A Single-Dose Intramuscular Nanoparticle Vaccine With or Without Prior Intrauterine Priming Triggers Specific Uterine and Colostral Mucosal Antibodies and Systemic Immunity in Gilts but Not Passive Protection for Suckling Piglets.

An effective single-dose vaccine that protects the dam and her suckling offspring against infectious disease would be widely beneficial to livestock animals. We assessed whether a single-dose intramuscular (i.m.) porcine epidemic diarrhea virus (PEDV) vaccine administered to the gilt 30 days post-breeding could generate mucosal and systemic immunity and sufficient colostral and mature milk antibodies to protect suckling piglets against infectious challenge. The vaccine was comprised of polymeric poly-(lactide-co-glycolide) (PGLA)-nanoparticle (NP) encapsulating recombinant PEDV spike protein 1 (PEDVS1) associated with ARC4 and ARC7 adjuvants, a muramyl dipeptide analog and a monophosphoryl lipid A (MPLA) analog, respectively (NP-PEDVS1). To establish whether prior mucosal exposure could augment the i.m. immune response and/or contribute to mucosal tolerance, gilts were immunized with the NP-PEDVS1 vaccine via the intrauterine route at breeding, followed by the i.m. vaccine 30 days later. Archived colostrum from gilts that were challenged with low-dose PEDV plus alum was used as positive reference samples for neutralizing antibodies and passive protection. On day 100 of gestation (70 days post i.m. immunization), both vaccinated groups showed significant PEDVS1-specific IgG and IgA in the serum, as well as in uterine tissue collected on the day of euthanasia. Anti-PEDVS1 colostral IgG antibody titers collected at farrowing were significantly higher relative to the negative control gilts indicating that the NP vaccine was effective in contributing to the colostral antibodies. The PEDVS1-specific colostral IgA and anti-PEDVS1 IgG and IgA antibodies in the mature milk collected 6 days after farrowing were low for both vaccinated groups. No statistical differences between the vaccinated groups were observed, suggesting that the i.u. priming vaccine did not induce mucosal tolerance. Piglets born to either group of vaccinated gilts did not receive sufficient neutralizing antibodies to protect them against infectious PEDV at 3 days of age. In summary, a single i.m. NP vaccine administered 30 days after breeding and a joint i.u./i.m. vaccine administered at breeding and 30 days post-breeding induced significant anti-PEDVS1 immunity in systemic and mucosal sites but did not provide passive protection in suckling offspring.

Amphiphilic desmuramyl peptides for the rational design of new vaccine adjuvants: Synthesis, in vitro modulation of inflammatory response and molecular docking studies.

Nucleotide-binding oligomerization domain 2 (NOD2) is cytosolic surveillance receptor of the innate immune system capable of recognizing the bacterial and viral infections. Muramyl dipeptide (MDP) is the minimal immunoreactive unit of murein. NOD2 perceives MDP as pathogen-associated molecular pattern, thereby triggering an immune response with undesirable side-effects. Beneficial properties of MDP, such as pro-inflammatory characteristics for the rational design of new vaccine adjuvants, can be harnessed by strategically re-designing the molecule. In this work, a new class of amphiphilic desmuramylpeptides (DMPs) were synthesized by replacing the carbohydrate moiety (muramic acid) of the parent molecule with hydrophilic arenes. A lipophilic chain was also introduced at the C-terminus of dipeptide moiety (alanine-isoglutamine), while conserving its L-D configuration. These novel DMPs were found to set off the release of higher levels of tumour necrosis factor alpha (TNF-α) than Murabutide, which is a well-known NOD2 agonist. Molecular docking studies indicate that all these DMPs bind well to NOD2 receptor with similar dock scores (binding energy) through a number of hydrogen bonding and hydrophobic/π interactions with several crucial residues of the receptor. More studies are needed to further assess their immunomodulatory therapeutic potential, as well as the possible involvement of NOD2 activation.

Combination of Innate Immune Modulators as Vaccine Adjuvants in Mice.

