Novel Structurally Designed Vaccine for S. aureus α-Hemolysin: Protection against Bacteremia and Pneumonia

Staphylococcus aureus (S. aureus) is a human pathogen associated with skin and soft tissue infections (SSTI) and life threatening sepsis and pneumonia. Efforts to develop effective vaccines against S. aureus have been largely unsuccessful, in part due to the variety of virulence factors produced by this organism. S. aureus alpha-hemolysin (Hla) is a pore-forming toxin expressed by most S. aureus strains and reported to play a key role in the pathogenesis of SSTI and pneumonia. Here we report a novel recombinant subunit vaccine candidate for Hla, rationally designed based on the heptameric crystal structure. This vaccine candidate, denoted AT-62aa, was tested in pneumonia and bacteremia infection models using S. aureus strain Newman and the pandemic strain USA300 (LAC). Significant protection from lethal bacteremia/sepsis and pneumonia was observed upon vaccination with AT-62aa along with a Glucopyranosyl Lipid Adjuvant-Stable Emulsion (GLA-SE) that is currently in clinical trials. Passive transfer of rabbit immunoglobulin against AT-62aa (AT62-IgG) protected mice against intraperitoneal and intranasal challenge with USA300 and produced significant reduction in bacterial burden in blood, spleen, kidney, and lungs. Our Hla-based vaccine is the first to be reported to reduce bacterial dissemination and to provide protection in a sepsis model of S. aureus infection. AT62-IgG and sera from vaccinated mice effectively neutralized the toxin in vitro and AT62-IgG inhibited the formation of Hla heptamers, suggesting antibody-mediated neutralization as the primary mechanism of action. This remarkable efficacy makes this Hla-based vaccine a prime candidate for inclusion in future multivalent S. aureus vaccine. Furthermore, identification of protective epitopes within AT-62aa could lead to novel immunotherapy for S. aureus infection

Knocking out Ornithine Decarboxylase Antizyme 1 (OAZ1) Improves Recombinant Protein Expression in the HEK293 Cell Line

Creating efficient cell lines is a priority for the biopharmaceutical industry, which produces biologicals for various uses. A recent approach to achieving this goal is the use of non-coding RNAs, microRNA (miRNA) and small interfering RNA (siRNA), to identify key genes that can potentially improve production or growth. The ornithine decarboxylase antizyme 1 (OAZ1) gene, a negative regulator of polyamine biosynthesis, was identified in a genome-wide siRNA screen as a potential engineering target, because its knock down by siRNA increased recombinant protein expression from human embryonic kidney 293 (HEK293) cells by two-fold. To investigate this further, the OAZ1 gene in HEK293 cells was knocked out using CRISPR genome editing. The OAZ1 knockout cell lines displayed up to four-fold higher expression of both stably and transiently expressed proteins, with comparable growth and metabolic activity to the parental cell line; and an approximately three-fold increase in intracellular polyamine content. The results indicate that genetic inactivation of OAZ1 in HEK293 cells is an effective strategy to improve recombinant protein expression in HEK293 cells

Knocking out Ornithine Decarboxylase Antizyme 1 (OAZ1) Improves Recombinant Protein Expression in the HEK293 Cell Line

Creating efficient cell lines is a priority for the biopharmaceutical industry, which produces biologicals for various uses. A recent approach to achieving this goal is the use of non-coding RNAs, microRNA (miRNA) and small interfering RNA (siRNA), to identify key genes that can potentially improve production or growth. The ornithine decarboxylase antizyme 1 (OAZ1) gene, a negative regulator of polyamine biosynthesis, was identified in a genome-wide siRNA screen as a potential engineering target, because its knock down by siRNA increased recombinant protein expression from human embryonic kidney 293 (HEK293) cells by two-fold. To investigate this further, the OAZ1 gene in HEK293 cells was knocked out using CRISPR genome editing. The OAZ1 knockout cell lines displayed up to four-fold higher expression of both stably and transiently expressed proteins, with comparable growth and metabolic activity to the parental cell line; and an approximately three-fold increase in intracellular polyamine content. The results indicate that genetic inactivation of OAZ1 in HEK293 cells is an effective strategy to improve recombinant protein expression in HEK293 cells

Structurally designed attenuated subunit vaccines for S. aureus LukS-PV and LukF-PV confer protection in a mouse bacteremia model.

