Abstract
The emergence of divergent SARS-CoV-2 variants has significantly compromised the effectiveness of first-generation COVID-19 vaccines. We investigated a prime-boost approach using bovine adenoviral (Ad) [BAd] and human Ad (HAd) vectors expressing the spike (S), membrane (M), or nucleocapsid (N) with the autophagy-inducing peptide C5 (AIP-C5) for enhanced antigen-specific immunity. The combinational vaccine formulation expressing three antigens demonstrated markedly elevated antigen-specific cell-mediated immune (CMI) responses compared to groups immunized with vectors expressing individual antigens. Furthermore, vaccinated animals exhibited 100% survival, significant reductions in lung viral titers, and no apparent signs of morbidity following challenges with Delta or Omicron variants in K18-hACE2 transgenic mice. Surprisingly, immunization with vectors expressing M and N resulted in immune suppression. However, including S with M and N overcomes this antagonistic interaction and significantly enhances immune responses and protection efficacy. Using the BAd vaccine platform in a multi-antigen approach complemented with AIP-C5 is a promising strategy for developing next-generation SARS-CoV-2 vaccines.
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Introduction
The coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in Wuhan, China in November 2019 and was declared a pandemic in March 2020, resulting in a major global public health crisis1,2. The pandemic has resulted in over 704 million cases, with more than 7 million deaths in the past four years3. In response to the coronavirus pandemic, mRNA4,5, human adenovirus (Ad) type 5 (HAd-5)6, HAd-267,8, chimpanzee Ad (ChAd)9,10, inactivated virus11, and subunit vaccines12,13 were developed and approved under the emergency use authorization (EUA). The use of billions of vaccine doses has provided significant protection for severe coronavirus disease14,15, thereby allowing various health agencies, including the Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO), to declare the end of the pandemic. Currently, SARS-CoV-2 has become an endemic pathogen and is still causing infections worldwide. While coronavirus disease severity has gone down significantly, the emergence of variants of concern (VOCs), including the highly transmissible and aggressive strain of the Delta variant, has been a vital issue since the pandemic’s beginning16. Recently, the Omicron variant has been the dominant VOC globally. Currently, the virus has further evolved into multiple subvariants, including BA.2, BA.4, BA.5, XBB, JN.1, KP.2, KP.3, BA.2.86, and others, leading to increased transmissibility and ability to evade vaccine-induced immunity17,18,19.
Most first-generation COVID-19 vaccines were formulated with the spike (S) protein derived from early isolates20. The S-based mRNA or Ad vectored vaccines showed over 90% protection from symptomatic disease against the ancestral strains5,21,22,23. However, emerging variants having multiple mutations in the S protein have led to immune evasion due to the reduced efficacy of vaccine-induced virus neutralization24,25,26. Vaccine efficacy against severe disease was reduced to about 70% against the Omicron variants, signifying the limitations of first-generation COVID-19 vaccines against the emerging variants. The protection efficacy of first-generation COVID-19 vaccines depends on developing S-specific neutralizing antibodies (NAbs), while the S-specific CD8+ T-cell response also plays a role in controlling viral infection27,28. The current COVID-19 vaccines used in the United States include two SARS-CoV-2 variants to elicit improved immunity against the circulating variants29. As the virus continues to evolve into subvariants that can evade vaccine protection, there is a vital need to design vaccines that retain protective efficacy against emerging variants, thereby eliminating the need to adjust vaccine formulation according to circulating variants.
Consequently, to develop the next generation of COVID-19 vaccine, we chose a novel bovine Ad (BAd) vector platform, expanded the antigens to include nucleocapsid (N) and membrane (M) with S, used an autophagy-inducing peptide C5 (AIP-C5), and evaluated the mucosal route of vaccine delivery. The BAd vector platform evades the preexisting Ad vector immunity that is prevalent in the majority of the human population30; activates innate immunity, leading to enhanced adaptive immune responses against the vaccine antigen/s31,32,33; and uses α(2,3)-linked and α(2,6)-linked sialic acid-binding proteins as receptors for internalization34. Including relatively conserved SARS-CoV-2 proteins such as N and M alongside S in the vaccine formulation may further broaden protective efficacy against emerging VOCs. AIP-C5 is a 21-amino-acid-long motif of the CFP10 protein of Mycobacterium tuberculosis that significantly enhances immunogen-specific cell-mediated immune (CMI) responses in mice when the immunogen is expressed with AIP-C5 in an Ad vector31. The mucosal route of immunization will elicit enhanced immune responses in the mucosal tract, leading to better protection against respiratory pathogens.
Here, we describe that inclusion of AIP-C5 with S, N, or M of Wuhan-Hu-1 (ancestral) strain in an Ad vector system resulted in significantly higher levels of CMI responses, and that vaccine formulation containing Ad vectors expressing all three immunogens elicited enhanced CMI responses in mice compared to the same antigen when expressed individually in an Ad vector with AIP-C5. Immunized animal groups with vaccine formulations containing S/C5 showed complete protection from morbidity and mortality in K18-ACE2-transgenic mice. Moreover, animals immunized with Ad expressing either N/C5 or M/C5 provided partial protection. Subsequently, we developed BAd or HAd vectors expressing the native form of S protein of a Delta variant, a stabilized form of S protein of an Omicron variant, N protein, or M protein of ancestral strain with AIP-C5. We used a heterologous vector prime-boost strategy to immunize mice with individual vectors or in combinations. The multi-antigen approach utilizing vectors expressing S/C5 (Delta or Omicron), M/C5, and N/C5 demonstrated excellent antigen-specific antibody titers and CMI responses. Immunized animal groups with vaccine formulations containing S/C5 showed a drastic reduction in lung virus titers in K18-ACE2-transgenic mice. Furthermore, animals immunized with Ad expressing either N/C5 or M/C5 yielded decreased lung virus titers.
