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Open Access Publications from the University of California

Scholarly Works (9 results)

  • Thesis
  • Peer Reviewed
UC Irvine Electronic Theses and Dissertations (2023)

Coxiella burnetii is an intracellular Gram-negative bacterium responsible for Q fever, a zoonotic disease with significant global health implications courtesy of its endemic status worldwide. This tier 2 select agent has the potential for use as a bioweapon due to low infectious dose and aerosol transmissibility. The development of a vaccine against C. burnetii is of great interest, particularly for people who are in close contact with ruminants and military personnel who may be exposed to the pathogen in the field. The Q-VAX vaccine, an inactivated whole-cell vaccine, has shown efficacy in preventing Q fever and has been used in high-risk populations. However, challenges remain in its widespread implementation due to limited availability and adverse reactions.Immunological studies have demonstrated the crucial role of macrophages in the host's defense against C. burnetii, with these cells playing a central role in phagocytosis, antigen presentation, and the production of pro-inflammatory cytokines. Interferon gamma (IFNγ), a key cytokine produced by T lymphocytes and natural killer cells, plays a significant role in controlling C. burnetii infection by activating macrophages and enhancing their antimicrobial activity. The use of nanoparticles as chemically programmable scaffolds in bead design shows promise in C. burnetii vaccine development. These nanoparticles can be engineered to display multiple proteins and epitopes, increasing immunogenicity and broadening the immune response. We showcased a high-throughput method of capturing expressed proteins onto bead scaffolds, which were directly administered to animals for in vivo evaluation. Additionally, the bacterial sacculus, the highly ordered peptidoglycan structure of bacteria, was explored as a vaccine scaffold and adjuvant. Muramyl dipeptide (MDP), the main component of the sacculus, acts as a pathogen-associated molecular pattern (PAMP) and activates innate immune responses through recognition by NOD-like receptors. The sacculus’s immunogenicity as an adjuvant was also evaluated in vivo. Adjuvants, such as toll-like receptor (TLR) agonists, are investigated here to enhance the immunogenicity of Q fever vaccines by increasing Th1 responses and IFNγ production. Challenge studies were conducted to evaluate subunit vaccine formulations containing downselected antigen candidates combined with optimized adjuvant combinations, including C. burnetii lipopolysaccharide. In conclusion, understanding the immunology of C. burnetii infection and the challenges associated with vaccine development is essential for effectively combating Q fever. Continued research into novel vaccine strategies, adjuvants, and delivery systems holds promise for improving vaccine efficacy, reducing the global burden of Q fever, and protecting at-risk individuals.

    Cover page: Vaccine-induced adaptive T cell immunity enhances protective responses against Coxiella burnetiiCreative Commons 'BY' version 4.0 license
    • Article
    Recent Work (2019)

    The lion’s share of media attention surrounding AB 5 has gone to the law’s effects on on-demand labor platforms like Uber and Lyft. However, these workers represent just a fraction of independent contractors, most of whom work across a diverse range of occupations such as janitors, hair stylists, and accountants.

