Ability of SpikoGen®, an Advax-CpG adjuvanted recombinant spike protein vaccine, to induce cross-neutralising antibodies against SARS-CoV-2 variants.

SpikoGen® vaccine is a subunit COVID-19 vaccine expressed in insect cells comprising recombinant spike protein extracellular domain formulated with Advax-CpG55.2™ adjuvant. A Phase 2 trial was conducted in 400 adult participants randomised 3:1 to receive two intramuscular doses of SpikoGen® vaccine or saline placebo 3 weeks apart. Some Phase 2 trial participants later enrolled in a separate booster study and received a third dose of SpikoGen® vaccine. This stored serum was used to assess the ability of SpikoGen® vaccine to induce cross-neutralising antibodies against SARS-CoV-2 variants of concern. Sera taken at baseline and 2 weeks after the second vaccine dose from baseline seronegative Phase 2 subjects was evaluated using a panel of spike pseudotype lentivirus neutralisation assays for the ability to cross-neutralise a wide range of SARS-CoV-2 variants, including Omicron BA.1, BA.2 and BA.4/5. Stored samples of subjects who participated in both the 2-dose Phase 2 trial and a third dose booster trial 6 months later were also analysed for changes in cross-neutralising antibodies over time and dose. Two weeks after the second dose, sera broadly cross-neutralised most variants of concern, albeit with titres against Omicron variants being ~10-fold lower. While Omicron titres fell to low levels 6 months after the second vaccine dose in most subjects, they showed a ~20-fold rise after the third dose booster, after which there was only a ~2-3-fold difference in neutralisation of Omicron and the ancestral strains. Despite being based on the ancestral Wuhan sequence, after two doses, SpikoGen® vaccine induced broadly cross-neutralising serum antibodies. Titres then reduced over time but were rapidly restored by a third dose booster. This resulted in high neutralisation including against the Omicron variants. This data supports ongoing use of SpikoGen® vaccine for protection against recent SARS-CoV-2 Omicron variants.

Immunogenicity and safety of SpikoGen®, an adjuvanted recombinant SARS-CoV-2 spike protein vaccine as a homologous and heterologous booster vaccination: A randomized placebo-controlled trial.

SpikoGen® is a subunit recombinant spike protein vaccine combined with Advax-CpG55.2™ adjuvant. This COVID-19 vaccine was shown to be safe, immunogenic and efficacious in previous clinical trials. This study aimed to assess the safety and immunogenicity of SpikoGen® vaccine as a homologous and heterologous booster vaccination. This double-blind and randomized placebo-controlled (5:1) trial was performed on 300 already vaccinated participants. SpikoGen® or saline placebo was administered as a booster dose to participants who had received a full two-dose COVID-19 vaccination course. Immunogenicity assessments were done 14 days after the booster dose with the primary immunogenicity outcome seroconversion rate of neutralizing antibodies. Safety outcomes included the incidence of solicited adverse events up to 7 days after the booster dose. SpikoGen® vaccine induced a robust humoral response both as a homologous and heterologous booster, when compared to the placebo. At Day 14, seroconversion of neutralizing antibodies was 76% (95% confidence interval [CI]: 69%-82%) in the SpikoGen® group versus 3% (95% CI: 0%-13%) in the placebo group. The most common local and systemic reported adverse events were injection site pain and fatigue. No serious adverse events were reported. The SpikoGen®-booster induced cross-neutralization of other SARS-CoV-2 variants. Irrespective of the primary vaccine course received, SpikoGen® vaccine showed promising effects as both a homologous and heterologous booster dose. This vaccine also had a good safety profile with no vaccine-associated serious adverse events. On the basis of these results, SpikoGen® vaccine has been approved as a booster dose.

Advax-SM™-Adjuvanted COBRA (H1/H3) Hemagglutinin Influenza Vaccines.

Adjuvants enhance immune responses stimulated by vaccines. To date, many seasonal influenza vaccines are not formulated with an adjuvant. In the present study, the adjuvant Advax-SM™ was combined with next generation, broadly reactive influenza hemagglutinin (HA) vaccines that were designed using a computationally optimized broadly reactive antigen (COBRA) methodology. Advax-SM™ is a novel adjuvant comprising inulin polysaccharide and CpG55.2, a TLR9 agonist. COBRA HA vaccines were combined with Advax-SM™ or a comparator squalene emulsion (SE) adjuvant and administered to mice intramuscularly. Mice vaccinated with Advax-SM™ adjuvanted COBRA HA vaccines had increased serum levels of anti-influenza IgG and IgA, high hemagglutination inhibition activity against a panel of H1N1 and H3N2 influenza viruses, and increased anti-influenza antibody secreting cells isolated from spleens. COBRA HA plus Advax-SM™ immunized mice were protected against both morbidity and mortality following viral challenge and, at postmortem, had no detectable lung viral titers or lung inflammation. Overall, the Advax-SM™-adjuvanted COBRA HA formulation provided effective protection against drifted H1N1 and H3N2 influenza viruses.