Results
Development and characterization of HAd vectors expressing SARS-CoV-2 S, M, or N protein with or without AIP-C5
The S, M, or N genes of SARS-CoV-2 (Ancestral) with or without AIP-C5 under the control of the immediate early promotor of human cytomegalovirus (HCMV) and bovine growth hormone (BGH) polyadenylation (PA) signal were cloned into the early region 1 (E1) of a HAd-ΔE1E3 to generate replication-defective vectors by a Cre recombinase-mediated recombination procedure (Fig. 1A). Cesium chloride gradient purified preparations of HAd-S (carrying S), HAd-S/C5 (carrying S with AIP-C5), HAd-M (carrying M), HAd-M/C5 (carrying M with AIP-C5), HAd-N (carrying N), and HAd-N/C5 (carrying N with AIP-C5) were used for extraction of vector DNA followed by restriction profile analyses and sequencing of the gene cassettes. The restriction profile and gene cassette sequence analyses confirmed the presence of the appropriate gene cassette in each vector. To confirm the expression of gene cassette carried by each vector, HAd-S, HAd-S/C5, HAd-M, HAd-M/C5, HAd-N, or HAd-N/C5 infected HEK293 cell extracts were utilized for immunoblotting using antigen-specific antibodies. Mock- or HAd5-ΔE1E3 (empty vector)-cell extracts served as negative controls. Immunoblotting with S-specific antibodies revealed about 240, 140, and 80 kDa bands corresponding with S or S/C5 and their S1 and S2 subunits, respectively in HAd-S- or HAd-S/C5-infected cell extracts (Fig. 1B, top panel). Immunoblotting with M-specific antibody revealed approximately 29 kDa bands representing M or M/C5 in HAd-M- or HAd-M/C5-infected cell extracts (Fig. 1B, middle panel). Similarly, immunoblotting with N-specific antibodies showed roughly 50 kDa bands of N or N/C5 in HAd-N- or HAd-N/C5-infected cell extracts (Fig. 1B, lower panel). There were noticeable differences in the molecular weights of bands corresponding to M and M/C5 or N and N/C5 due to the inclusion of AIP-C5. No significant bands were detected in mock- or HAd-ΔE1E3-infected cell extracts (Fig. 1B).
A Diagrammatic representation of human adenoviral (HAd) vectors. The gene cassettes of spike (S), membrane (M), or nucleocapsid (N) of SARS-CoV-2 Wuhan Hu 1 strain with or without autophagy-inducing peptide C5 (AIP-C5) were under the control of the cytomegalovirus (CMV) immediate early promotor and the bovine growth hormone (BGH) polyadenylation signal (PA). The diagram is not drawn to scale. ΔE1 deletion of E1 region, ΔE3 deletion of E3 region, HAd-ΔE1E3 HAd-5 having deletions of E1 and E3 regions, LITR left inverted terminal repeat, RITR right inverted terminal repeat. B Immunoblots confirming expression of S, M, or N with or without AIP-C5. 293Cre cells were mock-infected or infected with HAd-ΔE1E3 (empty vector), HAd-S, HAd-S/C5, HAd-M, HAd-M/C5, HAd-N, or HAd-N/C5. The cell pellets were processed for immunoblotting using S-, M-, or N-specific antibodies. To confirm equal loading, immunoblotting for β-actin was also performed. C Outline of the immunogenicity study in BALB/c mice.
Development of strong antigen-specific humoral immune responses in mice immunized with HAd vectors expressing SARS-CoV-2 S, M, or N protein with or without AIP-C5
In Study #1 BALB/c mice were intranasally immunized and boosted with HAd vector expressing SARS-CoV-2 S, M, or N proteins either alone or in combination, with or without AIP-C5 (Fig. 1C). Serum samples and lung washes were collected at four weeks post-boost and used for determining S-, M-, and N-specific IgG, IgG1, IgG2a, and IgA antibody titers by ELISA. Antibody titers were analyzed using area under the curve (AUC) as the quantitative metric (Fig. 2).
Seven-week-old BALB/c mice were intranasally (i.n.) mock-inoculated or inoculated with 5 × 107 plaque-forming units (PFU) of HAd-ΔE1E3, HAd-S, HAd-S/C5, HAd-M, HAd-M/C5, HAd-N, HAd-N/C5, HAd-S + HAd-M, HAd-S/C5 + HAd-M/C5, HAd-S + HAd-N, HAd-S/C5 + HAd-N/C5, HAd-M + HAd-N, HAd-M/C5 + HAd-N/C5, HAd-S + HAd-M + HAd-N, or HAd-S/C5 + HAd-M/C5 + HAd-N/C5 twice at four weeks interval. Blood samples were collected 4 weeks post-booster inoculation. A S-specific IgG, IgG1, IgG2a, and IgA levels were assessed in serum (top panel) and lung washes (bottom panel). B N-specific IgG, IgG1, IgG2a, and IgA responses in serum (top panel) and lung washes (bottom panel). C M-specific IgG, IgG1, IgG2a, and IgA responses in serum (top panel) and lung washes (bottom panel). ELISA data were shown as the area under curve (AUC) with a cut-off value calculated by the average of blank wells. Each symbol represents an individual animal. Median and interquartile range (IQR) are presented. Statistical significance was assessed using non-parametric one-way ANOVA with Kruskal–Wallis test.
For S-specific response, animals immunized with HAd-S or HAd-S/C5 alone or in various combinations demonstrated high levels of S-specific IgG, IgG1, or IgG2a, and low levels of IgA antibody titers in serum samples (Fig. 2A, top panel), whereas high levels of S-specific IgG, IgG1, IgG2a, or IgA (Fig. 2A, bottom panel) antibody titers were observed in lung washes. The inclusion of AIP-C5 did not significantly impact S-specific antibody levels in either compartment. There was a decreasing trend in S-specific antibody titers in both serum and lung washes of groups immunized with HAd-S or HAd-S/C5 when combined with vectors expressing M or M/C5 and N or N/C5 though these changes were not statistically significant. These observations suggest a potential dilutional effect when co-delivering M and N antigens alongside S, which warrants further investigation. Serum samples or lung washes from the mock- or HAd-ΔE1E3-infected group did not yield S-specific antibody titers above the background (Fig. 2A).
