Decoration of Burkholderia Hcp1 protein to virus-like particles as a vaccine delivery platform.

Virus-like particles (VLPs) are protein-based nanoparticles frequently used as carriers in conjugate vaccine platforms. VLPs have been used to display foreign antigens for vaccination and to deliver immunotherapy against diseases. Hemolysin-coregulated proteins 1 (Hcp1) is a protein component of the Burkholderia type 6 secretion system, which participates in intracellular invasion and dissemination. This protein has been reported as a protective antigen and is used in multiple vaccine candidates with various platforms against melioidosis, a severe infectious disease caused by the intracellular pathogen Burkholderia pseudomallei. In this study, we used P22 VLPs as a surface platform for decoration with Hcp1 using chemical conjugation. C57BL/6 mice were intranasally immunized with three doses of either PBS, VLPs, or conjugated Hcp1-VLPs. Immunization with Hcp1-VLPs formulation induced Hcp1-specific IgG, IgG1, IgG2c, and IgA antibody responses. Furthermore, the serum from Hcp1-VLPs immunized mice enhanced the bacterial uptake and opsonophagocytosis by macrophages in the presence of complement. This study demonstrated an alternative strategy to develop a VLPs-based vaccine platform against Burkholderia species.

Engineering Protein Nanoparticles Functionalized with an Immunodominant Coxiella burnetii Antigen to Generate a Q Fever Vaccine.

Coxiella burnetii is the causative agent of Q fever, for which there is yet to be an FDA-approved vaccine. This bacterial pathogen has both extra- and intracellular stages in its life cycle, and therefore both a cell-mediated (i.e., T lymphocyte) and humoral (i.e., antibody) immune response are necessary for effective eradication of this pathogen. However, most proposed vaccines elicit strong responses to only one mechanism of adaptive immunity, and some can either cause reactogenicity or lack sufficient immunogenicity. In this work, we aim to apply a nanoparticle-based platform toward producing both antibody and T cell immune responses against C. burnetii. We investigated three approaches for conjugation of the immunodominant outer membrane protein antigen (CBU1910) to the E2 nanoparticle to obtain a consistent antigen orientation: direct genetic fusion, high affinity tris-NTA-Ni conjugation to polyhistidine-tagged CBU1910, and the SpyTag/SpyCatcher (ST/SC) system. Overall, we found that the ST/SC approach yielded nanoparticles loaded with the highest number of antigens while maintaining stability, enabling formulations that could simultaneously co-deliver the protein antigen (CBU1910) and adjuvant (CpG1826) on one nanoparticle (CBU1910-CpG-E2). Using protein microarray analyses, we found that after immunization, antigen-bound nanoparticle formulations elicited significantly higher antigen-specific IgG responses than soluble CBU1910 alone and produced more balanced IgG1/IgG2c ratios. Although T cell recall assays from these protein antigen formulations did not show significant increases in antigen-specific IFN-γ production compared to soluble CBU1910 alone, nanoparticles conjugated with a CD4 peptide epitope from CBU1910 generated elevated T cell responses in mice to both the CBU1910 peptide epitope and whole CBU1910 protein. These investigations highlight the feasibility of conjugating antigens to nanoparticles for tuning and improving both humoral- and cell-mediated adaptive immunity against C. burnetii.

Tuning Subunit Vaccines with Novel TLR Triagonist Adjuvants to Generate Protective Immune Responses against Coxiella burnetii.

Coxiella burnetii is an obligate intracellular bacterium and the causative agent of Q fever. C. burnetii is considered a potential bioterrorism agent because of its low infectious dose; resistance to heat, drying, and common disinfectants; and lack of prophylactic therapies. Q-Vax, a formalin-inactivated whole-bacteria vaccine, is currently the only prophylactic measure that is protective against C. burnetii infections but is not U.S. Food and Drug Administration approved. To overcome the safety concerns associated with the whole-bacteria vaccine, we sought to generate and evaluate recombinant protein subunit vaccines against C. burnetii To accomplish this, we formulated C. burnetii Ags with a novel TLR triagonist adjuvant platform, which used combinatorial chemistry to link three different TLR agonists together to form one adjuvanting complex. We evaluated the immunomodulatory activity of a panel of TLR triagonist adjuvants and found that they elicited unique Ag-specific immune responses both in vitro and in vivo. We evaluated our top candidates in a live C. burnetii aerosol challenge model in C56BL/6 mice and found that several of our novel vaccine formulations conferred varying levels of protection to the challenged animals compared with sham immunized mice, although none of our candidates were as protective as the commercial vaccine across all protection criteria that were analyzed. Our findings characterize a novel adjuvant platform and offer an alternative approach to generating protective and effective vaccines against C. burnetii.

