Health
Protection against SARS-CoV-2 transmission by a parenteral prime—Intranasal boost vaccine strategy
Summary
Background
Licensed vaccines against SARS-CoV-2 effectively protect against severe disease, but display incomplete protection against virus transmission. Mucosal vaccines providing immune responses in the upper airways are one strategy to protect against transmission.
Methods
We administered Spike HexaPro trimer formulated in a cationic liposomal adjuvant as a parenteral (subcutaneous – s.c.) prime – intranasal boost regimen to elicit airway mucosal immune responses and evaluated this in a Syrian hamster model of virus transmission.
Findings
Parenteral prime – intranasal boost elicited high-magnitude serum neutralizing antibody responses and IgA responses in the upper respiratory tract. The vaccine strategy protected against virus in the lower airways and lung pathology, but virus could still be detected in the upper airways. Despite this, the parenteral prime – intranasal booster vaccine effectively protected against onward SARS-CoV-2 transmission.
Interpretation
This study suggests that parenteral-prime mucosal boost is an effective strategy for protecting against SARS-CoV-2 infection and highlights that protection against virus transmission may be obtained despite incomplete clearance of virus from the upper respiratory tract. It should be noted that protection against onward transmission was not compared to standard parenteral prime-boost, which should be a focus for future studies.
Funding
This work was primarily supported by the European Union Horizon 2020 research and innovation program under grant agreement no. 101003653.
Introduction
,
,
have proved highly effective against severe COVID-19. However, a major limitation of these vaccines is their lower effectiveness at protecting against virus transmission than against disease. Parenteral vaccines predominantly induce systemic IgG antibody responses, but respiratory viruses with pandemic potential, including coronaviruses, are mainly transmitted from person to person via respiratory droplets and infect the upper respiratory tract, which is not effectively protected by circulating IgG.
,
Failure to elicit sterilizing immunity can lead to local viral replication in respiratory tissues and potentially onward transmission, enabling the development and spread of resistant variants. Mucosal vaccination is an established strategy for induction of secretory IgA (sIgA) at the mucosal surfaces that, by blocking virus at the portal of entry, may prevent initial viral replication and thus potentially provide sterilizing immunity. Compared to the monomeric IgG, sIgA is multimeric, providing increased avidity and sIgA thus can be better at neutralizing SARS-CoV-2 than IgG.
,
Intranasal (i.n.) immunization also elicits local tissue resident (TRM) CD4 and CD8 T cell responses in nasal-associated lymphoid tissue (NALT).
,
For SARS-CoV-1, an i.n. vaccine induced respiratory CD4 T cells recruiting protective CD8 T cells to NALT via an IFN-γ dependent mechanism.
The Th17 cell subset has received particular focus in mucosal immune responses
and Th17-produced IL-17A upregulates polymeric immunoglobulin receptor (pIgR) to promote secretory IgA responses.
,
,
One strategy to facilitate both systemic immunity and mucosal immune responses in the upper airways is by a parenteral prime – i.n. boost regimen.
,
,
,
We tested this strategy for SARS-CoV-2, using a cationic liposome (CAF®01) adjuvanted spike subunit vaccine. Immunization by parenteral prime – i.n. boost induced anti-spike IgG and SARS-CoV-2 neutralizing antibody responses in serum and elicited IgA responses in the upper respiratory tract. In a transmission model in which vaccinated contacts were co-housed with SARS-CoV-2 infected index hamsters, the parenteral prime-mucosal boost strategy lowered virus titres in the upper airways and protected against onward transmission. Overall, a parenteral prime – i.n. boost vaccine strategy may be an effective means to limit virus spread in the population.
Methods
Ethics
Animal studies were conducted in accordance with European Community Directive 2010/63/EU. The experiments have been approved by, and conducted in compliance with, the governmental Animal Experiments Inspectorate under licenses 2017-15-0201-01363 and 2020-15-0201-00554.
Antigens and adjuvants
and HexaPro trimer
), and the RBD domain (RVQ-VNF) from the Wuhan-Hu-1 strain were produced by transient expression in freestyle 293-F cells, as reported previously.
,
CAF®01 (250 μg DDA/ 50 μg TDB) in 10mM TRIS buffer with 2.2% glycerol (pH 7.0) was produced as described previously.
