Methods
We administered ternary Spike HexaPro formulated in a cationic liposomal adjuvant as a parenteral (subcutaneous – sc) initial – intranasal booster regimen to elicit airway mucosal immune responses and evaluated it in a Syrian hamster virus transmission model.
Foundings
Parenteral priming – intranasal boosting induced high-magnitude neutralizing serum antibody and IgA responses in the upper respiratory tract. The vaccine strategy protected against virus in the lower airways and lung pathology, but virus could be detected in the upper airways. Nevertheless, parenteral prime-intranasal booster vaccine effectively protected against further transmission of SARS-CoV-2.
Interpretation
This study suggests that parenteral mucosal priming is an effective strategy to protect against SARS-CoV-2 infection and highlights that protection from viral transmission can be achieved despite incomplete clearance of the virus from the upper respiratory tract. It should be noted that protection against further transmission was not compared with standard parenteral initial boost, which should be the focus of future studies.
Financing
This work was mainly supported by the European Union’s Horizon 2020 research and innovation program under grant agreement no. 101003653.
Introduction
Licensed vaccines for Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) based on new technologies, including messenger RNA vaccines,1
Jackson LA Anderson EJ Rufail N.G et al.
SARS-CoV-2 mRNA vaccine – preliminary report.2
Erasmus JH Khandhar AP O’Connor MA et al.
An alphavirus-derived RNA replicon vaccine elicits SARS-CoV-2 neutralizing antibody and T cell responses in mice and nonhuman primates.3
Polish PP Thomas SJ Kitchen N et al.
Safety and efficacy of the BNT162b2 mRNA Covid-19 vaccine. have been shown to be highly effective against severe COVID-19. However, a major limitation of these vaccines is their lower effectiveness in protecting against transmission of the virus than against disease. Parenteral vaccines primarily elicit systemic IgG antibody responses, but respiratory viruses with pandemic potential, including coronaviruses, are primarily transmitted person-to-person via respiratory droplets and infect the upper respiratory tract, which is not effectively protected by circulating IgG. influenza A virus infection and vaccination.,5
Hou YJ Okuda K Edwards CE et al.
Reverse genetics of SARS-CoV-2 reveals a variable degree of infection in the respiratory tract. Failure to induce sterilizing immunity can lead to local viral replication in respiratory tissues and potentially future transmission, allowing the development and spread of resistant variants. Mucosal vaccination is an established strategy to induce secretory IgA (sIgA) on mucosal surfaces that, by blocking the virus at the portal of entry, can prevent initial viral replication and thus potentially provide sterilizing immunity. Compared to monomeric IgG, sIgA is multimeric, providing increased avidity and thus sIgA may be better at neutralizing SARS-CoV-2 than IgG.6
pound D Mathian A Miyara M et al.
IgA dominates the early neutralizing antibody response to SARS-CoV-2.,7
Wang Z Lorenzi JCC Muecksch F et al.
Enhanced neutralization of SARS-CoV-2 by IgA dimers. Intranasal (intra) immunization also elicits local tissue resident CD4 and CD8 T cell (TRM) responses in nasal lymphoid tissue (NALT).8
Pizza A IN by Nguyen Smith JM et al.
Permanent memory CD8(+) T cells in the upper respiratory tract prevent infection by pneumonic influenza virus.,9
Zens KD Chen J.K Farber DL.
Vaccine-generated lung tissue-resident memory T cells confer heterotypic protection against influenza infection. For SARS-CoV-1, a vaccine-induced respiratory CD4 T-cell recruits protective CD8 T cells to NALT through an IFN-γ-dependent mechanism.10
Zhao J Zhao J Mangalam AK et al.
Airway memory CD4(+) T cells mediate protective immunity against emerging respiratory coronaviruses. The Th17 cell subset has received particular focus in mucosal immune responses11
Christensen D Mortensen R Rosencrans I Dietrich J Andersen P.
Vaccine-induced Th17 cells establish as resident memory cells in the lung and promote local IgA responses. and Th17-produced IL-17A upregulates the immunoglobulin receptor (pIgR) to promote IgA secretory responses.12 Jaffar Z, Ferrini ME, Fau – Herritt LA, Herritt La Fau – Roberts K, Roberts K. Cutting edge: Th17-mediated lung mucosal responses induce polymeric Ig receptor expression by airway epithelium and increase secretory IgA levels. (1550–6606 (Electronic)). 13
Milpied PJ McHeyzer-Williams MG.
High-affinity IgA requires TH17 cell functional plasticity.14
Hirota K Turner I Villa M et al.
Plasticity of Th17 cells in Peyer’s patches is responsible for the induction of T cell-dependent IgA responses. One strategy to facilitate both systemic immunity and mucosal immune responses in the upper airways is parenteral priming—in the form of a boost.11
Christensen D Mortensen R Rosencrans I Dietrich J Andersen P.
