A platform technology for generating subunit vaccines against diverse viral pathogens.

The COVID-19 pandemic response has shown how vaccine platform technologies can be used to rapidly and effectively counteract a novel emerging infectious disease. The speed of development for mRNA and vector-based vaccines outpaced those of subunit vaccines, however, subunit vaccines can offer advantages in terms of safety and stability. Here we describe a subunit vaccine platform technology, the molecular clamp, in application to four viruses from divergent taxonomic families: Middle Eastern respiratory syndrome coronavirus (MERS-CoV), Ebola virus (EBOV), Lassa virus (LASV) and Nipah virus (NiV). The clamp streamlines subunit antigen production by both stabilising the immunologically important prefusion epitopes of trimeric viral fusion proteins while enabling purification without target-specific reagents by acting as an affinity tag. Conformations for each viral antigen were confirmed by monoclonal antibody binding, size exclusion chromatography and electron microscopy. Notably, all four antigens tested remained stable over four weeks of incubation at 40°C. Of the four vaccines tested, a neutralising immune response was stimulated by clamp stabilised MERS-CoV spike, EBOV glycoprotein and NiV fusion protein. Only the clamp stabilised LASV glycoprotein precursor failed to elicit virus neutralising antibodies. MERS-CoV and EBOV vaccine candidates were both tested in animal models and found to provide protection against viral challenge.

Dermal Delivery of a SARS-CoV-2 Subunit Vaccine Induces Immunogenicity against Variants of Concern.

The ongoing coronavirus disease 2019 (COVID-19) pandemic continues to disrupt essential health services in 90 percent of countries today. The spike (S) protein found on the surface of the causative agent, the SARS-CoV-2 virus, has been the prime target for current vaccine research since antibodies directed against the S protein were found to neutralize the virus. However, as new variants emerge, mutations within the spike protein have given rise to potential immune evasion of the response generated by the current generation of SARS-CoV-2 vaccines. In this study, a modified, HexaPro S protein subunit vaccine, delivered using a needle-free high-density microarray patch (HD-MAP), was investigated for its immunogenicity and virus-neutralizing abilities. Mice given two doses of the vaccine candidate generated potent antibody responses capable of neutralizing the parental SARS-CoV-2 virus as well as the variants of concern, Alpha and Delta. These results demonstrate that this alternative vaccination strategy has the potential to mitigate the effect of emerging viral variants.

Safety and immunogenicity of an MF59-adjuvanted spike glycoprotein-clamp vaccine for SARS-CoV-2: a randomised, double-blind, placebo-controlled, phase 1 trial.

