Weiguang Zeng

Highly Immunogenic and Totally Synthetic Lipopeptides as Self-Adjuvanting Immunocontraceptive Vaccines

In this study, we describe the synthesis of various lipopeptides based on the sequence of luteinizing hormone-releasing hormone (LHRH) and report on their abilities to induce Abs against this “self” hormone when inoculated into mice in the absence of additional adjuvant. The peptides consisted of a colinear CD4+ T helper cell epitope from the L chain of influenza virus hemagglutinin and LHRH, which has B cell epitopes but no T cell epitopes present in its sequence. Lipids were attached either at the N terminus or between the T cell epitope and LHRH, in the approximate center of the peptide. The lipopeptide constructs displayed different solubilities and immunological properties that depended not only on the lipid content but also on the position of attachment of the lipids. Some of these constructs were highly immunogenic, inducing high titers of Ab, which were capable of efficiently sterilizing female mice when administered in saline by s.c. or intranasal routes. The most effective vaccines were highly soluble, contained the dipalmitoyl-S-glyceryl cysteine moiety, and had this lipid attached at the center of the molecule. The relative ability of the lipopeptides to induce an Ab response in the absence of external adjuvant was reflected by their ability to up-regulate the surface expression of MHC class II molecules on immature dendritic cells. These results demonstrate that the composition and position within peptide vaccines of self-adjuvanting lipid groups can influence the ability to induce the maturation of dendritic cells and, in turn, the magnitude of the resulting Ab response.

New multi-determinant strategy for a group A streptococcal vaccine designed for the Australian Aboriginal population

Infection with group A streptococci can result in acute and post-infectious pathology, including rheumatic fever and rheumatic heart disease. These diseases are associated with poverty and are increasing in incidence, particularly in developing countries and amongst indigenous populations, such as Australias Aboriginal population, who suffer the highest incidence worldwide. Immunity to group A streptococci is mediated by antibodies against the M protein, a coiled-coil alpha helical surface protein of the bacterium. Vaccine development faces two substantial obstacles. Although opsonic antibodies directed against the N terminus of the protein are mostly responsible for serotypic immunity, more than 100 serotypes exist. Furthermore, whereas the pathogenesis of rheumatic fever is not well understood, increasing evidence indicates an autoimmune process. To develop a suitable vaccine candidate, we first identified a minimum, helical, non-host-cross-reactive peptide from the conserved C-terminal half of the protein and displayed this within a non-M-protein peptide sequence designed to maintain helical folding and antigenicity, J14 (refs. 8,9). As this region of the M protein is identical in only 70% of group A streptococci isolates, the optimal candidate might consist of the conserved determinant with common N-terminal sequences found in communities with endemic group A streptococci. We linked seven serotypic peptides with J14 using a new chemistry technique that enables the immunogen to display all the individual peptides pendant from an alkane backbone. This construct demonstrated excellent immunogenicity and protection in mice.

Protection against heterologous human papillomavirus challenge by a synthetic lipopeptide vaccine containing a broadly cross-neutralizing epitope of L2

Persistent infection with the high-risk subset of genitotropic human papillomavirus (HPV) genotypes is a necessary cause of cervical cancer. Given the global burden of cervical cancer, a low-cost, broadly protective vaccine is needed. RG-1 is a cross-neutralizing and protective monoclonal antibody that recognizes residues 17–36 of HPV16 minor capsid protein L2. Because this epitope is highly conserved in divergent HPV types, we determined whether vaccination with HPV16 L2 17–36 peptide is broadly protective. The peptide was administered to BALB/c mice three times at monthly intervals, either alone or in the context of a synthetic lipopeptide vaccine candidate (P25-P2C-HPV) produced by linkage of the HPV peptide with a broadly recognized T helper epitope (P25) and the Toll-like receptor-2 (TLR2) ligand dipalmitoyl-S-glyceryl cysteine (P2C). In contrast to vaccination with the L2 17–36 peptide or P25-P2C alone, a potent L2-specific antibody response was generated to the P25-P2C-HPV lipopeptide when delivered either s.c. or intranasally. Sera from mice vaccinated with the P25-P2C-HPV lipopeptide neutralized not only HPV16 pseudovirions but also other evolutionarily divergent oncogenic genital (HPV18, HPV45) and cutaneous (HPV5, BPV1) types. The L2-specific antibody response depended on MHC class II, CD40, and MyD88 signaling. Additionally, vaccination with the P25-P2C-HPV lipopeptide protected mice from homologous challenge with HPV16 pseudovirions at cutaneous and genital sites and heterologous challenge with HPV45 pseudovirions. If provided in the appropriate context, therefore, HPV16 L2 17–36 might be used in a totally synthetic cross-protective HPV vaccine.

