First-in-human use of a modular capsid virus-like vaccine platform: an open-label, non-randomised, phase 1 clinical trial of the SARS-CoV-2 vaccine ABNCoV2.

BACKGROUND: Capsid virus-like particles (cVLP) have proven safe and immunogenic and can be a versatile platform to counter pandemics. We aimed to clinically test a modular cVLP COVID-19 vaccine in individuals who were naive to SARS-CoV-2. METHODS: In this phase 1, single-centre, dose-escalation, adjuvant-selection, open-label clinical trial, we recruited participants at the Radboud University Medical Center in Nijmegen, Netherlands, and sequentially assigned them to seven groups. Eligible participants were healthy, aged 18-55 years, and tested negative for SARS-CoV-2 and anti-SARS-CoV-2 antibodies. Participants were vaccinated intramuscularly on days 0 and 28 with 6 µg, 12 µg, 25 µg, 50 µg, or 70 µg of the cVLP-based COVID-19 vaccine (ABNCoV2). A subgroup received MF59-adjuvanted ABNCoV2. Follow-up was for 24 weeks after second vaccination. The primary objectives were to assess the safety and tolerability of ABNCoV2 and to identify a dose that optimises the tolerability-immunogenicity ratio 14 days after the first vaccination. The primary safety endpoint was the number of related grade 3 adverse events and serious adverse events in the intention-to-treat population. The primary immunogenicity endpoint was the concentration of ABNCoV2-specific antibodies. The trial is registered with ClinicalTrials.gov, NCT04839146. FINDINGS: 45 participants (six to nine per group) were enrolled between March 15 and July 15, 2021. Participants had a total of 249 at least possibly related solicited adverse events (185 grade 1, 63 grade 2, and one grade 3) within a week after vaccination. Two serious adverse events occurred; one was classified as a possible adverse reaction. Antibody titres were dose-dependent with levels plateauing at a vaccination dose of 25-70 µg ABNCoV2. After second vaccination, live virus neutralisation activity against major SARS-CoV-2 variants was high but was lower with an omicron (BA.1) variant. Vaccine-specific IFNγ+ CD4+ T cells were induced. INTERPRETATION: Immunisation with ABNCoV2 was well tolerated, safe, and resulted in a functional immune response. The data support the need for additional clinical development of ABNCoV2 as a second-generation SARS-CoV-2 vaccine. The modular cVLP platform will accelerate vaccine development, beyond SARS-CoV-2. FUNDING: EU, Carlsberg Foundation, and the Novo Nordisk Foundation.

Freeze-Drying of a Capsid Virus-like Particle-Based Platform Allows Stable Storage of Vaccines at Ambient Temperature.

The requirement of an undisrupted cold chain during vaccine distribution is a major economic and logistical challenge limiting global vaccine access. Modular, nanoparticle-based platforms are expected to play an increasingly important role in the development of the next-generation vaccines. However, as with most vaccines, they are dependent on the cold chain in order to maintain stability and efficacy. Therefore, there is a pressing need to develop thermostable formulations that can be stored at ambient temperature for extended periods without the loss of vaccine efficacy. Here, we investigate the compatibility of the Tag/Catcher AP205 capsid virus-like particle (cVLP) vaccine platform with the freeze-drying process. Tag/Catcher cVLPs can be freeze-dried under diverse buffer and excipient conditions while maintaining their original biophysical properties. Additionally, we show that for two model cVLP vaccines, including a clinically tested SARS-CoV-2 vaccine, freeze-drying results in a product that once reconstituted retains the structural integrity and immunogenicity of the original material, even following storage under accelerated heat stress conditions. Furthermore, the freeze-dried SARS-CoV-2 cVLP vaccine is stable for up to 6 months at ambient temperature. Our study offers a potential solution to overcome the current limitations associated with the cold chain and may help minimize the need for low-temperature storage.

Seroprevalence of SARS-CoV-2 antibodies among children and adolescents recruited in a malariometric survey in north-eastern Tanzania July 2021.