      Cover page: Estimating the Coverage of California’s New AB 5 Law
      • Article
      • Peer Reviewed
      UC Irvine Previously Published Works (2023)
      Vaccines are among the most cost-effective public health measures for controlling infectious diseases. Coxiella burnetii is the etiological agent of Q fever, a disease with a wide clinical spectrum that ranges from mild symptoms, such as fever and fatigue, to more severe disease, such as pneumonia and endocarditis. The formalin-inactivated whole-cell vaccine Q-VAX® contains hundreds of antigens and confers lifelong protection in humans, but prior sensitization from infection or vaccination can result in deleterious reactogenic responses to vaccination. Consequently, there is great interest in developing non-reactogenic alternatives based on adjuvanted recombinant proteins. In this study, we aimed to develop a multivalent vaccine that conferred protection with reduced reactogenicity. We hypothesized that a multivalent vaccine consisting of multiple antigens would be more immunogenic and protective than a monovalent vaccine owing to the large number of potential protective antigens in the C. burnetii proteome. To address this, we identified immunogenic T and B cell antigens, and selected proteins were purified to evaluate with a combination adjuvant (IVAX-1), with or without C. burnetii lipopolysaccharide (LPS) in immunogenicity studies in vivo in mice and in a Hartley guinea pig intratracheal aerosol challenge model using C. burnetii strain NMI RSA 493. The data showed that multivalent vaccines are more immunogenic than monovalent vaccines and more closely emulate the protection achieved by Q-VAX. Although six antigens were the most immunogenic, we also discovered that multiplexing beyond four antigens introduces detectable reactogenicity, indicating that there is an upper limit to the number of antigens that can be safely included in a multivalent Q-fever vaccine. C. burnetii LPS also demonstrates efficacy as a vaccine antigen in conferring protection in an otherwise monovalent vaccine formulation, suggesting that its addition in multivalent vaccines, as demonstrated by a quadrivalent formulation, would improve protective responses.
        Cover page: Multivalent vaccines demonstrate immunogenicity and protect against Coxiella burnetii aerosol challenge.
        • Article
        • Peer Reviewed
        UC Irvine Previously Published Works (2021)
        Auranofin, a reprofiled FDA-approved drug originally designed to treat rheumatoid arthritis, has emerged as a promising anti-parasitic drug. It induces the accumulation of reactive oxygen species (ROS) in parasites, including Toxoplasma gondii. We generated auranofin resistant T. gondii lines through chemical mutagenesis to identify the molecular target of this drug. Resistant clones were confirmed with a competition assay using wild-type T. gondii expressing yellow fluorescence protein (YFP) as a reference strain. The predicted auranofin target, thioredoxin reductase, was not mutated in any of our resistant lines. Subsequent whole genomic sequencing analysis (WGS) did not reveal a consensus resistance locus, although many have point mutations in genes encoding redox-relevant proteins such as superoxide dismutase (TgSOD2) and ribonucleotide reductase. We investigated the SOD2 L201P mutation and found that it was not sufficient to confer resistance when introduced into wild-type parasites. Resistant clones accumulated less ROS than their wild type counterparts. Our results demonstrate that resistance to auranofin in T. gondii enhances its ability to abate oxidative stress through diverse mechanisms. This evidence supports a hypothesized mechanism of auranofin anti-parasitic activity as disruption of redox homeostasis.
          Cover page: Auranofin Resistance in Toxoplasma gondii Decreases the Accumulation of Reactive Oxygen Species but Does Not Target Parasite Thioredoxin Reductase
          • Article
          • Peer Reviewed
          UC Irvine Previously Published Works (2021)
          Combining variant antigens into a multivalent vaccine is a traditional approach used to provide broad coverage against antigenically variable pathogens, such as polio, human papilloma and influenza viruses. However, strategies for increasing the breadth of antibody coverage beyond the vaccine are not well understood, but may provide more anticipatory protection. Influenza virus hemagglutinin (HA) is a prototypic variant antigen. Vaccines that induce HA-specific neutralizing antibodies lose efficacy as amino acid substitutions accumulate in neutralizing epitopes during influenza virus evolution. Here we studied the effect of a potent combination adjuvant (CpG/MPLA/squalene-in-water emulsion) on the breadth and maturation of the antibody response to a representative variant of HA subtypes H1, H5 and H7. Using HA protein microarrays and antigen-specific B cell labelling, we show when administered individually, each HA elicits a cross-reactive antibody profile for multiple variants within the same subtype and other closely-related subtypes (homosubtypic and heterosubtypic cross-reactivity, respectively). Despite a capacity for each subtype to induce heterosubtypic cross-reactivity, broader coverage was elicited by simply combining the subtypes into a multivalent vaccine. Importantly, multiplexing did not compromise antibody avidity or affinity maturation to the individual HA constituents. The use of adjuvants to increase the breadth of antibody coverage beyond the vaccine antigens may help future-proof vaccines against newly-emerging variants.
            Cover page: Administration of Multivalent Influenza Virus Recombinant Hemagglutinin Vaccine in Combination-Adjuvant Elicits Broad Reactivity Beyond the Vaccine Components
            • Article
            • Peer Reviewed
            UC Irvine Previously Published Works (2021)
