Mucosal multivalent NDV-based vaccine provides cross-reactive immune responses against SARS-CoV-2 variants in animal models

Abstract

Introduction: A new generation of mucosal vaccine against the ever-evolving SARS-CoV-2 is of great value to fight COVID-19. In previous studies, our groups developed a viral vector vaccine based on an avirulent Newcastle disease virus (NDV) expressing the prefusion-stabilized spike protein of SARS-CoV-2 (NDV-HXP-S).

Methods: Here we characterized the in vivo biodistribution and immunogenicity of a live mucosal NDV-HXP-S vaccine in animal models.

Results: NDV showed restricted replication in mice and hamsters. Despite limited replication, intranasal live NDV-HXP-S provided protection against SARS-CoV-2 challenge and direct-contact transmission in hamsters. Importantly, a trivalent live NDV-HXP-S vaccine (Wuhan, Beta, Delta) induced more cross-reactive antibody responses against the phylogenetically distant Omicron variant than the ancestral vaccine. Furthermore, intranasal trivalent live NDV-HXP-S boosted systemic and mucosal immunity in mice pre-immunized with mRNA vaccine.

Discussion: Overall, a mucosal multivalent live NDV-HXP-S vaccine shows great promise as a safe, next-generation vaccine conferring broad mucosal and systemic immunity against future SARS-CoV-2 variants.

Keywords: COVID-19; Coronavirus; sterilizing immunity; transmission-proof; vector vaccine; viral shedding.

Figure 1

Biodistribution of live NDV-HXP-S vaccine in Golden Syrian hamsters. (a) Experimental design and vaccination groups. Golden Syrian hamsters (n=12) were immunized with a total dose of 10⁷ EID₅₀ of ancestral NDV-HXP-S via IN or IM route. Two more groups vaccinated with NDV LaSota (WT) via IN and PBS were used as controls. A total of 4 hamsters were analyzed per time point (b–e) At day 1 and day 7 after the vaccination, (b) lung homogenates, (c) nasal wash, (d) blood serum and (e) urine were collected and presence of infectious NDV was checked by injecting 100 µL of each biological fluid into each of the specific pathogen-free (SPF) embryonated chicken eggs. Viral titers were subsequently measured by EID₅₀. (limit of detection equals to 100 EID₅₀/mL; a titer of 10 EID₅₀/mL was assigned to HA negative samples, and a titer of 50 EID₅₀/mL was assigned to HA positive samples with no EID₅₀/mL titer). (f-i) Left lobe was fixed in 4% paraformaldehyde in PBS (4% PFA, v/v) overnight and analyzed by immunohistochemistry (IHC) at day 1 and day 7 post vaccination. Presence of NDV viral protein or spike expression was measured with a rabbit polyclonal anti-NDV serum 1:2000 (shown in brown) or a human anti-SARS-CoV-2 spike 1A9 antibody 1:2000 (shown in pink). Examples of positively stained regions are indicated with arrows. Golden Syrian hamster lungs infected with SARS-CoV-2 (USA-WA1/2020), were used as spike staining positive control. The percentage of (g) NDV and (i) spike positive cells in each slide and (h) the percentage of NDV positive cells by airway in the two lung samples from the 1-day post administration of WT NDV group were measured with HALO software.

Figure 2

Live intranasal NDV-HXP-S vaccination reduces SARS-CoV-2 viral shedding in the upper respiratory tract in direct-contact transmission study. (a) Experimental design. Eight- to ten-week-old female Golden Syrian hamsters (n= 3 or 4) were either vaccinated with 10⁷ EID₅₀ of live NDV-HXP-S intranasally (IN) or intramuscularly (IM), or WT NDV IN (negative control). Two immunizations were performed with 5-month interval. Twenty-eight days after second boost, naïve hamsters were challenged with 10⁵ PFU of ancestral (USA-WA1/2020) strain (Donors) and co-housed in pairs with vaccinated hamsters from different vaccinated groups (Recipient) and transmission was monitored in a direct-contact model. (b) Spike-specific IgG serum antibody titers were measured 5 months post-prime and 28 days post-boost, respectively. The geometric mean titer (GMT) of the area under the curve (AUC) ± the standard deviation (SD) is depicted. (c) Weight loss was monitored for 5 days. (d) Nasal washes were collected 1 and 3 days post challenge. At 5 days post challenge, (e) nasal turbinate and (f) right lung lobes were harvested and homogenized in 1 mL PBS and SARS-CoV-2 titer was measured by plaque assay. (D: Donor, R: recipient) (g) Left lobes were fixed in 4% PFA (v/v) and analyzed by IHC with a mouse anti-N monoclonal (1C7C7) (1:50, brown). Viral titers were measured by plaque assay on Vero E6 cells and were plotted as GMT of PFU/mL (limit of detection equals to 50 PFU/mL; a titer of 25 PFU/mL was assigned to negative samples).