BACKGROUND: African countries stand out globally as the region seemingly least affected by the COVID-19 pandemic, caused by the virus SARS-CoV-2. Besides a younger population and potential pre-existing immunity to a SARS-CoV-2-like virus, it has been hypothesized that co-infection or recent history of Plasmodium falciparum malaria may be protective of COVID-19 severity and mortality. The number of COVID-19 cases and deaths, however, may be vastly undercounted. Very little is known about the extent to which the Tanzanian population has been exposed to SARS-CoV-2. Here, we investigated the seroprevalence of IgG to SARS-CoV-2 spike protein in two Tanzanian rural communities 1½ years into the pandemic and the association of coinciding malaria infection and exposure. METHODS: During a malariometric survey in July 2021 in two villages in north-eastern Tanzania, blood samples were taken from 501 participants (0-19 years old). Malaria was detected by mRDT and microscopy. Levels of IgG against the spike protein of SARS-CoV-2 were measured by ELISA as well as IgG against five different antigens of P. falciparum; CIDRα1.1, CIDRα1.4 and CIDRα1.5 of PfEMP1 and GLURP and MSP3. RESULTS: The seroprevalence of SARS-CoV-2 IgG was 39.7% (106/267) in Kwamasimba and 32.5% (76/234) in Mkokola. In both villages the odds of being seropositive increased significantly with age (AOR = 1.12, 95% CI 1.07-1.17, p < 0.001). P. falciparum malaria prevalence by blood smear microscopy was 7.9% in Kwamasimba and 2.1% in Mkokola. 81.3% and 70.5% in Kwamasimba and Mkokola, respectively, showed recognition of minimum one malaria antigen. Residing in Kwamasimba was associated with a broader recognition (AOR = 1.91, 95% CI 1.34-2.71, p < 0.001). The recognition of malaria antigens increased significantly with age in both villages (AOR = 1.12; 95% CI 1.08-1.16, p < 0.001). Being SARS-CoV-2 seropositive did not associate with the breadth of malaria antigen recognition when adjusting for age (AOR = 0.99; 95% CI 0.83-1.18; p = 0.91). CONCLUSION: More than a third of the children and adolescents in two rural communities in Tanzania had antibodies to SARS-CoV-2. In particular, the adolescents were seropositive but being seropositive did not associate with the status of coinciding malaria infections or previous exposure. In Tanzania, natural immunity may have developed fast, potentially protecting a substantial part of the population from later variants.

SARS-CoV-2 RBD-Specific Antibodies Induced Early in the Pandemic by Natural Infection and Vaccination Display Cross-Variant Binding and Inhibition.

The development of vaccine candidates for COVID-19 has been rapid, and those that are currently approved display high efficacy against the original circulating strains. However, recently, new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have emerged with increased transmission rates and less susceptibility to vaccine induced immunity. A greater understanding of protection mechanisms, including antibody longevity and cross-reactivity towards the variants of concern (VoCs), is needed. In this study, samples collected in Denmark early in the pandemic from paucisymptomatic subjects (n = 165) and symptomatic subjects (n = 57) infected with SARS-CoV-2 were used to assess IgG binding and inhibition in the form of angiotensin-converting enzyme 2 receptor (ACE2) competition against the wild-type and four SARS-CoV-2 VoCs (Alpha, Beta, Gamma, and Omicron). Antibodies induced early in the pandemic via natural infection were cross-reactive and inhibited ACE2 binding of the VoC, with reduced inhibition observed for the Omicron variant. When examined longitudinally, sustained cross-reactive inhibitory responses were found to exist in naturally infected paucisymptomatic subjects. After vaccination, receptor binding domain (RBD)-specific IgG binding increased by at least 3.5-fold and inhibition of ACE2 increased by at least 2-fold. When vaccination regimens were compared (two doses of Pfizer-BioNTech BNT162b2 (n = 50), or one dose of Oxford-AstraZeneca ChAdOx1 nCoV-19 followed by Pfizer-BioNTech BNT162b2 (ChAd/BNT) (n = 15)), higher levels of IgG binding and inhibition were associated with mix and match (ChAd/BNT) prime-boosting and time since vaccination. These results are particularly relevant for countries where vaccination levels are low.

Freeze-Drying of a Capsid Virus-like Particle-Based Platform Allows Stable Storage of Vaccines at Ambient Temperature

The requirement of an undisrupted cold chain during vaccine distribution is a major economic and logistical challenge limiting global vaccine access. Modular, nanoparticle-based platforms are expected to play an increasingly important role in the development of the next-generation vaccines. However, as with most vaccines, they are dependent on the cold chain in order to maintain stability and efficacy. Therefore, there is a pressing need to develop thermostable formulations that can be stored at ambient temperature for extended periods without the loss of vaccine efficacy. Here, we investigate the compatibility of the Tag/Catcher AP205 capsid virus-like particle (cVLP) vaccine platform with the freeze-drying process. Tag/Catcher cVLPs can be freeze-dried under diverse buffer and excipient conditions while maintaining their original biophysical properties. Additionally, we show that for two model cVLP vaccines, including a clinically tested SARS-CoV-2 vaccine, freeze-drying results in a product that once reconstituted retains the structural integrity and immunogenicity of the original material, even following storage under accelerated heat stress conditions. Furthermore, the freeze-dried SARS-CoV-2 cVLP vaccine is stable for up to 6 months at ambient temperature. Our study offers a potential solution to overcome the current limitations associated with the cold chain and may help minimize the need for low-temperature storage.