Similarly, for N-specific responses (Fig. 2B) groups immunized with HAd vector/s expressing N or N-C5 alone or in combination generated high levels of N-specific IgG, IgG1, or IgG2a, and low levels of IgA antibody titers in serum samples (Fig. 2B, top panel). In lung washes, high levels of N-specific IgG, IgG1, or IgG2a, or IgA (Fig. 2B, bottom panel) were generated. AIP-C5 inclusion did not significantly change N-specific humoral responses though there was an increase that was not statistically significant. However, the group receiving the M + N combination showed a reduction in N-specific antibody titers, particularly in serum, suggesting possible immune interference when these internal antigens are co-delivered. The mock- or HAd-ΔE1E3-immunized groups did not produce N-specific antibody titers above the background in serum samples or lung washes (Fig. 2B).
Furthermore, M-specific antibody responses (Fig. 2C) were observed across mice immunized with HAd-M or HAd-M/C5 formulations. Modest levels of M-specific IgG, IgG1, or IgG2a, and low levels of IgA antibody titers in serum samples (Fig. 2C, top panel) were observed. In lung washes, M-specific IgG, IgG1, IgG2a, and IgA levels were detected (Fig. 2C, bottom panel). Similar to S- and N-specific responses, the inclusion of AIP-C5 had no significant effect on M-specific antibody production. M-specific titers in the combination groups remained consistent with single-vector formulations, without a clear trend of enhancement or suppression. M-specific antibody titers in serum samples or lung washes from the mock- or HAd-ΔE1E3-infected group were similar to the background (Fig. 2). These findings suggest that while all three antigens elicited antigen-specific humoral responses, AIP-C5 did not significantly enhance antibody titers, and co-administration of M and N did not generally improve nor severely compromise humoral responses. The elicitation of an adequate antigen-specific humoral response in the serum and respiratory tract is essential for mounting protective immunity against SARS-CoV-2.
Development of enhanced antigen-specific CMI responses in mice immunized with HAd vectors expressing SARS-CoV-2 S, M, or N protein with or without AIP-C5
In Study #1, splenocytes and lung mononuclear (MN) cells collected at 4 weeks post-boost were used to determine S-, M-, or N-specific CMI responses by ELISpot, enumerating interferon-gamma (IFN-γ) producing cells. Immunogen-specific responses were evident in all vaccination groups.
For S-specific responses (Fig. 3A, D), inclusion of AIP-C5 (HAd-S/C5) appeared to increase IFN-γ-producing cell numbers compared to HAd-S alone, particularly in splenocytes. However, this enhancement was not statistically significant in lung MN. Similarly, N- and M-specific CMI responses (Fig. 3B, C, E, F) were generally higher in groups receiving vectors co-expressing AIP-C5 (HAd-N/C5, HAd-M/C5) compared to their non-C5 counterparts, with more consistent increases observed in splenocytes.
Seven-week-old BALB/c mice (3 males + 3 females per group) were intranasally (i.n.) mock-inoculated, or inoculated with 5 × 107 plaque-forming units (PFU) of HAd-ΔE1E3, HAd-S, HAd-S/C5, HAd-M, HAd-M/C5, HAd-N, HAd-N/C5, HAd-S + HAd-M, HAd-S/C5 + HAd-M/C5, HAd-S + HAd-N, HAd-S/C5 + HAd-N/C5, HAd-M + HAd-N, HAd-M/C5 + HAd-N/C5, HAd-S + HAd-M + HAd-N, or HAd-S/C5 + HAd-M/C5 + HAd-N/C5 twice at four weeks interval. Four weeks post-booster inoculation, splenocytes (A–C) and lung mononuclear cells (D–F) were collected and analyzed for S- (A, D), N- (B, E), or M-specific (C, F) T cell-mediated immune responses by ELISpot assay. The numbers of IFN-γ-secreting cells per 106 splenocytes (A–C) or 105 lung mononuclear cells (D–F) are presented. Statistical analysis was performed using U-test Mann-Whitney for non-parametric data. Significance levels were set at *p < 0.05; and **p < 0.01. VN titers against SARS-CoV-2 variants in sera of mice immunized with different HAd vector formulations. Four weeks post-booster inoculation, the serum samples were collected and used to study the development of VN titers against SARS-CoV-2 variants: hCoV-19/USA-WA1/2020 (Wuhan) (G), hCoV-19/USA/PHC658/2021 (B.1.617.2, Delta) (H), or hCoV-19/USA/GR484A/2021 (B.1.1.529, Omicron) (I) by VN assay.
In the combinatorial groups, co-administration of HAd-S/C5 + HAd-N/C5 resulted in significantly higher N-specific CMI responses compared to single antigen groups, though this effect was not mirrored in S-specific response. Besides, this effect again was not observed uniformly across all tissues but was more localized in lung MN. Interestingly, co-immunization with HAd-N + HAd-M or HAd-N/C5 + HAd-M/C5 appeared to reduce N-specific IFN-γ-secreting cell numbers in both solenocytes and lung MN, suggesting potential immunological interference or suppression when these antigens are combined.
Interestingly, the addition of HAd-M or HAd-M/C5 to HAd-S or HAd-S/C5 seemed to significantly reduce S-specific CMI response in lung MN. This trend was also observed in the spleen but without statistical significance. These findings suggest that the M antigen may modulate or dampen cellular immunity in certain contexts.