Figure 3

Live intranasal trivalent NDV-HXP-S vaccination induces superior cross-reactive humoral immune responses against phylogenetically distant SARS-CoV-2 variants in mice. (a) Experimental design and (b) vaccination groups. Eight- to ten-week-old female BALB/c mice were vaccinated with 10⁶ EID₅₀ of live NDV-HXP-S monovalent or trivalent vaccines administered intranasally (IN) in a 1- or 2-dose regimen with a ten-week interval between doses. Mice vaccinated with WT NDV at a dose of 10⁶ EID₅₀ were used as negative control. (c-f) Spike-specific IgG serum antibody titer kinetic (n=5) against ancestral (Wuhan, blue) or Omicron BA.1(B.1.1.529, orange) spike was measured every other week by ELISA for thirty -weeks. Individual kinetics for (c) monovalent 1-dose, or (e) 2-doses, (d) trivalent 1-dose or (F) 2-doses, are shown respectively. Spike-specific serum IgG of NDV WT vaccinated group against ancestral (light grey dotted line) or Omicron BA.1 (dark grey line) are also depicted in these graphs for comparison. Ancestral serum IgG antibody titers against (g) RBD at 6-weeks, (h) Spike at 30-weeks, (i) RBD at 30-weeks and (j) microneutralization assays against ancestral virus (USA-WA1/2020) at 30-weeks. Omicron BA.1 serum IgG antibody titers against (k) RBD at 6-weeks, (l) Spike at 30-weeks, (m) RBD at 30-weeks and (n) microneutralization assays against Omicron BA.1. One-tailed unpaired t-test are depicted (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). Microneutralization assays against ancestral (USA-WA1/2020) (j) and Omicron BA.1 (n) were performed in technical duplicated from pooled sera. GMT ± SD is depicted.

Figure 4

Intranasal and intramuscular vaccination of live trivalent NDV-HXP-S confer better protections against BA.1 than the monovalent vaccines in hamsters. (a) Experimental design and (b) groups. Eight- to ten-week-old female Golden Syrian hamsters (n= 4) were either vaccinated with live monovalent (Mono) or trivalent (Triv) NDV-HXP-S or WT NDV (negative control), intranasally (IN, green) or intramuscularly (IM, blue). Two immunizations were performed with 12-week interval. Twelve weeks after the second boost, hamsters were challenged with 5x104 PFU of BA.1 (B.1.1.529) strain and infection was monitored for five days. (c) Ancestral Spike-specific (d) Omicron BA.1 Spike-specific or (e) RBD-specific serum IgG antibody titers, neutralizing antibodies against (f) ancestral-like and (g) Omicron BA.1strains were measured 12 weeks post-boost vaccinations, respectively. The geometric mean titer (GMT) of the area under the curve (AUC) ± the standard deviation (SD) is depicted. (h) Throat swabs were collected 1, 3 and 5 days post-challenge and SARS-CoV-2 N genomic copies per sample were measured by RT-qPCR (limit of detection [LoD] equals to 120 copies; limits of quantification [LoQ] equals 600 copies and upper limit of quantification [ULoQ] equals to 2.4 x107 copies). (i) Nasal turbinate and (j) lower right lung lobes were harvested and homogenized in 1 mL PBS. Viral titers were measured by plaque assay on Vero E6-TMPRSS2-ACE2 cells and were plotted as GMT of PFU/mL (limit of detection [LoD] equals to 50 PFU/mL; a titer of 10 PFU/mL was assigned to negative samples). Non-parametric Kluskal-Wallis test is depicted (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

Figure 5

Intranasal live NDV-HXP-S vaccines boost humoral and mucosal immunity after two doses of 0.25 µg mRNA in mice. (a) Experimental design and vaccination groups. Eight- to ten-week-old female 129 mice were vaccinated with two doses 0.25 µg of Comirnaty mRNA vaccine (Pfizer) and 10 months later, mice were intranasally (IN) boosted with either 1 µg of inactivated or 10⁶ EID₅₀ of live monovalent or trivalent NDV-HXP-S vaccine. Naïve age-matched mice were vaccinated in a two-dose regimen with 10⁶ EID₅₀ total dose of monovalent or trivalent NDV-HXP-S vaccine as positive vaccination controls and a no booster group and complete naïve mice were used as negative controls. Blood serum, nasal washes, BAL, feces and spleen were harvested 3-weeks after the boost. (b) Heatmap of mean spike and RBD-specific IgG serum antibody titers and (c-d) fold-increase in serum antibody titers of boosted groups over no booster control against ancestral (Wuhan) or Omicron BA.1 proteins (n=4) measured by ELISAs 21 days after last boost. Inactivated groups are shown in continuous lines whereas live are shown in discontinuous lines in Fig (c) The GMT of fold increase of serum antibody titers over two doses 0.25 µg (grey squares) of Comirnaty mRNA vaccine (Pfizer) are depicted. (e) Heatmap of mean spike-specific IgA antibody titers against ancestral (Wuhan) or Omicron BA.1 spike (n=4) measured 21 days after last boost by ELISAs in nasal washes (NW), bronchioalveolar lavage (BAL) and feces samples. (f-g) Neutralizing activity of post-boost pooled sera was tested in microneutralization (MNT) assays against USA-WA1/2020 strain, and Omicron BA.1variant in technical duplicates. GMT serum dilutions inhibiting 50% of the infection (ID₅₀) is plotted (limit of detection equals to 10 and a value of 5 and was assigned to negative samples). GMT ID₅₀ ± SD are depicted. (h-i) Omicron BA.1 serum IgG1 and IgG2a antibody titers (n=3-4).