Two-Component Nanoparticle Vaccine Displaying Glycosylated Spike S1 Domain Induces Neutralizing Antibody Response against SARS-CoV-2 Variants.

Vaccines pave the way out of the SARS-CoV-2 pandemic. Besides mRNA and adenoviral vector vaccines, effective protein-based vaccines are needed for immunization against current and emerging variants. We have developed a virus-like particle (VLP)-based vaccine using the baculovirus-insect cell expression system, a robust production platform known for its scalability, low cost, and safety. Baculoviruses were constructed encoding SARS-CoV-2 spike proteins: full-length S, stabilized secreted S, or the S1 domain. Since subunit S only partially protected mice from SARS-CoV-2 challenge, we produced S1 for conjugation to bacteriophage AP205 VLP nanoparticles using tag/catcher technology. The S1 yield in an insect-cell bioreactor was ∼11 mg/liter, and authentic protein folding, efficient glycosylation, partial trimerization, and ACE2 receptor binding was confirmed. Prime-boost immunization of mice with 0.5 µg S1-VLPs showed potent neutralizing antibody responses against Wuhan and UK/B.1.1.7 SARS-CoV-2 variants. This two-component nanoparticle vaccine can now be further developed to help alleviate the burden of COVID-19. IMPORTANCE Vaccination is essential to reduce disease severity and limit the transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Protein-based vaccines are useful to vaccinate the world population and to boost immunity against emerging variants. Their safety profiles, production costs, and vaccine storage temperatures are advantageous compared to mRNA and adenovirus vector vaccines. Here, we use the versatile and scalable baculovirus expression vector system to generate a two-component nanoparticle vaccine to induce potent neutralizing antibody responses against SARS-CoV-2 variants. These nanoparticle vaccines can be quickly adapted as boosters by simply updating the antigen component.

Site-Specific O-Glycosylation Analysis of SARS-CoV-2 Spike Protein Produced in Insect and Human Cells.

Enveloped viruses hijack not only the host translation processes, but also its glycosylation machinery, and to a variable extent cover viral surface proteins with tolerogenic host-like structures. SARS-CoV-2 surface protein S presents as a trimer on the viral surface and is covered by a dense shield of N-linked glycans, and a few O-glycosites have been reported. The location of O-glycans is controlled by a large family of initiating enzymes with variable expression in cells and tissues and hence is difficult to predict. Here, we used our well-established O-glycoproteomic workflows to map the precise positions of O-linked glycosylation sites on three different entities of protein S-insect cell or human cell-produced ectodomains, or insect cell derived receptor binding domain (RBD). In total 25 O-glycosites were identified, with similar patterns in the two ectodomains of different cell origin, and a distinct pattern of the monomeric RBD. Strikingly, 16 out of 25 O-glycosites were located within three amino acids from known N-glycosites. However, O-glycosylation was primarily found on peptides that were unoccupied by N-glycans, and otherwise had low overall occupancy. This suggests possible complementary functions of O-glycans in immune shielding and negligible effects of O-glycosylation on subunit vaccine design for SARS-CoV-2.

Capsid-like particles decorated with the SARS-CoV-2 receptor-binding domain elicit strong virus neutralization activity.

The rapid development of a SARS-CoV-2 vaccine is a global priority. Here, we develop two capsid-like particle (CLP)-based vaccines displaying the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. RBD antigens are displayed on AP205 CLPs through a split-protein Tag/Catcher, ensuring unidirectional and high-density display of RBD. Both soluble recombinant RBD and RBD displayed on CLPs bind the ACE2 receptor with nanomolar affinity. Mice are vaccinated with soluble RBD or CLP-displayed RBD, formulated in Squalene-Water-Emulsion. The RBD-CLP vaccines induce higher levels of serum anti-spike antibodies than the soluble RBD vaccines. Remarkably, one injection with our lead RBD-CLP vaccine in mice elicits virus neutralization antibody titers comparable to those found in patients that had recovered from COVID-19. Following booster vaccinations, the virus neutralization titers exceed those measured after natural infection, at serum dilutions above 1:10,000. Thus, the RBD-CLP vaccine is a highly promising candidate for preventing COVID-19.