Efficacy, safety, and immunogenicity of a booster regimen of Ad26.COV2.S vaccine against COVID-19 (ENSEMBLE2): results of a randomised, double-blind, placebo-controlled, phase 3 trial.

BACKGROUND: Despite the availability of effective vaccines against COVID-19, booster vaccinations are needed to maintain vaccine-induced protection against variant strains and breakthrough infections. This study aimed to investigate the efficacy, safety, and immunogenicity of the Ad26.COV2.S vaccine (Janssen) as primary vaccination plus a booster dose. METHODS: ENSEMBLE2 is a randomised, double-blind, placebo-controlled, phase 3 trial including crossover vaccination after emergency authorisation of COVID-19 vaccines. Adults aged at least 18 years without previous COVID-19 vaccination at public and private medical practices and hospitals in Belgium, Brazil, Colombia, France, Germany, the Philippines, South Africa, Spain, the UK, and the USA were randomly assigned 1:1 via a computer algorithm to receive intramuscularly administered Ad26.COV2.S as a primary dose plus a booster dose at 2 months or two placebo injections 2 months apart. The primary endpoint was vaccine efficacy against the first occurrence of molecularly confirmed moderate to severe-critical COVID-19 with onset at least 14 days after booster vaccination, which was assessed in participants who received two doses of vaccine or placebo, were negative for SARS-CoV-2 by PCR at baseline and on serology at baseline and day 71, had no major protocol deviations, and were at risk of COVID-19 (ie, had no PCR-positive result or discontinued the study before day 71). Safety was assessed in all participants; reactogenicity, in terms of solicited local and systemic adverse events, was assessed as a secondary endpoint in a safety subset (approximately 6000 randomly selected participants). The trial is registered with ClinicalTrials.gov, NCT04614948, and is ongoing. FINDINGS: Enrolment began on Nov 16, 2020, and the primary analysis data cutoff was June 25, 2021. From 34 571 participants screened, the double-blind phase enrolled 31 300 participants, 14 492 of whom received two doses (7484 in the Ad26.COV2.S group and 7008 in the placebo group) and 11 639 of whom were eligible for inclusion in the assessment of the primary endpoint (6024 in the Ad26.COV2.S group and 5615 in the placebo group). The median (IQR) follow-up post-booster vaccination was 36·0 (15·0-62·0) days. Vaccine efficacy was 75·2% (adjusted 95% CI 54·6-87·3) against moderate to severe-critical COVID-19 (14 cases in the Ad26.COV2.S group and 52 cases in the placebo group). Most cases were due to the variants alpha (B.1.1.7) and mu (B.1.621); endpoints for the primary analysis accrued from Nov 16, 2020, to June 25, 2021, before the global dominance of delta (B.1.617.2) or omicron (B.1.1.529). The booster vaccine exhibited an acceptable safety profile. The overall frequencies of solicited local and systemic adverse events (evaluated in the safety subset, n=6067) were higher among vaccine recipients than placebo recipients after the primary and booster doses. The frequency of solicited adverse events in the Ad26.COV2.S group were similar following the primary and booster vaccinations (local adverse events, 1676 [55·6%] of 3015 vs 896 [57·5%] of 1559, respectively; systemic adverse events, 1764 [58·5%] of 3015 vs 821 [52·7%] of 1559, respectively). Solicited adverse events were transient and mostly grade 1-2 in severity. INTERPRETATION: A homologous Ad26.COV2.S booster administered 2 months after primary single-dose vaccination in adults had an acceptable safety profile and was efficacious against moderate to severe-critical COVID-19. Studies assessing efficacy against newer variants and with longer follow-up are needed. FUNDING: Janssen Research & Development.

Cold-Adapted Viral Attenuation (CAVA): Highly Temperature Sensitive Polioviruses as Novel Vaccine Strains for a Next Generation Inactivated Poliovirus Vaccine.