Previous efforts towards S. aureus vaccine development have largely focused on cell surface antigens to induce opsonophagocytic killing aimed at providing sterile immunity, a concept successfully applied to other Gram-positive pathogens such as Streptococcus pneumoniae. However, these approaches have largely failed, possibly in part due to the remarkable diversity of the staphylococcal virulence factors such as secreted immunosuppressive and tissue destructive toxins. S. aureus produces several pore-forming toxins including the single subunit alpha hemolysin as well as bicomponent leukotoxins such as Panton-Valentine leukocidin (PVL), gamma hemolysins (Hlg), and LukED. Here we report the generation of highly attenuated mutants of PVL subunits LukS-PV and LukF-PV that were rationally designed, based on an octameric structural model of the toxin, to be deficient in oligomerization. The attenuated subunit vaccines were highly immunogenic and showed significant protection in a mouse model of S. aureus USA300 sepsis. Protection against sepsis was also demonstrated by passive transfer of rabbit immunoglobulin raised against LukS-PV. Antibodies to LukS-PV inhibited the homologous oligomerization of LukS-PV with LukF-PV as well heterologous oligomerization with HlgB. Importantly, immune sera from mice vaccinated with the LukS mutant not only inhibited the PMN lytic activity produced by the PVL-positive USA300 but also blocked PMN lysis induced by supernatants of PVL-negative strains suggesting a broad protective activity towards other bicomponent toxins. These findings strongly support the novel concept of an anti-virulence, toxin-based vaccine intended for prevention of clinical S. aureus invasive disease, rather than achieving sterile immunity. Such a multivalent vaccine may include attenuated leukotoxins, alpha hemolysin, and superantigens

Structural analysis of Hla.

<p>(<b>A</b>) The relative topology of 1–62 and 1–62(GGG)–(223–236) AT constructs on the protein surface of a subunit from the 7AHL heptameric hemolysin crystal structure. The protein surface for the 1–62 segment is colored green, the 223–236 sequence colored dark green, and the remaining structure colored purple. (<b>B</b>) Topology of the secondary structural elements in α-hemolysin for peptide segments examined in this study.</p

Passive protection with rabbit polyclonal AT-62aa (AT62-IgG) in bacteremia model.

<p>Protection from lethal challenge with <i>S. aureus</i> USA300 (<b>A</b>) or USA400 (<b>B</b>) after passive immunization with polyclonal rabbit antibodies AT62-IgG (black square) compared to mock-treated mice (grey diamond). Statistical analysis was performed using Log-Rank (Mantel-Cox) test, <i>P</i><0.0001.</p

Inhibition of toxin oligomerization with AT62-IgG.

<p>Rabbit RBCs were incubated with Hla alone or Hla pre-incubated with pAb. The mixtures were incubated with 10% rabbit RBC for 45 min at 37°C, cells were pelleted, washed, lysed, and loaded in SDS-PAGE without heating. Lane 1: boiled; lane 2 at 4°C, lane 3: Hla control without RBC; lanes 4–10: 15 µg/ml of Hla neutralized with decreasing concentration of anti AT-62aa pAb (two fold diluted from 400 to 6.25 ug/ml). Western blot was developed with sheep anti-Hla polyclonal antibody.</p

Comparative antibody response to vaccine candidates.

<p>(<b>A</b>) Individual serum antibody titers towards wild-type Hla determined after three immunizations (<b>B</b>) Neutralization titers of pooled sera towards wild-type Hla after three immunizations.</p

Comparative efficacy study of vaccine candidates in <i>S. aureus</i> bacteremia and pneumonia infection models.

<p>Survival of mice vaccinated with the three vaccine candidates and control mice after IP challenge with 5×10⁴ CFU of USA300 along with Hog Mucin (<b>A</b>) or IN challenge with 6×10⁷ CFU of <i>S. aureus</i> strain Newman (<b>B</b>). Survival of mice vaccinated with AT-62aa and challenged IN with 1.5×10⁸ CFU of <i>S. aureus</i> USA300 (<b>C</b>). Symbol key: AT-50aa (open circle), AT-62aa (black square), AT-79aa (open triangles) and mock-immunized mice (grey diamond). Statistical analysis was performed using Log-Rank (Mantel-Cox) test.</p