The triple vector formulations with HAd-S/C5 + HAd-N/C5 + HAd-M/C5 resulted in better CMI responses when compared with HAd-N/C5 or HAd-S/C5 alone, but this effect was not observed in HAd-M/C5 (Fig. 3A–F). The strongest enhancement in CMI responses was observed in mice receiving the triple vector formulation containing AIP-C5, particularly for S-specific and N-specific responses in both spleen and lungs. However, even in this group, M-specific responses were variable and not consistently elevated relative to the corresponding single-antigen groups (Fig. 3C, F). As expected, PBS- or HAd-∆E1E3-inoculated control groups did not elicit antigen-specific CMI responses above the background (Fig. 3).
Monitoring virus-neutralizing (VN) antibodies against SARS-CoV-2 variants in mice immunized with HAd vectors expressing SARS-CoV-2 S, M, or N protein with AIP-C5
For S-based vaccines, VN antibody levels against SARS-CoV-2 are considered a predictive parameter for protection35,36. Serum samples collected from immunized animals under Study #1 were utilized for evaluating the levels of VN antibodies against hCoV-19/USA-WA1/2020 (Wuhan or ancestral), hCoV-19/USA/PHC658/2021 (Lineage B.1.617.2; Delta), and hCoV-19/USA/GR484A/2021 (Lineage B.1.1.529; Omicron) by VN assay. Only groups that received HAd vectors expressing AIP-C5 coupled constructs were included in this analysis, as these groups elicited the strongest cell-mediated responses in the immunogenicity study. The HAd-S/C5 vaccinated group produced the highest VN titers (~10⁴) against the Wuhan strain (Fig. 3G). In comparison, the HAd-S/C5 + HAd-M/C5 and HAd-S/C5 + HAd-N/C5 groups showed reduced titers (~5 × 10³), while the triple vector group (HAd-S/C5 + HAd-M/C5 + HAd-N/C5) exhibited a further ~10-fold lower VN titer ( ~ 10³), representing more than a full log reduction relative to the HAd-S/C5 group alone. This reduction in neutralizing activity is expected, given that the triple formulation includes both N and M antigens, which are internal viral proteins and do not contribute to NAb production. Their inclusion may divert a portion of the immune response away from the S protein, effectively diluting the magnitude of S-directed humoral responses. VN titers against the Delta (B.1.617.2) and Omicron (B.1.1.529) variants were further reduced by ~0.5 and 1 log, respectively compared to Wuhan (Fig. 3G, H). Notably, the levels of VN titers in the sera of mice inoculated with PBS, HAd-∆E1E3, HAd-M/C5, HAd-N/C5, and HAd-M/C5 + HAd-N/C5 were below the detection limit against all three SARS-CoV-2 variants consistent with the absence of S antigen in these formulations (Fig. 3).
Protection efficacy in K18-hACE2 transgenic mice immunized with HAd vectors expressing S/C5, M/C5 or N/C5 following challenge with SARS-CoV-2 variants
Based on the findings of immunogenicity Study #1 which demonstrated that inclusion of AIP-C5 enhanced CMI responses without markedly impacting humoral immune responses, we proceeded to evaluate the protective efficacy of HAd vectors containing immunogen/s with AIP-C5. The K18-hACE2 transgenic mice were mock-inoculated with PBS or immunized with HAd-ΔE1E3 (empty vector), HAd-S/C5, HAd-M/C5, HAd-N/C5, or various combinations as described in the materials and methods section. Four weeks post-booster-inoculation, the vaccinated groups were challenged with hCoV-19/USA/PHC658/2021 (Lineage B.1.617.2; Delta) to evaluate the vaccines’ protective efficacies by monitoring morbidity and mortality for 14 days post-challenge (Fig. 4A). The Delta variant was chosen as the challenge virus because it was the predominant variant in the U.S. at the time of the study and provided a heterologous challenge to the Wuhan-based vaccine antigen. The groups immunized with the vector formulation containing S (HAd-S/C5, HAd-S/C5 + HAd-M/C5, HAd-S/C5 + HAd-N/C5 and HAd-S/C5 + HAd-M/C5 + HAd-N/C5) conferred complete protection from morbidity and mortality following challenge (Fig. 4B, C). In contrast, immunization with HAd-M/C5 or HAd-N/C5 provided 40% and 20% survival rates, respectively (Fig. 4C). The mock- or HAd-∆E1E3-inoculated group showed morbidity leading to the death of all animals in the group by day 8 post-challenge (Fig. 4B, C). Interestingly, the HAd-M/C5 + HAd-N/C5 group did not yield any protection from morbidity and mortality (Fig. 4B, C). Assessment of viral burden in lung tissues revealed reduced viral genome copy numbers in all S/C5-containing groups compared to controls, with the highest reductions found in the triple vector inoculated group (Fig. 4D).
A Outline of the protection study in K18-hACE2 mice. Five-week-old K18-hACE2 transgenic mice (3 males + 2 females per group) were intranasally (i.n.) mock-inoculated or inoculated with 5 × 107 plaque-forming units (PFU) of HAd-ΔE1E3, HAd-S/C5, HAd-M/C5, HAd-N/C5, HAd-S/C5 + HAd-M/C5, HAd-S/C5 + HAd-N/C5, HAd-M/C5 + HAd-N/C5, or HAd-S/C5 + HAd-M/C5 + HAd-N/C5 twice at four weeks interval (Created by BioRender.com). Four weeks post-booster, animals were challenged with 1 × 104 TCID50 of hCoV-19/USA/PHC658/2021 (Lineage B.1.617.2, Delta) and monitored for B morbidity (% body weight change) or C mortality 14 days post-challenge. Statistical analysis was performed using U-test Mann-Whitney for non-parametric data. Significance levels were set at *p < 0.05 and **p < 0.01. D Lungs were collected 5 days post-challenge and RT-PCR was conducted to quantify RNA copies. The data are shown as log10 RNA copy numbers and the detection limit was 500 copy numbers (2.5 log10). Each symbol represents an individual animal, and the error is shown as SD. Data were analyzed by one-way ANOVA with Tukey’s post-hoc test. *, significant at p ≤ 0.05; **, significant at p ≤ 0.01; ***, significant at p ≤ 0.001; and ****, significant at p ≤ 0.0001.
