Safety and Immunogenicity Analysis of a Newcastle Disease Virus (NDV-HXP-S) Expressing the Spike Protein of SARS-CoV-2 in Sprague Dawley Rats

Abstract

Despite global vaccination efforts, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve and spread globally. Relatively high vaccination rates have been achieved in most regions of the United States and several countries worldwide. However, access to vaccines in low- and mid-income countries (LMICs) is still suboptimal. Second generation vaccines that are universally affordable and induce systemic and mucosal immunity are needed. Here we performed an extended safety and immunogenicity analysis of a second-generation SARS-CoV-2 vaccine consisting of a live Newcastle disease virus vector expressing a pre-fusion stabilized version of the spike protein (NDV-HXP-S) administered intranasally (IN), intramuscularly (IM), or IN followed by IM in Sprague Dawley rats. Local reactogenicity, systemic toxicity, and post-mortem histopathology were assessed after the vaccine administration, with no indication of severe local or systemic reactions. Immunogenicity studies showed that the three vaccination regimens tested elicited high antibody titers against the wild type SARS-CoV-2 spike protein and the NDV vector. Moreover, high antibody titers were induced against the spike of B.1.1.7 (alpha), B.1.351 (beta) and B.1.617.2 (delta) variants of concern (VOCs). Importantly, robust levels of serum antibodies with neutralizing activity against the authentic SARS-CoV-2 USA-WA1/2020 isolate were detected after the boost. Overall, our study expands the pre-clinical safety and immunogenicity characterization of NDV-HXP-S and reinforces previous findings in other animal models about its high immunogenicity. Clinical testing of this vaccination approach is ongoing in different countries including Thailand, Vietnam, Brazil and Mexico.

Keywords: COVID-19; SARS-CoV-2; immunogenicity; newcastle disease virus; rat model; safety; vaccine.

Figure 1

Schematic representation of NDV-HXP-S production and testing in rats. (A) Vector design. The S/F chimera was created by fusing the spike protein ectodomain (S), containing HexaPro stabilizing mutations, to the transmembrane domain and cytoplasmic tail (TM/CT) of the Newcastle Disease Virus (NDV) fusion (F) protein via a short GGGGS linker. The three arginines (R) in the polybasic cleavage site (RRAR) were removed to eliminate the cleavage site. The construct was termed HXP-S, which is codon optimized for mammalian expression. The HXP-S nucleotide sequence was incorporated between the P and M genes of the La Sota NDV genome carrying the L289A mutation in the F protein. (B) Groups distribution. Rats were separated into five groups: intranasal (IN) control (group 1), intramuscular (IM) control (group 2), IN vaccinated (group 3), IM vaccinated (group 4), and IN-IM vaccinated (group 5). Rats receiving the vaccine by uneven administration routes first received the intranasal dose at day 0, followed by the intramuscular dose at day 15. Intranasal dose volume was administered to each nostril twice at 50 µL/occasion, 30 ± 5 minutes apart, for the total dose volume of 100 µL/naris, or 200 µL total. Intramuscular dose volume was administered to the thigh muscles of both hind legs, for the total dose volume of 100 µL/leg, or 200 µL total. (C) Experimental design. All the groups received a vaccine prime at day 0 followed by a boost at day 15. Serum samples were collected for immunogenicity analyses at days 0, 15, and 30 after the first vaccine dose administration. Weight measurements were performed on days 0, 1, 2, 8, 15, 16, and 17 post-vaccination. Body temperatures (°C) were measured at 0-, 6- and 24-hours post-dosage, after both the prime and boost vaccine administrations. Clinical pathology assessments were performed on days 2, 17, and 30 following the first vaccination. Rats were euthanized on day 17 and day 30 for the main and recovery groups, respectively.

Figure 2

Kinetics of body temperature in vaccinated rats. Individual body temperatures (°C) were taken at 0, 6 and 24 hours post vaccination, after both prime (left side) and boost (right side) vaccine doses, in both males (A) and females (B). Geometric mean of daily body temperature with geometric standard deviation (SD) for the IN control, IM control, IN vaccinated, IM vaccinated, and IN-IM vaccinated groups is shown. Statistically significant differences between the vaccinated groups and their respective controls are shown. The IN-IM administered group was compared with the IM control group. Statistical significance is indicated as follows: *P < 0.0413; **P < 0.0063.

Figure 3

Clinical and post-mortem evaluations in vaccinated rats. The percentage of initial weight following vaccination of male (A) or female (B) rats is shown. Weight measurements were performed on days 1, 2, 3, 8, 15, 16, and 17 after vaccination. Food consumption was measured daily and averaged over two different periods: 3-10 or 10-17 days after vaccination. The average food intake in males (C) and females (D) is shown. Weight of organs corresponding to excretory, urinary, nervous, respiratory, circulatory, lymphatic, reproductive and endocrine systems are shown (E). Geometric mean with geometric standard deviation (SD) for the IN control, IM control, IN vaccinated, IM vaccinated, and IN-IM vaccinated groups is shown in (A–E). No significant differences between the vaccinated groups and their respective controls were detected.

Figure 4

Immunogenicity of NDV-HXP-S after intranasal and intramuscular administration to rats. IgG antibody levels against wild type spike from SARS-CoV-2 (A) and NDV virus preps (B) were measured by ELISA in sera from vaccinated rats at days 0, 15 and 30 after the first vaccine dose administration. In both cases, antibody levels are expressed as area under the curve (AUC). The neutralization capacity of serum antibodies against the authentic USA-WA1/2020 SARS-CoV-2 isolate at day 30 after the first vaccine dose administration is shown in (C), and titers are expressed as inhibitory dilution 50% (ID₅₀, right panel). As a control, the inhibitory concentration 50% (IC₅₀ - μM) of the virus polymerase inhibitor remdesivir is shown (right panel). Geometric mean with geometric standard deviation (SD) for the IN control, IM control, IN vaccinated, IM vaccinated, and IN-IM vaccinated groups is shown in all panels. In (A–C), all vaccinated groups showed statistically significant differences vs their respective controls. The IN-IM administered group was compared with the IM control group. Statistically significant differences between the IN, IM and IN-IM groups are shown. Statistical significance is indicated as follows: *P ≤ 0.0270; **P ≤ 0.0032; ***P ≤ 0.002; ****P < 0.0001.

Figure 5

Reactivity of sera from vaccinated rats against the spike of variants of concern (VOCs). Antibody levels against the spike protein from B.1.1.7, B.1.351, and B.1.617.2 variants of concern (VOCs) and wild type (WT) spike were measured by ELISA in sera from vaccinated rats 30 days after the first vaccine dose administration (left panel). Antibody levels are expressed as area under the curve (AUC). As a control for antigen coating among the different spike proteins used, the reactivity towards the histidine tag present in all the recombinant proteins used is shown (right panel). Geometric mean with geometric standard deviation (SD) for the IN control, IM control, IN vaccinated, IM vaccinated, and IN-IM vaccinated groups is shown. Statistically significant differences between the reactivity against the spike of variants of concern (VOCs) and wild type spike are shown. Statistical significance is indicated as follows: ****P < 0.0001.