Activation of the NLRP3 Inflammasome by IAV Virulence Protein PB1-F2 Contributes to Severe Pathophysiology and Disease

The ability for a host to recognize infection is critical for virus clearance and often begins with induction of inflammation. The PB1-F2 of pathogenic influenza A viruses (IAV) contributes to the pathophysiology of infection, although the mechanism for this is unclear. The NLRP3-inflammasome has been implicated in IAV pathogenesis, but whether IAV virulence proteins can be activators of the complex is unknown. We investigated whether PB1-F2-mediated activation of the NLRP3-inflammasome is a mechanism contributing to overt inflammatory responses to IAV infection. We show PB1-F2 induces secretion of pyrogenic cytokine IL-1β by activating the NLRP3-inflammasome, contributing to inflammation triggered by pathogenic IAV. Compared to infection with wild-type virus, mice infected with reverse engineered PB1-F2-deficient IAV resulted in decreased IL-1β secretion and cellular recruitment to the airways. Moreover, mice exposed to PB1-F2 peptide derived from pathogenic IAV had enhanced IL-1β secretion compared to mice exposed to peptide derived from seasonal IAV. Implicating the NLRP3-inflammasome complex specifically, we show PB1-F2 derived from pathogenic IAV induced IL-1β secretion was Caspase-1-dependent in human PBMCs and NLRP3-dependent in mice. Importantly, we demonstrate PB1-F2 is incorporated into the phagolysosomal compartment, and upon acidification, induces ASC speck formation. We also show that high molecular weight aggregated PB1-F2, rather than soluble PB1-F2, induces IL-1β secretion. Furthermore, NLRP3-deficient mice exposed to PB1-F2 peptide or infected with PB1-F2 expressing IAV were unable to efficiently induce the robust inflammatory response as observed in wild-type mice. In addition to viral pore forming toxins, ion channel proteins and RNA, we demonstrate inducers of NLRP3-inflammasome activation may include disordered viral proteins, as exemplified by PB1-F2, acting as host pathogen ‘danger’ signals. Elucidating immunostimulatory PB1-F2 mediation of NLRP3-inflammasome activation is a major step forward in our understanding of the aetiology of disease attributable to exuberant inflammatory responses to IAV infection.

Induction of Long-Term Memory CD8+ T Cells for Recall of Viral Clearing Responses against Influenza Virus

ABSTRACT Induction of cytotoxic T-cell-mediated virus-clearing responses by influenza virus T cell determinant-containing peptide immunogens was examined. The most potent synthetic immunogens for eliciting pulmonary viral-clearing responses contained peptides representing determinants for CD4 and CD8 T cells (TH and CTL peptides, respectively) together with two or four palmitic acid (Pal) groups. Inoculated in adjuvant, these Pal2- or Pal4-CTL-TH lipopeptides and the nonlipidated CTL peptide induced equivalent levels of cytolytic activity in the primary effector phase of the response. The ability to recall lytic responses, however, diminished much more rapidly in CTL peptide-primed than in lipopeptide-primed mice. By 15 months postpriming, the recalled lytic activity in lipopeptide-inoculated mice remained potent, but the response induced by the CTL peptide was weak. Enumeration of specific gamma interferon-secreting CD8 T cells revealed that a greater number of these T cells had entered or remained in the memory pool in lipopeptide-primed mice, arguing for a quantitative rather than qualitative enhancement of the response on recall. Addition of either the lipid or the TH peptide to the CTL peptide was not sufficient to provide these long-lived antiviral responses, but inclusion of both components augmented the response. CD4 T cells elicited by the lipopeptides did not influence the rate of viral clearance upon challenge and most likely had a role in induction or maintenance of the memory response. It therefore appears that the lipopeptide immunogens, although not significantly superior at inducing primary effector CD8 T cells, elicit a much more effective memory population, the recall of which may account for their superiority in inducing pulmonary protection after viral challenge.

Intranasal lipopeptide primes lung-resident memory CD8+ T cells for long-term pulmonary protection against influenza

The longevity of the influenza virus‐specific CD8+ T cell response following intranasal delivery of a synthetic lipopeptide was investigated and the characteristics and location of the cells associated with viral clearance examined. The lipopeptide, incorporating an epitope for CD8+ T cells and another for CD4+ T cells with the lipid moiety S‐[2,3‐bis(palmitoyloxy)propyl]cysteine (Pam2Cys) attached, induced potent and long‐lived pulmonary protection. Both the lipopeptide and its largely unprotective non‐lipidated counterpart elicited comparable numbers of CD8+ T cells in the spleen, which was the main location of the memory pool. However, the lipopeptide, unlike the non‐lipidated peptide, also induced a substantial memory population that remained in the lungs and was rapidly activated upon viral challenge months later. These lipopeptide‐induced lung‐resident CD8+ T cells were also very similar in number and IFN‐γ‐secreting potential to those induced by prior exposure to the virus itself and are likely mediators of initial viral clearance prior to recruitment from the expanding lymph node T cell pool. Significant clearing responses were demonstrated as late as 9 months post‐lipopeptide vaccination. This study shows that CD8+ T cells primed by the lipopeptide are not only long‐lived but can take up residence in the lung where they are important early mediators of pulmonary protection.