Characterization of formulations
A compatibility study of the spike HexaPro trimer in CAF®01 was performed at room temperature. Formulations were analysed visually for potential flocculation and then characterized for particle size and polydispersity index (PDI) by dynamic light scattering, using the photon correlation spectroscopy technique. The zeta potential was measured by laser-Doppler electrophoresis. For the size measurements, the samples were diluted 10 times, whereas for the zeta potential measurements, the samples were diluted 100 times in milli-Q water. The measurements were performed at 25 °C, using a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK) with a 633 nm laser and 173° detection optics. Malvern Zetasizer software v.8.01 was used for analysis.
Animals
Female C57Bl/6 (C57BL/6JOlaHsd) wild type mice, 7–9 weeks old, were obtained Envigo (The Netherlands). Male Syrian golden hamsters (Mesocricetus auratus), nine weeks old, were obtained from Janvier. Both species were housed in the animal facilities at Statens Serum Institut, Denmark during the studies and maintained in rooms with controlled environment (20–23 °C; relative humidity 52 ± 10%; 12/12 h light/dark cycle). The mice were randomly allocated to cages (type III polycarbonate cages (820 cm2)) with up to eight mice per cage and the hamsters were housed in polycarbonate cages type IV (1820 cm2) with high lid (in total app. 30 cm. high) with up to four animals/cage. A total of 28 mice and 51 hamsters were used for the studies. All animals were offered Aspen bedding and bricks (Tapvei), Sizzelnest (Datesand) and tunnels or houses made of polycarbonate. Besides, mice were offered DesRes paper houses (LBS) while hamsters had twisted paper rolls (“Diamond Twist” Envigo Teclad) hanging in their cage lids. Irradiated sunflower seeds, corn grains and peanuts or bits of carrots were offered all the animals once a week.
Pelleted diet (Envigo Teclad 2916) and tap water was provided ad libitum.
Immunizations
as described previously.
The spike S-2P variant was used in initial mouse studies, whilst the HexaPro variant was used in all hamster studies and in mouse studies where indicated. The vaccine was either given as two subcutaneous (s.c.) immunizations (s.c./s.c.) or as s.c. immunization followed by intranasal (i.n.) boost (s.c./i.n.). The s.c. immunizations were given at the base of tail (mice) or in the scruff of the neck (hamsters) at a final volume of 200 μl per immunization. The i.n. immunizations were performed under isofluorane anaesthesia. Mice were given a final volume of 20 μl (10 μl per nostril). Hamsters were placed in supine position and given 50 μl (25 μl per nostril). The number of animals in each group is stated in the figure legends. Power calculations were based on levels of antigen-specific IgA (against another protein antigen) in s.c./i.n. immunized compared to unvaccinated controls in a previous mouse study with effect size (OD450) of 0.58 and SD of 0.297. An analysis plan was prepared for each study as part of the study protocol.
Sampling and organ preparation from mice
Mice were euthanized by CO2 (80%)/O2 (20%), after anaesthetization with Zoletil-mix (Zolazepam, Tiletamin, Xylazin and Butorphanol). Lungs and spleens were filtered through a 70 μm nylon mesh (BD Biosciences). The cells were washed and re-suspended in cell culture medium (RPMI-1640 supplemented with 2-mercaptoethanol, 1% pyruvate, 1% HEPES, 1% (v/v) premixed penicillin-streptomycin solution (Invitrogen Life Technologies), 1 mM glutamine, and 10% (v/v) fetal calf serum (FCS)). For Meso Scale Discovery (MSD) cytokine profiling, cells were added in 200 μl of cell culture medium into 96-well cell culture plates at a density of 2 × 105 cells/well. Nasal washes were sampled by flushing with 350 µl phosphate buffered saline (PBS) +0.05% bovine serum albumin.
