Dry and liquid formulations of IBT-V02, a novel multi-component toxoid vaccine, are effective against Staphylococcus aureus isolates from low-to-middle income countries

Staphylococcus aureus is the leading cause of skin and soft tissue infections (SSTIs) in the U.S. as well as more serious invasive diseases, including bacteremia, sepsis, endocarditis, surgical site infections, osteomyelitis, and pneumonia. These infections are exacerbated by the emergence of antibiotic-resistant clinical isolates such as methicillin-resistant S. aureus (MRSA), highlighting the need for alternatives to antibiotics to treat bacterial infections. We have previously developed a multi-component toxoid vaccine (IBT-V02) in a liquid formulation with efficacy against multiple strains of Staphylococcus aureus prevalent in the industrialized world. However, liquid vaccine formulations are not compatible with the paucity of cold chain storage infrastructure in many low-to-middle income countries (LMICs). Furthermore, whether our IBT-V02 vaccine formulations are protective against S. aureus isolates from LMICs is unknown. To overcome these limitations, we developed lyophilized and spray freeze-dried formulations of IBT-V02 vaccine and demonstrated that both formulations had comparable biophysical attributes as the liquid formulation, including similar levels of toxin neutralizing antibodies and protective efficacy against MRSA infections in murine and rabbit models. To enhance the relevancy of our findings, we then performed a multi-dimensional screen of 83 S. aureus clinical isolates from LMICs (e.g., Democratic Republic of Congo, Palestine, and Cambodia) to rationally down-select strains to test in our in vivo models based on broad expression of IBT-V02 targets (i.e., pore-forming toxins and superantigens). IBT-V02 polyclonal antisera effectively neutralized toxins produced by the S. aureus clinical isolates from LMICs. Notably, the lyophilized IBT-V02 formulation exhibited significant in vivo efficacy in various preclinical infection models against the S. aureus clinical isolates from LMICs, which was comparable to our liquid formulation. Collectively, our findings suggested that lyophilization is an effective alternative to liquid vaccine formulations of our IBT-V02 vaccine against S. aureus infections, which has important implications for protection from S. aureus isolates from LMICs

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

Comparison of Adhesion and Virulence of Two Predominant Hospital-Acquired Methicillin-Resistant Staphylococcus aureus Clones and Clonal Methicillin-Susceptible S. aureus Isolates▿

The virulence of SCCmec type IV hospital-acquired methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates belonging to the major sequence type 8 (ST8 [Lyon clone]) and to a minor upcoming clone, ST5, was compared with that of methicillin-susceptible S. aureus (MSSA) isolates of matching sequence types. In vitro adhesion to human airway epithelial cells (HAECs) as an indicator of dissemination and mortality in a murine sepsis model as an indicator of virulence were evaluated. Ten MRSA isolates and 8 MSSA isolates of ST8 and 8 MRSA isolates and 8 MSSA isolates of ST5 were characterized with respect to multilocus sequence type; agr, spa, and capsule typing; in vitro doubling time; toxin and adhesin gene profiles; and adherence to HAECs. Adherence was significantly lower in the MRSA ST5 group than in the ST8 groups. Infections with MRSA and MSSA isolates ST8 and ST5 were compared. No change in virulence related to the presence of SCCmec was observed, since ST8 but not ST5 caused a significantly lower mortality in its presence. Despite their similar genetic backgrounds, individual clonal MRSA and MSSA isolates were heterogeneous in adherence and virulence. No one of these specific virulence factors determined in vitro was related to mouse mortality. In conclusion, in a bacteremic model, mortality was dependent on the ST and was differentially modulated by SCCmec; within an ST, clonality was not associated with a homogenous outcome

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

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

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

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

Passive protection against bacterial dissemination in pneumonia model.

<p>Bacterial burden in organs and blood was determined after passive immunization with polyclonal AT62-IgG or naïve IgG followed by IN infection with <i>S. aureus</i> USA300. Mann-Whitney Test with two-tailed P value (<i>P</i> values as indicated in figures).</p