Lung tissue samples collected from the immunized mice challenged with SARS-CoV-2 were analyzed for SARS-CoV-2 N antigen expression by immunohistochemistry. There was a widespread distribution of N protein expression in the bronchioles and alveoli of the HAd-∆E1E3-inoculated group (Fig. S1). The lung tissue from animals vaccinated with HAd-N/C5 or HAd-M/C5 had comparatively lower distribution of detectable antigen than the HAd-∆E1E3-inoculated group. The groups immunized with the vector expressing S/C5 alone or in combination showed undetectable to minimum antigen detection, whereas animals vaccinated with HAd-N/C5 exhibited lesser antigen expression compared to the group inoculated with HAd-M/C5 (Fig. S1). The HAd-M/C5 + HAd-N/C5 immunized group revealed a somewhat higher number antigen-positive cells compared to the HAd-N/C5 group (Fig. S1), consistent with the levels of M- or N-specific immune responses generated.
Taken together, these results suggest that while M/C5 and N/C5 individually elicited partial protection, the inclusion of S/C5 - particularly in the triple vector formulation - was associated with greater reduction in lung viral burden and improved survival outcomes. Interestingly, the combination of HAd-M/C5 + HAd-N/C5 failed to confer any protection, resulting in 100% mortality, comparable to the control groups (Fig. 4B, C). This unexpected outcome suggests that co-administration of M and N antigens may interfere with or suppress protective immune mechanisms. These findings support a potential role for multivalent vaccine strategies, though further studies are needed to optimize antigen combinations and better understand mechanisms governing these interactions.
Development and characterization of BAd and HAd vectors expressing SARS-CoV2 S, M, or N protein with AIP-C5
For Study #2 (Fig. 5A), we utilized a novel replication-defective BAd vaccine platform for expressing S, M, or N gene cassettes with AIP-C5, and updated the S gene cassette from the Delta or Omicron variant. We used the native form of the Delta S gene cassette and the stabilized form of the Omicron S gene cassette.
A Diagrammatic representation of bovine adenoviral (BAd) or human adenoviral (HAd) vectors. The gene cassettes of the native spike (S) protein of the Delta variant or the stabilized form of S protein of the Omicron variant, membrane (M) of Wuhan Hu 1, or nucleocapsid (N) of Wuhan Hu 1 with autophagy-inducing peptide C5 (AIP-C5) were under the control of the cytomegalovirus (CMV) immediate early promotor and the bovine growth hormone (BGH) polyadenylation signal (PA) (Created by BioRender.com). The diagram is not drawn to scale. ΔE1 deletion of E1 region, ΔE3 deletion of E3 region, Ad-ΔE1E3 adenovirus having deletions of E1 and E3 regions, LITR left inverted terminal repeat, RITR right inverted terminal repeat. B Immunoblots confirming expression of S, M, or N with AIP-C5. 293Cre cells were mock-infected or infected with HAd-ΔE1E3, HAd-S/C5(Delta), HAd-S/C5(Omicron/St), HAd-M/C5, or HAd-N/C5. BHHF5 cells infected with BAd-S/C5(Delta), BAd-S/C5(Omicron/St), BAd-M/C5, or BAd-N/C5. At 48 h post-infection, the cell pellets were collected and processed for immunoblotting using S-, M-, or N-specific antibodies. To confirm equal loading, immunoblotting for β-actin was also performed. C An outline of the immunogenicity & protection studies in BALB/c & k18-hACE2 mice, respectively, is depicted (Created by BioRender.com).
The native form of the S gene of the Delta variant, the stabilized form of the S gene of the Omicron variant, the M gene of the Ancestral variant, or the N gene of the Ancestral variant with AIP-C5 under the control of the CMV promoter and the BGH PA signal were cloned into the E1 of BAd-ΔE1E3 vector to generate replication-defective vectors using I-SceI-mediated procedure (Fig. 5A). Similarly, the native form of S gene of the Delta variant or stabilized-form of S gene of the Omicron variant with AIP-C5 under the control of the CMV promoter and the BGH PA signal were cloned into the E1 of a HAd-ΔE1E3 vector to generate replication-defective vectors using a Cre recombinase-mediated protocol (Fig. 5A). The generation of HAd-N/C5 and HAd-M/C5 is described in Study #1 (Fig. 1).
Cesium chloride density-gradient purified preparations of BAd-S(Delta)/C5 (carrying the native-form of S of the Delta variant with AIP-C5), BAd-S(Omicron 22E/St)/C5 (carrying the stabilized-form of S of the Omicron variant with AIP-C5), BAd-M/C5 (carrying the M of the Wuhan Hu 1 variant with AIP-C5), BAd-N/C5 (carrying the N of the Wuhan Hu 1variant with AIP-C5), HAd-S(Delta)/C5 (HAd vector carrying the native-form of S of the Delta variant with AIP-C5), and HAd-S(Omicron 22E/St)/C5 (HAd vector carrying the stabilized-form of S of Omicron variant with AIP-C5) were used to extract vector DNA for restriction profile analyses, and sequencing of the gene cassettes. The restriction profile and gene cassette sequence analyses confirmed the presence of the appropriate gene cassette in each vector. To confirm the expression of gene cassette carried by each vector, BAd-S(Delta)/C5, BAd-S(Omicron 22E/St)/C5, BAd-M/C5, BAd-N/C5, or BAd-ΔE1E3 (empty vector) were used to infect BHH/F5 cells, and cell extracts were utilized for immunoblotting using antigen-specific antibodies. Similarly, HAd-S(Delta)/C5, HAd-S(Omicron 22E/St)/C5, HAd-M/C5, HAd-N/C5, or HAd-ΔE1E3 (empty vector) were used to infect HEK293 cells, and cell extracts were utilized for immunoblotting using antigen-specific antibodies. Immunoblotting with an S-specific antibody revealed about 180, 140, and 80 kDa bands corresponding with S/C5(Delta) in HEK293-infected cell extracts and 180 and 140 kDa bands in BHHF5-infected cells, whereas S/C5(22E/St) showed a single band at approximately 180 kDa in both cell lines (Fig. 5B, top panel). Meanwhile, immunoblotting with an M-specific antibody revealed approximately 29 kDa bands representing M/C5 in both cell line extracts (Fig. 5B, middle panel). Similarly, immunoblotting with an N-specific antibody showed roughly 50 kDa bands of N/C5 (Fig. 5B, lower panel). There were noticeable differences in the band molecular weights corresponding to M and M/C5 or N and N/C5 due to the cleavage of AIP-C5. No significant bands were detected in BAd-ΔE1E3- or HAd-ΔE1E3-infected cell extracts (Fig. 5B).