Totally synthetic lipid-containing polyoxime peptide constructs are potent immunogens

A synthetic peptide corresponding to a sequence from influenza hemagglutinin was used as a model antigen to study the immunogenicity of polyoxime constructs. In the absence of any adjuvant, tetrameric forms of different polyoxime constructs did not elicit an antibody response. High and long-lasting levels of antibody were induced, however, by polyoxime constructs to which Pam3Cys (tripalmitoyl-S-glyceryl cysteine) was attached. Comparable serum antibody levels were achieved with Tetraoxime-Pam3Cys administered by the intraperitoneal or intranasal routes to those obtained when the monomeric peptide was administered by the intraperitoneal route in complete Freunds adjuvant (CFA). Mice receiving Tetraoxime-Pam3Cys and Pam3Cys-peptide intranasally developed peptide-specific antibody secreting cells (ASCs) in their lungs and mediastinal lymph nodes. At low dose, the Tetraoxime-Pam3Cys induced higher levels of antibody compared to those elicited by the monomeric Pam3Cys-peptide delivered by either route. These results show that lipo-tetraoxime constructs assembled by polyoxime chemistry can be potent inducers of systemic and mucosal immunity.

Intranasal vaccination with a lipopeptide containing a conformationally constrained conserved minimal peptide, a universal T cell epitope, and a self-adjuvanting lipid protects mice from group A streptococcus challenge and reduces throat colonization

Infection with group A streptococcus (GAS) may result in a number of human diseases, including potentially life-threatening postinfectious sequelae. In the present study, J14, a conformationally constrained conserved minimal peptide from the M protein, was incorporated into a lipopeptide construct to which a universal T cell epitope and a self-adjuvanting lipid moiety, Pam(2)Cys, were also attached. We demonstrate that this lipopeptide construct, when administered intranasally (inl) without additional adjuvants, protects outbred mice from lethal respiratory GAS challenge. In addition, the lipopeptide was capable of inducing J14-specific mucosal immunoglobulin A, which coincided with reduced throat colonization after respiratory GAS challenge. These preclinical experiments show that this lipopeptide could form the basis of an antidisease and transmission-blocking inl GAS vaccine.

Chitosan Microparticles and Nanoparticles as Biocompatible Delivery Vehicles for Peptide and Protein-Based Immunocontraceptive Vaccines

It has become increasingly recognized that polymer particle size can have a profound effect on the interactions of particle-based vaccines with antigen presenting cells (APCs) thereby influencing and modulating ensuing immune responses. With the aim of developing chitosan particle-based immunocontraceptive vaccines, we have compared the use of chitosan-based nanoparticles and chitosan-based microparticles as vaccine delivery vehicles for vaccine candidates based on luteinizing hormone-releasing hormone (LHRH). Particles, functionalized with chloroacetyl groups, which allows the covalent attachment of thiol-containing antigens, were able to adsorb ~60-70% of their weight of peptide-based antigen and 10-20% of their weight of protein-based antigen. Quantitation by amino acid analysis of antigen associated with particles demonstrated a correlation between associated antigen and the degree of chloracetylation of particles. Visualization of fluorescently labeled antigen-loaded particles by confocal microscopy indicated that the majority of antigen was localized at the particle surface with a smaller amount located in the interior. We also found that uptake of both fluoresceinated nanoparticles and microparticles by dendritic cells occurred in a manner dependent on particle concentration. Nanoparticles trafficked from the injection site to draining lymph nodes faster than microparticles; high numbers of nanoparticle-bearing cells appeared in draining lymph nodes on day 3 and microparticles on day 4. This difference in trafficking rate did not, however, appear to have any significant impact on the ensuing immune response because inoculation with both peptide-conjugated and protein-conjugated particles induced high levels of LHRH-specific antibodies. In the case of protein-conjugated particles, the levels of antibodies elicited were similar to those elicited following inoculation with antigen emulsified with complete Freunds adjuvant. The approach to vaccine design that we have described here could represent another useful method for inducing immune responses against microbial, viral and tumorigenic protein antigens.

Assembly of synthetic peptide vaccines by chemoselective ligation of epitopes : influence of different chemical linkages and epitope orientations on biological activity

In this paper, we describe the assembly of synthetic peptide vaccines composed of a T helper cell epitope and a B cell epitope that were synthesized separately and then attached using three different chemoselective ligation methods: oxime bond formation, thioether bond formation and disulfide bond formation. The resulting vaccines were tested in animals to investigate their efficacy. We found that thioether bond formation gave the highest yield of material and that the chemistry involved did not adversely affect immunogenicity and biological activity of the peptide vaccine. Ligation of epitopes by oxime bond formation did not diminish biological activity either, but the yields of peptide vaccine were lower than when thioether bond formation was used. The vaccines in which a disulfide bond was used to attach the two epitopes resulted in the lowest yield and produced vaccines that also generated a weaker immune response with sub-optimal biological activity. Connecting the T helper epitope via its N-terminus or its C-terminus to the N-terminus of the B cell epitope had little influence on resulting immunogenicity and biological activity.