Burkholderia pseudomallei Complex Subunit and Glycoconjugate Vaccines and Their Potential to Elicit Cross-Protection to Burkholderia cepacia Complex.

Burkholderia are a group of Gram-negative bacteria that can cause a variety of diseases in at-risk populations. B. pseudomallei and B. mallei, the etiological agents of melioidosis and glanders, respectively, are the two clinically relevant members of the B. pseudomallei complex (Bpc). The development of vaccines against Bpc species has been accelerated in recent years, resulting in numerous promising subunits and glycoconjugate vaccines incorporating a variety of antigens. However, a second group of pathogenic Burkholderia species exists known as the Burkholderia cepacia complex (Bcc), a group of opportunistic bacteria which tend to affect individuals with weakened immunity or cystic fibrosis. To date, there have been few attempts to develop vaccines to Bcc species. Therefore, the primary goal of this review is to provide a broad overview of the various subunit antigens that have been tested in Bpc species, their protective efficacy, study limitations, and known or suspected mechanisms of protection. Then, we assess the reviewed Bpc antigens for their amino acid sequence conservation to homologous proteins found in Bcc species. We propose that protective Bpc antigens with a high degree of Bpc-to-Bcc sequence conservation could serve as components of a pan-Burkholderia vaccine capable of protecting against both disease-causing groups.

Unraveling the role of toxin-antitoxin systems in Burkholderia pseudomallei: exploring bacterial pathogenesis and interactions within the HigBA families.

Burkholderia pseudomallei (Bpm) is a Gram-negative intracellular pathogen that causes melioidosis in humans, a neglected, underreported, and lethal disease that can reach a fatal outcome in over 50% of the cases. It can produce both acute and chronic infections, the latter being particularly challenging to eliminate because of the intracellular life cycle of the bacteria and its ability to generate a "persister" dormant state. The molecular mechanism that allows the switch between growing and persister phenotypes is not well understood but it is hypothesized to be due at least in part to the participation of toxin-antitoxin (TA) systems. We have previously studied the link between one of those systems (defined as HigBA) with specific expression patterns associated with levofloxacin antibiotic exposure. Through in silico methods, we predicted the presence of another three pairs of genes encoding for additional putative HigBA systems. Therefore, our main goal was to establish which mechanisms are conserved as well as which pathways are specific among different Bpm TA systems from the same family. We hypothesize that the high prevalence, and sometimes even redundancy of these systems in the Bpm chromosomes indicates that they can interact with each other and not function as only individual systems, as it was traditionally thought, and might be playing an undefined role in Bpm lifecycle. Here, we show that both the toxin and the antitoxin of the different systems contribute to bacterial survival and that toxins from the same family can have a cumulative effect under environmental stressful conditions. IMPORTANCE: Toxin-antitoxin (TA) systems play a significant role in bacterial persistence, a phenomenon where bacterial cells enter a dormant or slow-growing state to survive adverse conditions such as nutrient deprivation, antibiotic exposure, or host immune responses. By studying TA systems in Burkholderia pseudomallei, we can gain insights into how this pathogen survives and persists in the host environment, contributing to its virulence and ability to cause melioidosis chronic infections.

Protein Nanoparticle-Mediated Delivery of Recombinant Influenza Hemagglutinin Enhances Immunogenicity and Breadth of the Antibody Response.

The vast majority of seasonal influenza vaccines administered each year are derived from virus propagated in eggs using technology that has changed little since the 1930s. The immunogenicity, durability, and breadth of response would likely benefit from a recombinant nanoparticle-based approach. Although the E2 protein nanoparticle (NP) platform has been previously shown to promote effective cell-mediated responses to peptide epitopes, it has not yet been reported to deliver whole protein antigens. In this study, we synthesized a novel maleimido tris-nitrilotriacetic acid (NTA) linker to couple protein hemagglutinin (HA) from H1N1 influenza virus to the E2 NP, and we evaluated the HA-specific antibody responses using protein microarrays. We found that recombinant H1 protein alone is immunogenic in mice but requires two boosts for IgG to be detected and is strongly IgG1 (Th2) polarized. When conjugated to E2 NPs, IgG2c is produced leading to a more balanced Th1/Th2 response. Inclusion of the Toll-like receptor 4 agonist monophosphoryl lipid A (MPLA) significantly enhances the immunogenicity of H1-E2 NPs while retaining the Th1/Th2 balance. Interestingly, broader homo- and heterosubtypic cross-reactivity is also observed for conjugated H1-E2 with MPLA, compared to unconjugated H1 with or without MPLA. These results highlight the potential of an NP-based delivery of HA for tuning the immunogenicity, breadth, and Th1/Th2 balance generated by recombinant HA-based vaccination. Furthermore, the modularity of this protein-protein conjugation strategy may have utility for future vaccine development against other human pathogens.