Development of strong antigen-specific humoral immune responses in mice immunized following a prime-boost approach with BAd and HAd vectors expressing SARS-CoV-2 S, M, or N protein with AIP-C5
Immunogenicity Study #2 in mice immunized following a prime-boost approach with BAd and HAd vectors expressing SARS-CoV-2 S, M, or N protein with AIP-C5 was conducted (Fig. 5). BALB/c mice were primed i.n. with BAd vectors followed by boost with HAd vectors individually or in various combinations as described in Material & Methods and shown in Fig. 5C. Serum samples (Fig. 6A) and lung washes (Fig. 6B) collected at four weeks post-boost were used for determining S-, M-, and N-specific IgG, IgG1, IgG2a, and IgA antibody titers by ELISA. Antibody titers were analyzed using AUC as the quantitative metric. Animals immunized with Ad vector/s expressing S/C5 alone or in various combinations demonstrated high levels of S-specific IgG, IgG1, or IgG2a, and low levels of IgA antibody titers in serum samples (Fig. 6A). In lung washes, high levels of S-specific IgG, IgG1, IgG2a, or IgA (Fig. 6B) antibody titers were observed in mice immunized with Ad vector/s expressing S/C5 alone or in various combinations. Serum samples or lung washes from the mock- or Ad-ΔE1E3-infected group did not yield S-specific antibody titers above the background (Fig. 6).
Seven-week-old BALB/c mice (3 males + 3 females per group) were intranasally (i.n.) mock-inoculated or inoculated with BAd vector individually or various combinations as shown in Fig. 5C with a dose of 1 × 107 plaque-forming units (PFU) (each vector). At four weeks post-prime, each group received a booster i.n. inoculation with HAd vector individually or various combinations as shown in Fig. 5C with a dose of 1 × 107 PFU (each vector). Blood samples were collected 4 weeks post-booster inoculation. A S-specific IgG, IgG1, IgG2a, and IgA levels were assessed in serum (top panel) and lung washes (bottom panel). B N-specific IgG, IgG1, IgG2a, and IgA responses in serum (top panel) and lung washes (bottom panel). C M-specific IgG, IgG1, IgG2a, and IgA responses in serum (top panel) and lung washes (bottom panel). ELISA data were shown as the area under curve (AUC) with a cut-off value calculated by the average of blank wells. Each symbol represents an individual animal. Median and interquartile range (IQR) are presented. Statistical significance was assessed using non-parametric one-way ANOVA with Kruskal–Wallis test.
Similarly, groups immunized with Ad vector/s expressing N/C5 alone or with various combinations produced high levels of N-specific IgG, IgG1, or IgG2a, and low levels of IgA antibody titers in serum samples (Fig. 6A), whereas high levels of N-specific IgG, IgG1, IgG2a, or IgA (Fig. 6B) antibody titers were noticed in lung washes of animals vaccinated with Ad vector/s expressing N-C5 alone or various combinations. There was a marked overall decrease in N-specific antibody titers in the group immunized with Ad-N/C5 in combination with M/C5, reaching statistical significance in serum IgG (p < 0.5 compared to Ad-S(Delta)/C5 + Ad-M/C5 + Ad-N/C5, or Ad-S(Delta)/C5 + Ad-S(Omicron)/C5 + Ad-M/C5 + Ad-N/C5, respectively) and lung wash IgA (p < 0.5 compared to Ad-S(Omicron)/C5 + Ad-N/C5, Ad-S(Omicron)/C5 + Ad-M/C5 + Ad-N/C5, or Ad-S(Delta)/C5 + Ad-S(Omicron)/C5 + Ad-M/C5 + Ad-N/C5, respectively). The mock- or Ad-ΔE1E3-infected group did not produce N-specific antibody titers above the background in serum samples or lung washes (Fig. 6).
Furthermore, animal groups vaccinated by the prime-boost approach with BAd/HAd vector/s expressing M/C5 alone or in various combinations elicited moderate levels of M-specific IgG, IgG1, or IgG2a, and low levels of IgA antibody titers in serum samples (Fig. 6A) and moderate levels of M-specific IgG, IgG1, IgG2a, or IgA antibody titers in lung washes (Fig. 6B). M-specific antibody titers in serum samples or lung washes from the mock- or HAd-ΔE1E3-infected group were close to the background (Fig. 6). These findings highlight that the prime-boost approach using BAd and HAd vectors expressing S, N, or M proteins, with the inclusion of AIP-C5, effectively induces strong antigen-specific humoral responses.