Vaccine-induced Th17 cells establish as resident memory cells in the lung and promote local IgA responses.,15
Abraham S Joel HB Bang P et al.
Safety and immunogenicity of the chlamydial vaccine candidate CTH522 adjuvanted with CAF01 liposomes or aluminum hydroxide: a first-in-human, randomized, double-blind, placebo-controlled, phase 1 trial.16
Ozberk V Reynolds S Huo G et al.
Primary immunization with a CAF(R)01 liposome-boosted M-protein and spy-CEP bivalent vaccine induces both mucosal and peripheral protection against CovR/S Mutant Streptococcus pyogenes.17
Sui Y Lee J Zhang R et al.
Protection against SARS-CoV-2 infection by mucosal vaccine in rhesus macaques. We tested this strategy for SARS-CoV-2, using a cationic liposome spike subunit vaccine (CAF®01). Immunization with parenteral prime – in boost induced IgG and SARS-CoV-2 neutralizing antibody responses in serum and induced IgA responses in the upper respiratory tract. In a transmission model in which vaccinated contacts were co-housed with SARS-CoV-2-infected marker hamsters, the parenteral primary mucosal boost strategy reduced viral titers in the upper airways and protected against further transmission. Overall, a parenteral primary vaccine strategy may be an effective means of limiting the spread of the virus in the population.
Methods
Morality
Animal studies were conducted in accordance with European Community Directive 2010/63/EU. Experiments have been approved by the government’s Animal Experimentation Inspection and have been conducted under permits 2017-15-0201-01363 and 2020-15-0201-00554.
Antigens and adjuvants
Recombinant spike exodomain stabilized in SARS-CoV-2 prefusion (S-2P18
Wrap D Wang N Corbett KS et al.
Cryo-EM structure of the 2019-nCoV spike in the prefusion configuration. and HexaPro trimer19
Hsieh CL Goldsmith JA Schaub JM et al.
Structure-based design of injection-stabilized SARS-CoV-2 spikes.), and the RBD region (RVQ-VNF) from the Wuhan-Hu-1 strain were produced by transient expression in free-style 293-F cells as previously reported.20
Sheward DJ Madolesi M Urgard E et al.
Beta RBD amplification broadens antibody-mediated protection against SARS-CoV-2 variants in animal models.,21
Hanke L Vidakovic Pereth L Sheward DJ et al.
An alpaca nanobody neutralizes SARS-CoV-2 by blocking receptor interaction. CAF®01 (250 μg DDA/ 50 μg TDB) in 10 mM TRIS buffer with 2.2% glycerol (pH 7.0) was prepared as previously described.22
Davidsen J Rosencrans I Christensen D et al.
Characterization of cationic liposomes based on dimethyldioctadecylammonium and synthetic cord factor from M. tuberculosis (trehalose 6,6′-divehenate) – a novel adjuvant that elicits potent CMI and antibody responses.
Characterization of preparations
A compatibility study of the HexaPro tripartite spike in CAF®01 was performed at room temperature. The formulations were visually analyzed for possible flocculation and then characterized for particle size and polydispersity index (PDI) by dynamic light scattering using the photon correlation spectroscopy technique. Zeta potential was measured by laser-Doppler electrophoresis. For size measurements, samples were diluted 10-fold, while for zeta potential measurements, samples were diluted 100-fold in milli-Q water. Measurements were performed at 25 °C, using a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK) with a 633 nm laser and 173 ° optical detection. Malvern Zetasizer v.8.01 software was used for analysis.
Animals
Female C57Bl/6 (C57BL/6JOlaHsd) wild-type mice, 7–9 weeks old, were obtained from Envigo (The Netherlands). Nine-week-old male Syrian golden hamsters (Mesocricetus auratus) were obtained from Janvier. Both species were housed in the animal facilities at the Statens Serum Institut, Denmark during the studies and maintained in controlled environment rooms (20–23 °C, 52 ± 10% relative humidity; 12/12 h light/dark cycle). Mice were randomly assigned to cages (type III polycarbonate cages (820 cm2)) with up to eight mice per cage, and hamsters were housed in type IV polycarbonate cages (1820 cm2) with high lids (total height approx. 30 cm. ) with up to four animals/cage. A total of 28 mice and 51 hamsters were used for the studies. All animals were offered Aspen (Tapvei) bedding and bricks, Sizzelnest (Datesand) and polycarbonate tunnels or houses. In addition, mice were offered DesRes paper houses (LBS) while hamsters had twisted paper rolls (‘Diamond Twist’ Envigo Teclad) hung from their cage lids. Irradiated sunflower seeds, corn kernels and peanuts or pieces…