BACKGROUND: Given the scale of the ongoing COVID-19 pandemic, the development of vaccines based on different platforms is essential, particularly in light of emerging viral variants, the absence of information on vaccine-induced immune durability, and potential paediatric use. We aimed to assess the safety and immunogenicity of an MF59-adjuvanted subunit vaccine for COVID-19 based on recombinant SARS-CoV-2 spike glycoprotein stabilised in a pre-fusion conformation by a novel molecular clamp (spike glycoprotein-clamp [sclamp]). METHODS: We did a phase 1, double-blind, placebo-controlled, block-randomised trial of the sclamp subunit vaccine in a single clinical trial site in Brisbane, QLD, Australia. Healthy adults (aged ≥18 to ≤55 years) who had tested negative for SARS-CoV-2, reported no close contact with anyone with active or previous SARS-CoV-2 infection, and tested negative for pre-existing SARS-CoV-2 immunity were included. Participants were randomly assigned to one of five treatment groups and received two doses via intramuscular injection 28 days apart of either placebo, sclamp vaccine at 5 µg, 15 µg, or 45 µg, or one dose of sclamp vaccine at 45 µg followed by placebo. Participants and study personnel, except the dose administration personnel, were masked to treatment. The primary safety endpoints included solicited local and systemic adverse events in the 7 days after each dose and unsolicited adverse events up to 12 months after dosing. Here, data are reported up until day 57. Primary immunogenicity endpoints were antigen-specific IgG ELISA and SARS-CoV-2 microneutralisation assays assessed at 28 days after each dose. The study is ongoing and registered with ClinicalTrials.gov, NCT04495933. FINDINGS: Between June 23, 2020, and Aug 17, 2020, of 314 healthy volunteers screened, 120 were randomly assigned (n=24 per group), and 114 (95%) completed the study up to day 57 (mean age 32·5 years [SD 10·4], 65 [54%] male, 55 [46%] female). Severe solicited reactions were infrequent and occurred at similar rates in participants receiving placebo (two [8%] of 24) and the SARS-CoV-2 sclamp vaccine at any dose (three [3%] of 96). Both solicited reactions and unsolicited adverse events occurred at a similar frequency in participants receiving placebo and the SARS-CoV-2 sclamp vaccine. Solicited reactions occurred in 19 (79%) of 24 participants receiving placebo and 86 (90%) of 96 receiving the SARS-CoV-2 sclamp vaccine at any dose. Unsolicited adverse events occurred in seven (29%) of 24 participants receiving placebo and 35 (36%) of 96 participants receiving the SARS-CoV-2 sclamp vaccine at any dose. Vaccination with SARS-CoV-2 sclamp elicited a similar antigen-specific response irrespective of dose: 4 weeks after the initial dose (day 29) with 5 µg dose (geometric mean titre [GMT] 6400, 95% CI 3683-11 122), with 15 µg dose (7492, 4959-11 319), and the two 45 µg dose cohorts (8770, 5526-13 920 in the two-dose 45 µg cohort; 8793, 5570-13 881 in the single-dose 45 µg cohort); 4 weeks after the second dose (day 57) with two 5 µg doses (102 400, 64 857-161 676), with two 15 µg doses (74 725, 51 300-108 847), with two 45 µg doses (79 586, 55 430-114 268), only a single 45 µg dose (4795, 2858-8043). At day 57, 67 (99%) of 68 participants who received two doses of sclamp vaccine at any concentration produced a neutralising immune response, compared with six (25%) of 24 who received a single 45 µg dose and none of 22 who received placebo. Participants receiving two doses of sclamp vaccine elicited similar neutralisation titres, irrespective of dose: two 5 µg doses (GMT 228, 95% CI 146-356), two 15 µg doses (230, 170-312), and two 45 µg doses (239, 187-307). INTERPRETATION: This first-in-human trial shows that a subunit vaccine comprising mammalian cell culture-derived, MF59-adjuvanted, molecular clamp-stabilised recombinant spike protein elicits strong immune responses with a promising safety profile. However, the glycoprotein 41 peptide present in the clamp created HIV diagnostic assay interference, a possible barrier to widespread use highlighting the criticality of potential non-spike directed immunogenicity during vaccine development. Studies are ongoing with alternative molecular clamp trimerisation domains to ameliorate this response. FUNDING: Coalition for Epidemic Preparedness Innovations, National Health and Medical Research Council, Queensland Government, and further philanthropic sources listed in the acknowledgments.

Complete protection by a single-dose skin patch-delivered SARS-CoV-2 spike vaccine.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 160 million people and resulted in more than 3.3 million deaths, and despite the availability of multiple vaccines, the world still faces many challenges with their rollout. Here, we use the high-density microarray patch (HD-MAP) to deliver a SARS-CoV-2 spike subunit vaccine directly to the skin. We show that the vaccine is thermostable on the patches, with patch delivery enhancing both cellular and antibody immune responses. Elicited antibodies potently neutralize clinically relevant isolates including the Alpha and Beta variants. Last, a single dose of HD-MAP­delivered spike provided complete protection from a lethal virus challenge in an ACE2-transgenic mouse model. Collectively, these data show that HD-MAP delivery of a SARS-CoV-2 vaccine was superior to traditional needle-and-syringe vaccination and may be a significant addition to the ongoing COVID-19 (coronavirus disease 2019) pandemic.

Combinatorial F-G Immunogens as Nipah and Respiratory Syncytial Virus Vaccine Candidates.