            Q fever is caused by the obligate intracellular bacterium, Coxiella burnetii, a designated potential agent of bioterrorism because of its route of transmission, resistance to disinfectants, and low infectious dose. The only vaccine licensed for human use is Q-VAX® (Seqirus, licensed in Australia), a formalin-inactivated whole-cell vaccine, which produces severe local and systemic reactogenic responses in previously sensitized individuals. Accordingly, the U.S. Food and Drug Administration and other regulatory bodies around the world, have been reluctant to approve Q-VAX for widespread use. To obviate these adverse reactions, we prepared recombinant protein subunit vaccine candidates containing purified CBU1910, CBU0307, CBU0545, CBU0612, CBU0891, and CBU1398 proteins and TLR triagonist adjuvants. TLR triagonist adjuvants combine different TLR agonists to enhance immune responses to vaccine antigens. We tested both the protective efficacy and reactogenicity of our vaccine candidates in Hartley guinea pigs using intratracheal infection with live C. burnetii. While all of our candidates showed varying degrees of protection during challenge, local reactogenic responses were significantly reduced for one of our vaccine candidates when compared with a formalin-inactivated whole-cell vaccine. Our findings show that subunit vaccines combined with novel TLR triagonist adjuvants can generate protective immunity to C. burnetii infection while reducing reactogenic responses.
              Cover page: Subunit Vaccines Using TLR Triagonist Combination Adjuvants Provide Protection Against Coxiella burnetii While Minimizing Reactogenic Responses
              • Article
              • Peer Reviewed
              UC Irvine Previously Published Works (2019)
              The influenza virus remains a significant cause of mortality worldwide due to the limited effectiveness of currently available vaccines. A key challenge to the development of universal influenza vaccines is high antigenic diversity resulting from antigenic drift. Overcoming this challenge requires novel research tools to measure the breadth of serum antibodies directed against many virus strains across different antigenic subtypes. Here, we present a protocol for analyzing the breadth of serum antibodies against diverse influenza virus strains using a protein microarray of influenza antigens. This influenza antigen microarray is constructed by printing purified hemagglutinin and neuraminidase antigens onto a nitrocellulose-coated membrane using a microarray printer. Human sera are incubated on the microarray to bind antibodies against the influenza antigens. Quantum-dot-conjugated secondary antibodies are used to simultaneously detect IgG and IgA antibodies binding to each antigen on the microarray. Quantitative antibody binding is measured as fluorescence intensity using a portable imager. Representative results are shown to demonstrate assay reproducibility in measuring subtype-specific and cross-reactive influenza antibodies in human sera. Compared to traditional methods such as ELISA, the influenza antigen microarray provides a high throughput multiplexed approach capable of testing hundreds of sera for multiple antibody isotypes against hundreds of antigens in a short time frame, and thus has applications in sero-surveillance and vaccine development. A limitation is the inability to distinguish binding antibodies from neutralizing antibodies.
                Cover page: Use of an Influenza Antigen Microarray to Measure the Breadth of Serum Antibodies Across Virus Subtypes.
                • Article
                • Peer Reviewed
                UC Irvine Previously Published Works (2022)
                The effects of adjuvants for increasing the immunogenicity of influenza vaccines are well known. However, the effect of adjuvants on increasing the breadth of cross-reactivity is less well understood. In this study we have performed a systematic screen of different toll-like receptor (TLR) agonists, with and without a squalene-in-water emulsion on the immunogenicity of a recombinant trimerized hemagglutinin (HA) vaccine in mice after single-dose administration. Antibody (Ab) cross-reactivity for other variants within and outside the immunizing subtype (homosubtypic and heterosubtypic cross-reactivity, respectively) was assessed using a protein microarray approach. Most adjuvants induced broad IgG profiles, although the response to a combination of CpG, MPLA and AddaVax (termed 'IVAX-1') appeared more quickly and reached a greater magnitude than the other formulations tested. Antigen-specific plasma cell labeling experiments show the components of IVAX-1 are synergistic. This adjuvant preferentially stimulates CD4 T cells to produce Th1>Th2 type (IgG2c>IgG1) antibodies and cytokine responses. Moreover, IVAX-1 induces identical homo- and heterosubtypic IgG and IgA cross-reactivity profiles when administered intranasally. Consistent with these observations, a single-cell transcriptomics analysis demonstrated significant increases in expression of IgG1, IgG2b and IgG2c genes of B cells in H5/IVAX-1 immunized mice relative to naïve mice, as well as significant increases in expression of the IFNγ gene of both CD4 and CD8 T cells. These data support the use of adjuvants for enhancing the breath and durability of antibody responses of influenza virus vaccines.
                  Cover page: Magnitude and breadth of antibody cross-reactivity induced by recombinant influenza hemagglutinin trimer vaccine is enhanced by combination adjuvants
                  • Article
                  • Peer Reviewed
                  UC Irvine Previously Published Works (2023)
                  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.
                    Cover page: Engineering Protein Nanoparticles Functionalized with an Immunodominant Coxiella burnetii Antigen to Generate a Q Fever Vaccine
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