ELISA for spike-ACE2 binding
ACE2 binding ELISA was performed to assess if spike-ACE2 binding was intact after formulation of spike in CAF®01 liposomes. Maxisorp Plates (Nunc, Denmark) were coated overnight with recombinant human ACE2 (ACE-HM101, Kactus Biosystems) at 2 µg/ml in carbonate buffer pH 9.6. Plates were washed with PBS containing 0.2% Tween 20 and blocked with 2% skimmed-milk powder (SM). After blocking, spike protein alone or spike protein formulated in CAF®01 were added serially diluted in PBS with 1% SM, followed by incubation with hamster-anti-spike protein serum (generated in-house by immunizing hamsters with recombinant spike protein) as primary antibody. HRP-conjugated goat anti-hamster IgG antibody (Invitrogen, AB_2536572) was used as secondary antibody. TMB-PLUS (Kem-En-TEC, Denmark) was used as substrate, and the absorbance was recorded at 450 nm with subtraction of the absorbance value measured at 620 nm.
ELISA for antibody responses
Maxisorb Plates (Nunc) were coated overnight with 0.05 μg/well SARS-CoV-2 trimer or RBD (4 °C). For antigen-specific IgG, plates were blocked with 2% BSA and serum was added diluted in PBS with 1% BSA. Polyclonal HRP-conjugated secondary rabbit anti-mouse IgG (Thermofisher, RRID:AB_138451) or goat anti-hamster IgG antibody (Invitrogen, AB_2536572), was diluted in PBS with 1% BSA. For antigen-specific IgA, plates were blocked with 2% milk, and serum and nasal washes were added in PBS with 1% milk. Biotin-coupled goat anti-mouse IgA (Southern Biotech, AB_2794374) diluted in 1% milk was used as primary antibody, followed by streptavidin-HRP (BD, AB_2868972) diluted in PBS containing 0.2% Tween 20.
After incubation of secondary antibodies, Spike- or RBD-specific antibodies were detected using TMB substrate as described by the manufacturer (Kem-En-Tec Diagnostics), and the reaction was stopped with H2SO4. The absorbance was measured at 450 nm with subtraction of the absorbance value measured at 620 nm.
Cytokine profiling
The Mouse U-plex (IFN-γ, IL-17A, IL-5, IL-13 and IL-10) was performed according to the manufacturer’s instructions (Meso Scale Discovery) to measure CD4 T cell profiles after ex vivo re-stimulation of splenocytes with SARS-CoV-2 trimer antigen (2 µg/ml cell culture medium incubated for 72 h at 37 °C and 5% CO2). The plates were analysed on a Sector Imager 2400 system (Meso Scale Discovery) and calculation of cytokine concentrations was performed by 4-parameter logistic non-linear regression analysis of the standard curve.
Neutralization assay
ID50 values were estimated by fitting a logistic curve in Prism 5 (GraphPad Software), bounded between 0% and 100%, and interpolating the dilution at which luciferase expression was reduced by 50% relative to wells in the absence of serum.
,
This isolate is closely related to the Wu-Hu-1 sequence (with E309K and D614G substitutions in the spike protein). For cross-neutralization studies, a B.1.617.2 delta-variant (DK-AHH3 isolate) and a B.1.1.529 omicron-variant (DK-AHH4 isolate) were used. In brief, SARS-CoV-2 at a multiplicity of infection (MOI) of 0.01 to 0.15 was incubated 1h at room temperature with serially diluted heat inactivated (at 56 °C for 30 min) plasma (1/100 to 1/51200 dilution) at a 1:1 ratio. Following incubation, plasma/virus mixtures were added to naïve Vero E6 cells in quadruplicates in 96-well plates. After incubation for 48 h at 37 °C and 5% CO2, cells were fixed and stained as described,
but using SARS-CoV-2 spike chimeric monoclonal primary antibody (( 40150-D004, Sino Biological) and F(ab’)2-goat anti-human IgG Fc cross-adsorbed secondary antibody conjugated to HRP (A24476, Invitrogen) for the B.1.617.2 variant and anti-SARS-CoV-2 spike rabbit polyclonal primary antibody (no. ab272504, Abcam) and goat anti-Rabbit IgG (H+L) secondary antibody conjugated to HRP (no. 31460, ThermoFisher) for the B.1.1.529 variant). Single spots representing virus infected cells were counted by an Immunospot series 5 UV analyser, and percent neutralization calculated. Neutralization curves were constructed and the ID50 of plasma was calculated using non-linear regression (Log [inhibitor] vs normalized response [variable slope]), using GraphPad Prism. A mouse derived SARS-CoV-2 spike neutralizing antibody (Sino Biological #40592-MM57, RRID: AB_2857935) was the positive control. Four replicates of a 1/800 dilution (1.25 µg/mL) of a mouse derived SARS-CoV-2 spike neutralizing antibody (Sino Biological #40592-MM57, RRID: AB_2857935) was used as positive control for each plate, giving an average of 81% neutralization for the homologous Wu-Hu-1 variant and 96% for the B.1.617.2 variant. A 1/80 dilution of plasma from a COVID-19 vaccinated individual from the Clinical, Virological and Immunological (CVIC) study
,
was used in the same conditions for the B.1.1.529 variant, giving an average of 95% neutralization. The investigators performing the neutralization assays were blinded to the groups.