The development of new, effective, and safe vaccines necessarily requires the identification of new adjuvant(s) to enhance the potency and longevity of antigen-specific immune responses. In the present study, we compare the antibody-mediated and cell-mediated immune (CMI) responses within groups of mice vaccinated subcutaneously with ovalbumin (OVA; as an experimental antigen) plus polyphosphazene (an innate immune modulator), Polyinosinic:polycytidylic acid (poly-I:C; (an RNA mimetic) and glycopeptide ARC5 (which is a Toll-like receptor (TLR), TLR2 ligand and PAM3CSK4 analogue) formulated together in a soluble vaccine. We also investigated the effect of a polymeric nanoparticle of ARC4 and ARC7 (which are a novel muramyl dipeptide analogue and a monophosophoryl lipid A (MPLA) analogue, respectively) plus OVA +/- ARC5 as a subcutaneous vaccine in mice. OVA+ARC4/ARC7 nanoparticle +/- ARC5 triggered a robust and balanced Th1/Th2-type humoral response with significant anti-OVA IgA in serum, and significant interferon (IFN)-γ and interleukin (IL)-17 production in splenocytes after 35 days relative to the controls. Formulation of OVA with ARC4/ARC7 nanoparticles should be investigated for inducing protective immunity against infectious pathogens in mice and other species.

Design, synthesis and immunological evaluation of novel amphiphilic desmuramyl peptides.

Muramyl dipeptide (MDP) - an essential bacterial cell wall component - is recognized by our immune system as pathogen-associated molecular pattern (PAMP) which results in immune responses with adverse toxic effects. In order to harness the beneficial properties from the pro-inflammatory characteristics of the bacterial cell wall motif, MDP was strategically re-designed while conserving the L-D configurations of the dipeptide moiety. The muramic acid was replaced with a hydrophilic arene and lipophilic chain was introduced at peptide end to give the amphiphilic desmuramyl peptides (DMPs). The novel DMPs were found to modulate the immune response by amplifying the LPS-induced surface glycoprotein (ICAM-1) expression in THP-1 cells without showing significant toxicity. Furthermore, these compounds were able to trigger the secretion of higher levels of pro-inflammatory cytokine (TNF-α) than the well-studied NOD2 agonist, Murabutide.

Monophosphoryl lipid A analogues with varying 3-O-substitution: synthesis and potent adjuvant activity.

Structurally defined immunostimulatory adjuvants play important roles in the development of new generation vaccines. Here described are the syntheses of three monophosphoryl lipid A analogues (1-3) with different substitution at 3-O-position of the reducing sugar and their potent immunostimulatory adjuvant activity. The syntheses involve the preparation of glycosylation acceptors benzyl 3,4-di-O-benzyl-2-deoxy-2-[(R)-3-tetradecanoyloxytetradecanamido]-beta-D-glucopyranoside (16) and benzyl 3-O-allyl-4-O-benzyl-2-deoxy-2-[(R)-3-tetradecanoyloxytetradecanamido]-beta-D-glucopyranoside (17). The glycosylation reactions between the donor 4,6-di-O-benzylidene-2-deoxy-2-(2',2',2'-trichloroethoxycarbonylamino)-alpha-d-glucopyranosyl trichloroacetimidate (21) and acceptors 16 and 17 provide the desired beta-(1-->6)-linked disaccharides 22 and 23, respectively. Selective reductive ring opening of the 4,6-di-O-benzylidene group, installation of a phosphate group to the 4'-hydroxyl group, and the final global debenzylation produce the designed monophosphoryl lipid A analogues 1-3. All three synthetic analogues induce antigen specific T-cell proliferation and interferon-gamma (IFN-gamma) production in ex vivo experiments with a totally synthetic liposomal vaccine system. The immunostimulatory potency of compound 1-3 is in the same order of magnitude as that of the detoxified natural lipid A product isolated from Salmonella minnesota R595 (R595 lipid A). The substituent at the 3-O-position of the reducing sugar does not have much effect on the adjuvant activity of monophosphoryl lipid A analogues. The preliminary lethal toxicity study indicates that the 3-O-acylated hepta-acyl monophosphoryl lipid A may not be more toxic than its 3-O-deacylated hexa-acyl analogue.