The poliovirus vaccine field is moving towards novel vaccination strategies. Withdrawal of the Oral Poliovirus Vaccine and implementation of the conventional Inactivated Poliovirus Vaccine (cIPV) is imminent. Moreover, replacement of the virulent poliovirus strains currently used for cIPV with attenuated strains is preferred. We generated Cold-Adapted Viral Attenuation (CAVA) poliovirus strains by serial passage at low temperature and subsequent genetic engineering, which contain the capsid sequences of cIPV strains combined with a set of mutations identified during cold-adaptation. These viruses displayed a highly temperature sensitive phenotype with no signs of productive infection at 37°C as visualized by electron microscopy. Furthermore, decreases in infectious titers, viral RNA, and protein levels were measured during infection at 37°C, suggesting a block in the viral replication cycle at RNA replication, protein translation, or earlier. However, at 30°C, they could be propagated to high titers (9.4-9.9 Log10TCID50/ml) on the PER.C6 cell culture platform. We identified 14 mutations in the IRES and non-structural regions, which in combination induced the temperature sensitive phenotype, also when transferred to the genomes of other wild-type and attenuated polioviruses. The temperature sensitivity translated to complete absence of neurovirulence in CD155 transgenic mice. Attenuation was also confirmed after extended in vitro passage at small scale using conditions (MOI, cell density, temperature) anticipated for vaccine production. The inability of CAVA strains to replicate at 37°C makes reversion to a neurovirulent phenotype in vivo highly unlikely, therefore, these strains can be considered safe for the manufacture of IPV. The CAVA strains were immunogenic in the Wistar rat potency model for cIPV, inducing high neutralizing antibody titers in a dose-dependent manner in response to D-antigen doses used for cIPV. In combination with the highly productive PER.C6 cell culture platform, the stably attenuated CAVA strains may serve as an attractive low-cost and (bio)safe option for the production of a novel next generation IPV.

A virosomal respiratory syncytial virus vaccine adjuvanted with monophosphoryl lipid A provides protection against viral challenge without priming for enhanced disease in cotton rats.

BACKGROUND: Non-replicating respiratory syncytial virus (RSV) vaccine candidates could potentially prime for enhanced respiratory disease (ERD) due to a T-cell-mediated immunopathology, following RSV infection. Vaccines with built-in immune response modifiers, such as Toll-like receptor (TLR) ligands, may avoid such aberrant imprinting of the immune system. METHODS: We developed reconstituted RSV envelopes (virosomes) with incorporated TLR4 ligand, monophosphoryl lipid A (RSV-MPLA virosomes). Immune responses and lung pathology after vaccination and challenge were investigated in ERD-prone cotton rats and compared with responses induced by live virus and formaldehyde-inactivated vaccine (FI-RSV), a known cause of ERD upon RSV challenge. RESULTS: Vaccination with RSV-MPLA virosomes induced higher levels of virus-neutralizing antibodies than FI-RSV or live virus infection and provided protection against infection. FI-RSV, but not RSV-MPLA virosomes, primed for increases in expression of Th2 cytokines IL-4, IL-5, IL-13, and Th1 cytokine IL-1b, 6 hour-5 days after infection. By contrast, RSV-MPLA virosomes induced IFN-γ transcripts to similar levels as induced by live virus. Animals vaccinated with FI-RSV, but not RSV-MPLA virosomes showed alveolitis, with prominent neutrophil influx and peribronchiolar and perivascular infiltrates. CONCLUSION: These results show that RSV-MPLA virosomes represent a safe and immunogenic vaccine candidate that warrants evaluation in a clinical setting.

Efficacy and safety of an intranasal virosomal respiratory syncytial virus vaccine adjuvanted with monophosphoryl lipid A in mice and cotton rats.

Respiratory syncytial virus infection remains a serious health problem, not only in infants but also in immunocompromised adults and the elderly. An effective and safe vaccine is not available due to several obstacles: non-replicating RSV vaccines may prime for excess Th2-type responses and enhanced respiratory disease (ERD) upon natural RSV infection of vaccine recipients. We previously found that inclusion of the Toll-like receptor 4 (TLR4) ligand monophosphoryl lipid A (MPLA) in reconstituted RSV membranes (virosomes) potentiates vaccine-induced immunity and skews immune responses toward a Th1-phenotype, without priming for ERD. As mucosal immunization is an attractive approach for induction of RSV-specific systemic and mucosal antibody responses and TLR ligands could potentiate such responses, we explored the efficacy and safety of RSV-MPLA virosomes administered intranasally (IN) to mice and cotton rats. In mice, we found that incorporation of MPLA in IN-administered RSV virosomes increased both systemic IgG and local secretory-IgA (S-IgA) antibody levels and resulted in significantly reduced lung viral titers upon live virus challenge. Also, RSV MPLA virosomes induced more Th1-skewed responses compared to responses induced by FI-RSV. Antibody responses and Th1/Th2-cytokine responses induced by RSV-MPLA virosomes were comparable to those induced by live RSV infection. By comparison, formalin-inactivated RSV (FI-RSV) induced serum IgG that inhibited viral shedding upon challenge, but also induced Th2-skewed responses. In cotton rats, similar effects of incorporation of MPLA in virosomes were observed with respect to induction of systemic antibodies and inhibition of lung viral shedding upon challenge, but mucosal sS-IgA responses were only moderately enhanced. Importantly, IN immunization with RSV-MPLA virosomes, like live virus infection, did not lead to any signs of ERD upon live virus challenge of vaccinated animals, whereas IM immunization with FI-RSV did induce severe lung immunopathology under otherwise comparable conditions. Taken together, these data show that mucosally administered RSV-MPLA virosomes hold promise for a safe and effective vaccine against RSV.