Meso Scale discovery ACE2 competition assay
The V-PLEX SARS-CoV-2 Panel 19 (ACE2) Kit was run according to the manufacturer’s instructions. Briefly, serum samples (diluted x25, x250 and x2500) and nasal washes (diluted x5) were added to pre-coated V-plex plates followed by ACE2 binding detection using a Sector Imager 2400 system (Meso Scale Discovery).
SARS-CoV-2 challenge in Syrian hamsters
Index hamsters were anesthetized with isofluorane and inoculated i.n. with 1.8 × 105 TCID50 in 50 µl of the Danish SARS-CoV-2 isolate SARS-CoV-2/Hu/DK/SSI-H5 (GenBank accession number: ON809567). This isolate is closely related to the Wu-Hu-1 sequence (with V367F and E990A substitutions in the spike protein and a G251V substitution in ORF3a). In the transmission model each infected hamster was isolated for six hours and then placed in a cage containing one hamster immunized s.c/s.c., one immunized s.c./i.n. and one unvaccinated control (six cages in total). In the onwards transmission model, index hamsters were isolated for 24 h and then housed with s.c./i.n. vaccinated or unvaccinated contacts. 24 h later the contacts were co-housed with another set of naïve hamsters for the rest of the study (five days) to assess onwards transmission. Weight and signs of disease (lethargy, ruffled fur, hunched posture) were monitored until the study was terminated at seven days post infection of the index hamsters. Humane endpoints included weight loss >20% (none of the animals reached humane endpoints). Virus titres were determined in nasal washes at days two and seven post challenge and in lungs at the time of termination.
RT-qPCR for SARS-CoV-2 detection
Cycling conditions were 55°C for 10 min, denaturation at 95 °C for 3 min, and 45 cycles of 95 °C (15 s) and 58 °C (30 s). To generate standard curves, synthetic SARS-CoV-2 RNA ctr1 (MT007544.1) (Twist Bioscience) of a known copy number was serially diluted. A stabilized RNA for 2019-nCoV E gene (EVAg) was used as positive control. RT-qPCR for detection of subgenomic RNA for the SARS-CoV-2 E-gene was performed as previously described
using 50 °C for 10 min, 95 °C for 3 min, and 45 cycles of 95 °C (10 s), 56 °C (15 s) and 72 °C (5 s). Reactions were carried out using a Lightcycler-480 Real-Time PCR System (Roche). Results were expressed as log10-transformed numbers of genome equivalent copies per ml (nasal washes) or gram (lungs).
Pathology
From animals euthanized on day seven post challenge, the right diaphragmatic lung lobe was pseudo-perfused fixed in 10% neutral buffered formalin and then placed in histo-cassettes for immersion fixation in 10% neutral buffered formalin for 24 h before being transferred to ethanol. Paraffin embedded tissues were sectioned (4 μm) and stained with haematoxylin and eosin (H&E) for histopathological examination. Sections were evaluated for infiltration of inflammatory cells (neutrophils and macrophages). In all sections the inflammatory reaction was scored as 0: absent, 1: few sporadic inflammatory cells present, and 2: numerous accumulated inflammatory cells. The inflammatory cells were identified from their characteristic morphology. The presence of type-II pneumocyte hyperplasia was determined from the presence of this cell type laying the alveolar lumen in a row morphology. Type-II pneumocyte hyperplasia was examined by H&E staining and confirmed by cytokeratin staining using a mouse anti-cytokeratin antibody (clone AE1/AE3, DAKO M3515). The investigators performing the histological examination were blinded to the groups.