Nipah virus (NiV) and respiratory syncytial virus (RSV) possess two surface glycoproteins involved in cellular attachment and membrane fusion, both of which are potential targets for vaccines. The majority of vaccine development is focused on the attachment (G) protein of NiV, which is the immunodominant target. In contrast, the fusion (F) protein of RSV is the main target in vaccine development. Despite this, neutralising epitopes have been described in NiV F and RSV G, making them alternate targets for vaccine design. Through rational design, we have developed a vaccine strategy applicable to phylogenetically divergent NiV and RSV that comprises both the F and G proteins (FxG). In a mouse immunization model, we found that NiV FxG elicited an improved immune response capable of neutralising pseudotyped NiV and a NiV mutant that is able to escape neutralisation by two known F-specific antibodies. RSV FxG elicited an immune response against both F and G and was able to neutralise RSV; however, this was inferior to the immune response of F alone. Despite this, RSV FxG elicited a response against a known protective epitope within G that is conserved across RSV A and B subgroups, which may provide additional protection in vivo. We conclude that inclusion of F and G antigens within a single design provides a streamlined subunit vaccine strategy against both emerging and established pathogens, with the potential for broader protection against NiV.

A versatile reverse genetics platform for SARS-CoV-2 and other positive-strand RNA viruses.

The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We demonstrate that despite the large size of the viral RNA genome (~30 kb), infectious full-length cDNA is readily assembled in vitro by a circular polymerase extension reaction (CPER) methodology without the need for technically demanding intermediate steps. Overlapping cDNA fragments are generated from viral RNA and assembled together with a linker fragment containing CMV promoter into a circular full-length viral cDNA in a single reaction. Transfection of the circular cDNA into mammalian cells results in the recovery of infectious SARS-CoV-2 virus that exhibits properties comparable to the parental virus in vitro and in vivo. CPER is also used to generate insect-specific Casuarina virus with ~20 kb genome and the human pathogens Ross River virus (Alphavirus) and Norovirus (Calicivirus), with the latter from a clinical sample. Additionally, reporter and mutant viruses are generated and employed to study virus replication and virus-receptor interactions.

An Optimized High-Throughput Immuno-Plaque Assay for SARS-CoV-2.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has been identified as the causative agent of coronavirus disease 2019 and is capable of human-to-human transmission and rapid global spread. The rapid emergence and global spread of SARS-CoV-2 has encouraged the establishment of a rapid, sensitive, and reliable viral detection and quantification methodology. Here, we present an alternative assay, termed immuno-plaque assay (iPA), which utilizes a combination of plaque assay and immunofluorescence techniques. We have extensively optimized the conditions for SARS-CoV-2 infection and demonstrated the great flexibility of iPA detection using several antibodies and dual-probing with two distinct epitope-specific antibodies. In addition, we showed that iPA could be utilized for ultra-high-throughput viral titration and neutralization assay within 24 h and is amenable to a 384-well format. These advantages will significantly accelerate SARS-CoV-2 research outcomes during this pandemic period.

Adjuvant Selection for Influenza and RSV Prefusion Subunit Vaccines.

Subunit vaccines exhibit favorable safety and immunogenicity profiles and can be designed to mimic native antigen structures. However, pairing with an appropriate adjuvant is imperative in order to elicit effective humoral and cellular immune responses. In this study, we aimed to determine an optimal adjuvant pairing with the prefusion form of influenza haemagglutinin (HA) or respiratory syncytial virus (RSV) fusion (F) subunit vaccines in BALB/c mice in order to inform future subunit vaccine adjuvant selection. We tested a panel of adjuvants, including aluminum hydroxide (alhydrogel), QS21, Addavax, Addavax with QS21 (AdQS21), and Army Liposome Formulation 55 with monophosphoryl lipid A and QS21 (ALF55). We found that all adjuvants elicited robust humoral responses in comparison to placebo, with the induction of potent neutralizing antibodies observed in all adjuvanted groups against influenza and in AdQS21, alhydrogel, and ALF55 against RSV. Upon HA vaccination, we observed that none of the adjuvants were able to significantly increase the frequency of CD4+ and CD8+ IFN-γ+ cells when compared to unadjuvanted antigen. The varying responses to antigens with each adjuvant highlights that those adjuvants most suited for pairing purposes can vary depending on the antigen used and/or the desired immune response. We therefore suggest that an adjuvant trial for different subunit vaccines in development would likely be necessary in preclinical studies.