Nanoaggregates of micropurified lipopolysaccharide identified using dynamic light scattering, zeta potential measurement, and TLR4 signaling activity.

Nanoaggregates composed of selected glycoforms from Escherichia coli 055:B5 lipopolysaccharide (LPS) were prepared by combining sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, zinc-imidazole reverse staining, zinc chelation after cutting gel slices, elution with either 0.5% triethylamine (TEA) or 0.4% to 0.5% surfactant (SDS or deoxycholate [DOC]) from extrusion-generated gel microparticles, and centrifugal diafiltration after appropriate surfactant dilution. Dynamic light scattering allows detecting these aggregates, giving a size distribution from 10 to 100nm in diameter. The formation of the aggregates prepared with selected DOC-eluted LPS glycoforms was notably improved over those prepared with TEA-eluted glycoforms. As the O-side chain length increased in the composition of the former aggregates, there was a gradual decrease in the electrophoretic mobility (from -1.2 to 0.0110(-8)m(2)/Vs), giving a calculated zeta potential from -15 to 0.1mV at pH6.8. These aggregates were further characterized for their abilities to elicit agonistic effects on human Toll-like receptor 4, as shown by in vitro activation of nuclear factor kappa light chain enhancer of activated B cells (NF-κB) in engineered HEK293 cells.

Immunogenicity and protective capacity of a virosomal respiratory syncytial virus vaccine adjuvanted with monophosphoryl lipid A in mice.

Respiratory Syncytial Virus (RSV) is a major cause of viral brochiolitis in infants and young children and is also a significant problem in elderly and immuno-compromised adults. To date there is no efficacious and safe RSV vaccine, partially because of the outcome of a clinical trial in the 1960s with a formalin-inactivated RSV vaccine (FI-RSV). This vaccine caused enhanced respiratory disease upon exposure to the live virus, leading to increased morbidity and the death of two children. Subsequent analyses of this incident showed that FI-RSV induces a Th2-skewed immune response together with poorly neutralizing antibodies. As a new approach, we used reconstituted RSV viral envelopes, i.e. virosomes, with incorporated monophosphoryl lipid A (MPLA) adjuvant to enhance immunogenicity and to skew the immune response towards a Th1 phenotype. Incorporation of MPLA stimulated the overall immunogenicity of the virosomes compared to non-adjuvanted virosomes in mice. Intramuscular administration of the vaccine led to the induction of RSV-specific IgG2a levels similar to those induced by inoculation of the animals with live RSV. These antibodies were able to neutralize RSV in vitro. Furthermore, MPLA-adjuvanted RSV virosomes induced high amounts of IFNγ and low amounts of IL5 in both spleens and lungs of immunized and subsequently challenged animals, compared to levels of these cytokines in animals vaccinated with FI-RSV, indicating a Th1-skewed response. Mice vaccinated with RSV-MPLA virosomes were protected from live RSV challenge, clearing the inoculated virus without showing signs of lung pathology. Taken together, these data demonstrate that RSV-MPLA virosomes represent a safe and efficacious vaccine candidate which warrants further evaluation.

Lipopeptide-adjuvanted respiratory syncytial virus virosomes: A safe and immunogenic non-replicating vaccine formulation.

Respiratory syncytial virus (RSV) causes severe respiratory disease in children and the elderly. There is no registered RSV vaccine. Early experimental non-replicating vaccines have been found to exacerbate RSV symptoms upon infection causing enhanced respiratory disease. Here we show that immunization of mice with reconstituted virosomes produced from RSV envelopes and containing the lipopeptide adjuvant (P3CSK4), induces high-titer virus-neutralizing antibodies, and the secretion of IFN-gamma through both MHC-I and MHC-II presentation of antigen, with a balanced Th1/Th2 profile. Immunization with RSV virosomes provides sterilizing immunity to virus challenge in mice and cotton rats, while not producing symptoms of enhanced disease. Therefore, these virosomes represent a promising candidate inactivated RSV vaccine formulation.