Development of enhanced antigen-specific CMI responses in mice immunized following a prime-boost approach with BAd and HAd vectors expressing SARS-CoV-2 S, M, or N protein with AIP-C5
Following priming with BAd and boosting with HAd vectors as shown in (Fig. 5A) splenocytes, MLN cells, and lung MN cells collected at four weeks post-boost were used to determine S-, M-, or N-specific CMI responses by ELISpot by enumerating IFN-γ-producing cells. In the Ad-S(Delta)/C5 or Ad-S(Omicron 22E/St)/C5 inoculated group, there were significantly high numbers of S-specific IFN-γ-producing cells in the spleen (Fig. 7A), MLN (Fig. 7D), and lungs (Fig. 7G) compared to control groups. Similarly, in the Ad-N/C5 inoculated group, there were significant increases in the numbers of N-specific IFN-γ-producing cells in the spleen (Fig. 7B), MLN (Fig. 7E), and lungs (Fig. 7H). Furthermore, there were significant increases in the numbers of M-specific IFN-γ-producing cells in the spleen (Fig. 7C), MLN (Fig. 7F), and lungs (Fig. 7I) in the Ad-M/C5 inoculated group. The quadruple vector formulation group produced superior CMI responses compared to single formulations against each antigen (p < 0.5) in all tested tissues. In MLN, the triple formulation groups along with the Ad-S(Delta)/C5 and Ad-S(Omicron 22E/St)/C5 groups showed significant enhancement for S-specific CMI response compared to single S formulation groups (p < 0.05). In agreement with Study #1, immunization with Ad-M/C5+Ad-N/C5 led to greater reductions in the number of M- and N-specific IFN-γ-secreting cells in the spleen (p > 0.9), MLN (p > 0.9 and p < 0.05 for M and N, respectively) and lungs (p > 0.9 and p < 0.05 for M and N, respectively) than groups that received either antigen alone (Fig. 7B, C, E, F, H, I). The PBS- or Ad-∆E1E3-inoculated control groups did not elicit antigen-specific CMI responses above the background (Fig. 7A–I). Overall, the formulation of the three antigens had an additive effect on CMI, with the most significant enhancement in the numbers of S-, M-, and N-IFN-γ-expressing cells in the spleen, MLN and lungs was observed in the quadruple vector group vaccinated with Ad-S(Delta)/C5 + Ad-S(Omicron 22E/St)/C5 + Ad-M/C5 + Ad-N/C5 (Fig. 7A–I).
Seven-week-old BALB/c mice (3 males + 3 females per group) were intranasally (i.n.) mock-inoculated or inoculated with BAd vector individually or various combinations as shown in Fig. 5C with a dose of 1 × 107 plaque-forming units (PFU) (each vector). At four weeks post-prime, each group received a booster i.n. inoculation with HAd vector individually or various combinations as shown in Fig. 5C with a dose of 1 × 107 PFU (each vector). Four weeks post-booster inoculation, splenocytes (A–C), mediastinal lymph nodes (MLN) (D–F), and lung mononuclear (MN) cells (G–I) were collected and analyzed for S- (A, D, G), N- (B, E, H), or M-specific (C, F, I) T-cell-mediated immune responses by ELISpot assay using antigen-specific peptides. Numbers of IFN-γ-secreting cells per 106 splenocytes (A–C), 106 MLN cells (D–F), or 105 lung MN cells (G–I) are presented. *, significant at p < 0.05; and **, significant at p < 0.01. VN titers against SARS-CoV-2 variants in sera of mice immunized by a prime-boost approach using BAd and HAd vector formulations. Four weeks post-booster inoculation, the serum samples were collected and used to study the development of VN titers against SARS-CoV-2 variants: J hCoV-19/USA/PHC658/2021 (B.1.617.2, Delta) and K hCoV-19/USA/GR484A/2021 (B.1.1.529, Omicron) by VN assay.
We also investigated the numbers of S-, M-, or N-specific IL-2-producing cells by ELISpot since IL-2 helps in T-cell activation, expansion, and differentiation of CD8+ T-cells into effector and memory T-cells. In the Ad-S(Delta)/C5 or Ad-S (Omicron 22E/St)/C5 inoculated group, there was a significant boost in numbers of IL-2-producing cells in the spleen, MLN, and lungs (Fig. S2). These results demonstrate that the prime-boost approach with BAd and HAd vectors, particularly the triple or quadruple vector combination, significantly enhances antigen-specific IFN-γ and IL-2 responses in the spleen, MLN, and lungs, suggesting robust CMI responses are crucial for effective protection against SARS-CoV-2.
Monitoring VN antibodies against SARS-CoV-2 variants in mice immunized following a prime-boost approach with BAd and HAd vectors expressing SARS-CoV-2 S, M, or N protein with AIP-C5
Serum samples collected from immunized animals in Study #2 were analyzed for VN antibody titers against hCoV-19/USA/PHC658/2021 (Lineage B.1.617.2; Delta) and hCoV-19/USA/GR484A/2021 (Lineage B.1.1.529; Omicron) using VN assays. The levels of VN titers in the sera of mouse groups inoculated with PBS, ∆E1E3, M/C5, N/C5, or M/C5 + N/C5 were either below the detection limit or negligible against both SARS-CoV-2 variants. Meanwhile, all animals vaccinated with formulations expressing either S(Delta)/C5 or S(Omicron 22E/St)/C5 elicited elevated VN titers against both tested strains with titers increasing against the corresponding strain (p < 0.5 between S(Delta)/C5 and S(Omicron 22E/St)/C5) (Fig. 7J, K). S(Delta)/C5 and S(Delta)/C5 + N/C5 had a median VN titer of ~7.0 × 103 and ~1.0 × 104 against the Delta strain, respectively (Fig. 7J). Groups receiving S(Delta)/C5, S(Omicron 22E/St)/C5, S(Delta)/C5 + S(Omicron 22E/St)/C5 had ~7.0 × 103, ~1.3 × 103, and ~2.0 × 103 VN titers against the Omicron strain (Fig. 7J), suggesting that S(Delta)/C5 alone provides significant VN titers against Delta and Omicron variants. Moreover, sera from the single Ad-S(Delta)/C5 formulation group showed significantly higher VN titer against the Delta strain than the double formulation group Ad-S/C5(Delta)+Ad-S/C5(Omicron 22E/St) and the quadruple formulation group (p < 0.05 both), suggesting a dilution effect when having both S(Delta)/C5 and S(Omicron 22E/St)/C5 in one formulation (Fig. 7J). Interestingly, adding N/C5 and M/C5 to the Ad-S/C5(Delta) formulation significantly enhanced VN titers against the heterologous Omicron strain compared to Ad-S/C5(Delta) alone (p < 0.5) (Fig. 7K). In summary, these results show an overall strong induction of VN titers by all S/C5-containing vector groups against the Delta and Omicron variants (Fig. 7J, K).