An Advax-CpG55.2 adjuvanted recombinant hemagglutinin vaccine provides immunity against H7N9 influenza in adult and neonatal mice.

There is a major unmet need for strategies to improve the immunogenicity and effectiveness of pandemic influenza vaccines, particularly in poor responder populations such as neonates. Recombinant protein approaches to pandemic influenza offer advantages over more traditional inactivated virus approaches, as they are free of problems such as egg adaptation or need for high level biosecurity containment for manufacture. However, a weakness of recombinant proteins is their low immunogenicity. We asked whether the use of an inulin polysaccharide adjuvant (Advax) alone or combined with a TLR9 agonist (CpG55.2) would enhance the immunogenicity and protection of a recombinant hemagglutinin vaccine against H7N9 influenza (rH7HA), including in neonatal mice. Advax adjuvant induced predominantly IgG1 responses against H7HA, whereas Advax-CpG55.2 adjuvant also induced IgG2a, IgG2b and IgG3 responses, consistent with the TLR9 agonist component inducing a Th1 bias. Advax-CpG55.2 adjuvanted rH7HA induced high serum neutralizing antibody titers in adult mice. In newborns it similarly overcame immune hypo-responsiveness and enhanced serum anti-rH7HA IgG levels in 7-day-old BALB/C and C57BL/6 mice. Immunized adult mice were protected against a lethal H7N9 virus challenge. When formulated with Advax-CpG55.2 adjuvant, greater protection was seen with rH7HA than with inactivated H7 whole virus antigen. Advax-CpG55.2 adjuvanted rH7HA represents a promising influenza vaccine platform for further development.

An Advax-CpG55.2™ adjuvanted recombinant spike protein vaccine protects cynomolgus macaques from a homologous SARS-CoV-2 virus challenge.

Traditional protein-based vaccine approaches to COVID-19 were overshadowed by the new mRNA and adenoviral vector vaccine approaches which were first to receive marketing authorization. The current study tested for the first time in repurposed aged (median 15.4 years) cynomolgus macaques, a novel Advax-CpG55.2™ adjuvanted recombinant extracellular domain spike protein trimer antigen for immunogenicity, protection and safety. Nine animals received two intramuscular injections 10 days apart of recombinant spike protein (25 µg) with Advax-CpG55.2™ (10 mg/200 µg) and 5 controls received saline injections. Serum antibody levels were followed for 3 months and then the animals were challenged with SARS-CoV-2 virus. Clinical signs, local reactions, body weight, food consumption and antibody levels were monitored till termination on either day 3 or 7 post-infection. Two weeks after the second dose, 8/9 immunized macaques had high serum spike and receptor binding domain binding antibodies that were able to cross-neutralize Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2) and, to a lesser extent, Omicron variants (B.1.1.529 ). Antibody levels decayed over the subsequent 3 months, and minimal neutralizing antibody was detectable immediately prior to the challenge which used a vaccine-homologous Wuhan-like ancestral virus. Of the nine vaccinated animals, only one 18-year-old female sacrificed at d3 had low levels of lung virus, versus 100 % of the control animals. Four of 5 (80 %) control animals had positive lung staining for SARS-CoV-2 virus versus just 1 of 9 (11 %) in the immunized group. The immunized animals exhibited better maintenance of appetite post-challenge. Neutralizing antibody levels rebounded rapidly in immunized animals, post-challenge. This data supports the benefits of Advax-CpG adjuvanted recombinant spike protein vaccine in protecting against a homologous SARS-CoV-2 infection.

Fusion of PspA to detoxified pneumolysin enhances pneumococcal vaccine coverage.