Statistics
Differences between groups were analysed by one-way ANOVA comparing the mean of each column with each other column. Differences between naïve and s.c./i.n. immunized groups in the onward transmission model were analysed by unpaired student’s t test (GraphPad v8.2.1). Sample size determination is described under immunizations.
Role of funders
The funding source had no role in study design, collection, analysis or interpretation of data or in the writing of the publication.
Discussion
,
which may allow for virus replication and onward transmission. To protect against emerging viruses with pandemic potential, a vaccine approach that can effectively prevent virus transmission is desired. One key strategy to this aim is via induction of mucosal immune responses in the upper airways. Whilst parenteral vaccines provide only low immunity at this site, vaccines applied in the nasal cavity can induce a number of immune mediators that can block viral entry or virus replication, including sIgA
and local nasal-associated lymphoid tissue (NALT) CD4 and CD8 T cell responses.
In the present study we tested a combination of parenteral priming with mucosal boosting to elicit both systemic and local airway immunity and evaluated this in a Syrian hamster transmission model.
,
Previous studies have demonstrated that hamsters immunized i.n. with chimpanzee adenovirus encoding prefusion-stabilized spike protein had reduced virus titres in the upper respiratory tract compared to unvaccinated controls.
Similarly, hamsters immunized i.n. with the adenovirus-vectored vaccine ChAdOx1 nCoV-19/AZD1222 had significantly lower virus titres in the upper airways than unvaccinated controls, after being co-housed with SARS-CoV-2 infected animals.
A recombinant spike-based protein vaccine linked to outer membrane vesicles (OMV) was also found to protect hamsters against pathology after i.n. administration, but did not reduce virus titres in nasal turbinates.
Here we have demonstrated that the prefusion stabilized (HexaPro) SARS-CoV-2 spike protein,
adjuvanted with CAF®01 and given as parenteral prime – i.n. boost protects against lower respiratory tract infection and pathology upon SARS-CoV-2 challenge in Syrian hamsters. Furthermore, the vaccine significantly reduced viral load in the upper airways. It is possible that other vaccine strategies, such as parental prime followed by a mucosal boost with a heterologous vaccine,
would be even more beneficial at inducing mucosal immunity. As application of the mucosal booster vaccine in humans would require special devices, e.g. nasal spray syringe or nebulizer, the methods of delivery could also influence the effectiveness of mucosal booster strategies.
Another intriguing possibility is that virus may be kept at bay by vaccine-induced mucosal antibodies, thereby preventing transmission. Since detection of virus in the upper respiratory tract has been directly associated with transmission, our studies indicating that virus in the upper respiratory tract of mucosally vaccinated animals does not readily transmit, therefore raises the question if detection of SARS-CoV-2 in the upper respiratory tract is necessarily indicative for risk of onwards transmission. Several SARS-CoV-2 vaccine strategies, including i.n. vaccine regimens, have demonstrated complete protection against SARS-CoV-2 in the lower airways, but incomplete clearance in the upper airways.
,
,
,
To what extent these strategies protect against onwards transmission is an important question, which should be further studied for different pandemic vaccine candidates. An important limitation of the current study is that, although parenteral prime – i.n. boost protected against onward transmission, we did not compare this regimen head-to-head with parenterally administered vaccines. Future studies should therefore include parenteral only vaccines, e.g. the licensed NVX-CoV2373
or Comirnaty®3, in the onward transmission model to gauge the benefit of parenteral prime-intranasal boost over standard parenteral vaccines.
,
,
and proof-of-concept has also been demonstrated in humans where volunteers vaccinated parenterally with cationic liposome (CAF®01) adjuvanted Chlamydia trachomatis subunit vaccine and boosted i.n. with antigen alone, developed mucosal IgA antibody responses in the airways and the female reproductive tract.
