play important roles in preventing cancer growth, and IFN-γ, in addition to IL-12 and type I interferon, is critical for activating CD8⁺ cytotoxic T cells. We recently identified the capability of the amino-terminus region of dense granule protein 6 (GRA6Nt) of Toxoplasma gondii, an intracellular protozoan parasite, to activate IFN-γ production of microglia, a tissue-resident macrophage population. Therefore, in the present study, we examined whether recombinant GRA6Nt protein (rGRA6Nt) functions as an effective adjuvant to potently activate cancer-specific protective immunity using a murine model of MC38 colorectal cancer (CRC). When mice were immunized with non-replicable (either treated with mitomycin C or irradiated by X-ray) MC38 CRC cells in combination with rGRA6Nt adjuvant and received a challenge implantation of replication-capable MC38 tumor cells, those mice markedly inhibited the growth of the implanted tumors in association with a two-fold increase in CD8⁺ T cell density within the tumors. In addition, CD8⁺ T cells of the immunized mice secreted significantly increased amounts of granzyme B, a key mediator of the cytotoxic activity of CD8⁺ T cells, and IFN-γ in response to MC38 CRC cells in vitro when compared to the T cells from unimmunized mice. Notably, the protective effects of the immunization were specific to MC38 CRC cells, as the immunized mice did not exhibit a significantly inhibited growth of EL4 lymphoma tumors. These results indicate that rGRA6Nt is a novel and effective protein adjuvant when used in immunizations with non-replicable cancer cells to potently activate the protective immunity specifically against the cancer cells employed in the immunization.