Abstract: We investigated the efficacy of a novel microparticle (MP) based vaccine formulation consisting of pertussis toxoid (PTd), polyphosphazene (PCEP), CpG ODN 10101 and synthetic cationic innate defense regulator peptide 1002 (IDR) against Bordetella pertussis in mice. We studied whether encapsulation of these IDR-CpG ODN complexes into polyphosphazene-based microparticles further enhanced their immunomodulatory activity compared to soluble formulations containing PCEP (SOL), or without PCEP (AQ). In vitro stimulation of murine macrophages showed MP induced significantly higher levels of pro-inflammatory cytokines. When assessed in a B. pertussis infection challenge model, a single immunization with MP formulation led to significantly lower bacterial loads compared to other formulations and non-vaccinated animals. ELISPOT of splenocytes showed that MP group mice had significantly higher number of antigen-specific IL-17 secreting cells. The cytokine profile in lung homogenates of MP group mice after challenge showed significantly higher amounts of MCP-1, TNF-α, IFN-γ, IL-12 and IL-17 and significantly lowered IL-10 levels suggesting a strong Th1 shift. Protection was observed against challenge infection with B. pertussis. On the other hand protective immune responses elicited in Quadracel(®) immunized mice were Th2 skewed. Hence, we conclude that formulation of PTd, PCEP, CpG ODN and IDR into MP generates a protective immune response in mice against pertussis emphasizing the potential of MP as a delivery vehicle for the potential development of single-shot vaccines.
Abstract: Adjuvants are critical components of many vaccines. The majority of existing vaccines contain a single adjuvant. Owing to their inherent limitations, no single adjuvant is capable of inducing all the protective immune responses required in the many different vaccines. Consequently, investigators are exploring the potential of using formulations with multiple adjuvants in a vaccine. An emerging paradigm is that careful selection of adjuvant combinations can result in complementary and even synergistic enhancement of immune responses to vaccines. This approach is promising and presents tremendous opportunities for vaccinologists to tailor immune responses to specific vaccines. In this article, adjuvant combinations at different stages of development will be reviewed.
Abstract: Since foot-and-mouth disease virus (FMDV) serotypes display a great genetic and antigenic diversity, there is a constant requirement to monitor the performance of FMDV vaccines in the field with respect to their antigenic coverage. To avoid possible antigenic changes in field FMDV isolates during their adaptation to BHK-21 cells, a standard step used in production of conventional FMDV vaccines, the custom-made chimeric conventional or DNA vaccines, in which antigenic determinants are replaced with those of appropriate field strains, should be constructed. Using this approach, we made a plasmid-based chimeric FMDV DNA vaccine containing structural genes of serotype O in the genome backbone of serotype Asia 1, all under the control of Human cytomegalovirus (HCMV) immediate early gene promoter. BHK-21 cells transfected with the chimeric DNA vaccine did not show cytopathic effect (CPE), but expressed virus-specific proteins as demonstrated by 35S-methionine labeling and immunoprecipitation. Guinea pigs immunized with the chimeric DNA vaccine produced virus-specific antibodies assayed by ELISA and virus neutralization test (VNT), respectively. The chimeric DNA vaccine showed a partial protection of guinea pigs challenged with the virulent FMDV. Although the chimeric DNA vaccine, in general, was not as effective as a conventional one, this study encourages further work towards the development of genetically engineered custom-made chimeric vaccines against FMDV.
Abstract: We generated poly[di(carboxylatophenoxy)-phosphazene] (PCPP) microparticles encapsulating ovalbumin (OVA) and CpG of 0.5-2.5 μm in diameter with an encapsulation efficiency of approximately 63% and 95% respectively. In mice the microparticles generated high antigen-specific IgG, IgG1 and IgG2a titers with higher IgG2a/IgG1 ratios. Whole body in vivo imaging of mice subcutaneously injected with MPs showed several fold increase of OVA and CpG in draining inguinal lymph nodes compared to soluble formulations. We conclude that PCPP MPs are more effective in enhancing immune responses compared to soluble formulations, due to co-delivery of OVA and CpG resulting in a Th1 type of immune response.
Abstract: Polyphosphazene polyelectrolytes are synthetic, biodegradable polymers that have shown great potential in vaccine and drug delivery applications. Numerous investigations in laboratory animals have revealed that polyphosphazenes are also potent immunological adjuvants that can dramatically enhance the magnitude, quality and duration of immune responses to a variety of bacterial and viral vaccine antigens. Evidence is accumulating that these polymers have potent adjuvant activity in large animals as well. Interestingly, polyphosphazenes can be combined with novel immune modulatory agents resulting in even more potent immune activity and protection against experimental infection. While most reports are on the activity of polyphosphazenes in aqueous formulations, these polymers can also be easily made into microparticles, making them especially attractive for mucosal delivery. The mechanisms which mediate the adjuvant activity of polyphosphazenes are not fully understood, but there is evidence to suggest that activation of innate immunity may be involved. Further research and development of polyphosphazene adjuvants is warranted to fully explore their potential in the delivery of vaccines and immunotherapeutic agents.
Abstract: Adjuvants are important components of vaccine formulations. Their functions include the delivery of antigen, recruitment of specific immune cells to the site of immunization, activation of these cells to create an inflammatory microenvironment, and maturation of antigen-presenting cells for enhancement of antigen-uptake and -presentation in secondary lymphoid tissues. Adjuvants include a large family of molecules and substances, many of which were developed empirically and without knowledge of their specific mechanisms of action. The discovery of pattern recognition receptors including Toll-like-, nucleotide-binding oligomerization domain (NOD)- and mannose-receptors, has significantly advanced the field of adjuvant research. It is now clear that effective adjuvants link innate and adaptive immunity by signaling through a combination of pathogen recognition receptors (PRRs). Research in our lab is focused towards the development of novel adjuvants and immunomodulators that can be used to improve neonatal vaccines for humans and animals. Using a neonatal pig model for pertussis, we are currently analyzing the effectiveness of host defence peptides (HDPs), bacterial DNA and polyphosphazenes as vaccine adjuvants.
Abstract: The coding sequence of the bovine (Bos indicus) Glucose-6-phosphate-dehydrogenase (G6PD) gene was amplified by Reverse Transcriptase-PCR (RT-PCR), cloned, sequenced and characterized. The deduced amino acid sequence clustered the bovine G6PD sequence with the other mammalian G6PD proteins into a monophyletic group. The bovids (B. indicus and B. taurus) clustered clearly from the rodent (rat, mouse and hamster) subcluster and from humans. The multiple sequence alignment of the bovine G6PD with the mammalian species clearly revealed conservation of the substrate, coenzyme, catalytic and the dimer binding sites with the solved X-ray crystallographic structure of Homo sapiens. Also, four fragments of bovine (Bos indicus) G6PD gene viz. 118, 319, 683 and 408 bp were amplified and sequenced for the first time. A G/A and G/C single nucleotide polymorphisms in intron-9 and exon-10 were detected on PCR-RFLP of the 319 bp amplicon with Hae III and Pst I, respectively. This work is the first study on Bos indicus G6PD gene at the nucleotide level.