Although unadjuvanted protein given i.n. can boost parenterally primed responses (e.g. after mRNA-LNP or protein in adjuvant immunization),
,
a more feasible strategy, particularly in a future pandemic mass-vaccination setting, would be to use similar vaccine formulations for both the systemic priming and mucosal boosting. We demonstrate here that spike protein formulated in CAF®01 and given as a parenteral prime – i.n. boost regimen completely protected hamsters against lung pathology following SARS-CoV-2 infection. Furthermore, the vaccine reduced virus loads in the upper airways and protected against onward transmission, thus highlighting the promise of this approach for vaccination against respiratory viruses of pandemic potential.
Contributors
D.C., C.P., G.M., B.M., H.E.J., G.J., K.I., L.K.I., I.R., F.F., J.B. and G.K.P. designed research D.J.S., L.H., A.M.M., K.T.H., R.F.J., J.S.H., C.F., S.R. performed experiments D.C., C.P, H.E.J., K.T.H., R.F.J., J. S. H., C.F., S.R. and G.K.P. analyzed data. D.C., J.B., G.K.P. wrote the paper. All authors read and approved the final version of the manuscript, and have had access to the raw data. D.C and G.K.P. can verify the accuracy of the raw data for the study.
|
Sources 2/ https://www.thelancet.com/journals/ebiom/article/PIIS2352-3964(22)00430-3/fulltext The mention sources can contact us to remove/changing this article |
What Are The Main Benefits Of Comparing Car Insurance Quotes Online
LOS ANGELES, CA / ACCESSWIRE / June 24, 2020, / Compare-autoinsurance.Org has launched a new blog post that presents the main benefits of comparing multiple car insurance quotes. For more info and free online quotes, please visit https://compare-autoinsurance.Org/the-advantages-of-comparing-prices-with-car-insurance-quotes-online/ The modern society has numerous technological advantages. One important advantage is the speed at which information is sent and received. With the help of the internet, the shopping habits of many persons have drastically changed. The car insurance industry hasn't remained untouched by these changes. On the internet, drivers can compare insurance prices and find out which sellers have the best offers. View photos The advantages of comparing online car insurance quotes are the following: Online quotes can be obtained from anywhere and at any time. Unlike physical insurance agencies, websites don't have a specific schedule and they are available at any time. Drivers that have busy working schedules, can compare quotes from anywhere and at any time, even at midnight. Multiple choices. Almost all insurance providers, no matter if they are well-known brands or just local insurers, have an online presence. Online quotes will allow policyholders the chance to discover multiple insurance companies and check their prices. Drivers are no longer required to get quotes from just a few known insurance companies. Also, local and regional insurers can provide lower insurance rates for the same services. Accurate insurance estimates. Online quotes can only be accurate if the customers provide accurate and real info about their car models and driving history. Lying about past driving incidents can make the price estimates to be lower, but when dealing with an insurance company lying to them is useless. Usually, insurance companies will do research about a potential customer before granting him coverage. Online quotes can be sorted easily. Although drivers are recommended to not choose a policy just based on its price, drivers can easily sort quotes by insurance price. Using brokerage websites will allow drivers to get quotes from multiple insurers, thus making the comparison faster and easier. For additional info, money-saving tips, and free car insurance quotes, visit https://compare-autoinsurance.Org/ Compare-autoinsurance.Org is an online provider of life, home, health, and auto insurance quotes. This website is unique because it does not simply stick to one kind of insurance provider, but brings the clients the best deals from many different online insurance carriers. In this way, clients have access to offers from multiple carriers all in one place: this website. On this site, customers have access to quotes for insurance plans from various agencies, such as local or nationwide agencies, brand names insurance companies, etc. "Online quotes can easily help drivers obtain better car insurance deals. All they have to do is to complete an online form with accurate and real info, then compare prices", said Russell Rabichev, Marketing Director of Internet Marketing Company. CONTACT: Company Name: Internet Marketing CompanyPerson for contact Name: Gurgu CPhone Number: (818) 359-3898Email: [email protected]: https://compare-autoinsurance.Org/ SOURCE: Compare-autoinsurance.Org View source version on accesswire.Com:https://www.Accesswire.Com/595055/What-Are-The-Main-Benefits-Of-Comparing-Car-Insurance-Quotes-Online View photos
to request, modification Contact us at Here or [email protected]
 |
 |
 |