Despite the implementation of conjugate vaccines in several countries, S. pneumoniae continues to pose a great burden worldwide, causing around 1 million annual deaths. Pneumococcal proteins have long been investigated as serotype-independent vaccines against this pathogen, with promising results. However, it is a consensus that one antigen alone will not be sufficient to provide long-term protection with wide coverage. Amongst the most well studied pneumococcal proteins are PspA and pneumolysin (Ply), two major virulence factors required by the bacterium for successful invasion of host tissues. PspA is highly immunogenic and protective, but it is structurally variable; pneumolysin is conserved among different pneumococci, but it is toxic to the host. To overcome these limitations, N-terminal PspA fragments have been genetically fused to non-toxic pneumolysin derivatives (PlD) to create PspA_PlD chimeras. Mouse immunization with these fusions confers protection against pneumococcal strains expressing heterologous PspAs, which correlates with antibody-induced complement C3 deposition on the surface of multiple pneumococcal strains. Analysis of mutant strains lacking PspA or Pneumolysin shows that both proteins contribute to the antibody-mediated enhancement in complement deposition induced by the fusion. These results expand previous data evaluating PspA_PlD and demonstrate that the fusion combines the protective traits of both proteins, inducing antibodies that efficiently promote complement deposition on multiple strains and cross-protection.

Protective role of PhtD and its amino and carboxyl fragments against pneumococcal sepsis.

The implementation of polysaccharide-based vaccines has massively reduced the incidence of invasive pneumococcal diseases. However, there is great concern regarding serotype replacement and the increase in antibiotic resistant strains expressing non-vaccine capsular types. In addition, conjugate vaccines have high production costs, a limiting factor for their implementation in mass immunization programs in developing countries. These limitations have prompted the development of novel vaccine strategies for prevention of Streptococcus pneumoniae infections. The use of conserved pneumococcal antigens such as recombinant proteins or protein fragments presents an interesting serotype-independent alternative. Pht is a family of surface-exposed proteins which have been evaluated as potential vaccine candidates with encouraging results. The present work investigated the immune responses elicited by subcutaneous immunization of mice with the polyhistidine triad protein D (PhtD) and its amino and carboxyl terminal fragments. The proteins were immunogenic and protective against pneumococcal sepsis in mice. Antibodies raised against PhtD increased complement C3b deposition on the pneumococcal surface, mainly mediated by the alternative pathway. Sera from mice immunized with PhtD and PhtD_Cter promoted an increase in bacterial uptake by mouse phagocytes. The interaction of PhtD with the complement system regulator factor H was investigated in silico and in vitro by ELISA and western blot, confirming PhtD as a factor-H binding protein. Our results support the inclusion of PhtD and more specifically, its C-terminal fragment in a multicomponent serotype independent vaccine and suggests a role for the complement system in PhtD-mediated protection.

Immunization with PhtD truncated fragments reduces nasopharyngeal colonization by Streptococcus pneumoniae.

Despite the undeniable success of polysaccharide vaccines against Streptococcus pneumoniae infections, there is a consensus on the scientific field that this approach should be revised in order to overpass the problems related with these formulations, such as serotype replacement and high production costs. The study of conserved pneumococcal proteins or its truncated fragments has emerged as a serotype independent alternative. In this work, we have characterized the immune response elicited by systemic immunization of mice with the Histidine triad protein D (PhtD) and its' amino and carboxyl terminal fragments. The proteins were shown to be immunogenic and protective against pneumococcal colonization, with increased IL-17 production, and induction of antibodies able to limit pneumococcal adhesion to human respiratory cells. Antiserum against PhtD_Nter, but not C_ter or PhtD, promoted an increase in bacterial phagocytosis in vitro. Interestingly, antibodies against the PhtD_Nter displayed cross-reactivity with two other pneumococcal proteins, PspA and PspC, due to sequence similarities in the proline rich region of the molecules. On a whole, our results support the inclusion of PhtD, and more specifically, its N-terminal fragment, in a multicomponent serotype independent vaccine.

Role of Streptococcus pneumoniae Proteins in Evasion of Complement-Mediated Immunity.

The complement system plays a central role in immune defense against Streptococcus pneumoniae. In order to evade complement attack, pneumococci have evolved a number of mechanisms that limit complement mediated opsonization and subsequent phagocytosis. This review focuses on the strategies employed by pneumococci to circumvent complement mediated immunity, both in vitro and in vivo. At last, since many of the proteins involved in interactions with complement components are vaccine candidates in different stages of validation, we explore the use of these antigens alone or in combination, as potential vaccine approaches that aim at elimination or drastic reduction in the ability of this bacterium to evade complement.