Protection efficacy of prime-boost approach in K18-hACE2 transgenic mice following challenge with SARS-CoV-2 variants
The K18-hACE2 transgenic mouse groups immunization and challenge with hCoV-19/USA/PHC658/2021 (Lineage B.1.617.2; Delta) (Fig. 8A) or hCoV-19/USA/MD-HP20874/2021, (Lineage B.1.1.529; Omicron) (Fig. 8B) is outlined (Fig. 5C). To capture the peak of infection, vaccine protection efficacy was monitored by determining lung virus loads five days post-challenge by TCID50 assay. Groups immunized with S(Delta)/C5- or S(Omicron 22E/St)/C5-expressing vector in a single formulation or in any combination reduced lung viral loads to below detection limits for both variants (Fig. 8A, B). Mouse groups vaccinated with vectors expressing N/C5 or M/C5 led to reductions in titers against the Delta and Omicron variants (Fig. 8A, B). Surprisingly, the M/C5 + N/C5 group yielded lower protection efficacy compared to the groups vaccinated with vectors expressing M/C5 or N/C5 individually (Fig. 8A, B).
A, B Protection of mice immunized by a prime-boost approach using BAd and HAd vector formulations. Five-week-old K18-hACE2 transgenic mice (3 males + 2 females per group) were intranasally (i.n.) mock-inoculated or inoculated with BAd vector individually or various combinations as shown in Fig. 5C with a dose of 1 × 107 plaque-forming units (PFU) (each vector). At four weeks post-prime, each group received a booster i.n. inoculation with HAd vector individually or in various combinations, as shown in Fig. 10C with a dose of 1 × 107 PFU (each vector). Three weeks post-booster inoculation, animal groups were challenged with 105 TCID50 of A SARS-CoV-2 (B.1.617.2; Delta) or B (B.1.1.529; Omicron) variant. Five days post-challenge, mice were euthanized, and lung samples were collected and processed for lung virus titration. Data is shown as log10 TCID50/g and detection limit is 0.5 log10 TCID50/g. Each symbol represents an individual animal, and the error is shown as SD. Data were analyzed by one-way ANOVA with Tukey’s post-hoc test. *, significant at p ≤ 0.05; **, significant at p ≤ 0.01; ***, significant at p ≤ 0.001; and ****, significant at p ≤ 0.0001. C–F Monitoring numbers of IL-10- or IL-4-secreting cells in the spleen of immunized mice. Splenocytes from Study #2 were revived and analyzed for N- (C, E) and M-specific (D, F) IL-10- (C, D) or IL-4- (E, F) secreting cells by ELISpot assay using antigen-specific peptides. Numbers of IL-10- or IL-4-secreting cells per 106 splenocytes are presented. Data were analyzed by one-way ANOVA with Tukey’s post-hoc test. *, significant at p ≤ 0.05; **, significant at p ≤ 0.01; ***, significant at p ≤ 0.001; and ****, significant at p ≤ 0.0001.
Immunosuppressive effect of vaccine regimen expressing M/C5 and N/C5
In both Study #1 and Study #2, we observed that the administration of the vaccine regimen expressing M/C5 + N/C5 resulted in a marked reduction in antigen-specific humoral and CMI responses compared to groups immunized with Ad vector expressing M/C5 or N/C5, leading to higher lung viral titers following the virus challenge. These findings suggest an antagonistic interaction between M/C5 and N/C5 proteins when expressed together, resulting in immune suppression. However, inclusion of S/C5 with M/C5 + N/C5 not only overcomes this antagonistic interaction but also significantly enhances immune responses and protection efficacy.
IL-10 has been shown to modulate the Th1 response both directly and indirectly, polarizing a Th2 response marked by IL-4 expression37, impacting the decline in the magnitude and quality of immune responses, and thus serving as a marker of immune suppression37,38,39,40. To explore the role of IL-10 and IL-4 in the observed immune suppression, we evaluated the numbers of IL-10- and IL-4-producing cells in the spleen of immunized groups in response to stimulation with S, M, or N protein. Significant increases in IL-10- (Fig. 8C, D) or IL-4- (Fig. 8E, F) secreting cells in the spleens of groups vaccinated with a combination of vectors expressing M/C5 and N/C5 were observed compared to the groups immunized with vectors expressing M/C5 or N/C5 alone or M/C5 + N/C5 with S/C5. These findings indicate that co-administration of vectors expressing M/C5 and N/C5 induces an immunosuppressive response. Interestingly, this immunosuppressive effect of M/C5 and N/C5 appears to be mitigated by including the vector expressing S/C5 in the vaccine formulation, suggesting a potential immunomodulatory role of the S/C5 protein.
Discussion
The continuous evolution of SARS-CoV-2 has compromised the efficacy of first-generation vaccines, which predominantly target the S protein41,42. This has resulted in the development of second-generation COVID-19 vaccines incorporating S proteins from recent variants29,43. If SARS-CoV-2 continues to circulate endemically, it is likely to evolve further, necessitating tailor-made updated or multivalent vaccines such as seasonal influenza vaccines. The evolutionary conservation and immunogenicity of SARS-CoV-2 N and M make them attractive candidates for inclusion in multivalent vaccine formulations. Additionally, we utilized AIP-C5 to enhance antigen-specific CMI based on prior studies showing its ability to boost T-cell responses via autophagy pathway. In this study, we evaluated the next generation of COVID-19 vaccine formulations, incorporating the relatively conserved N and M proteins alongside S protein aiming to broaden T-cell immunity and enhance protection. The N protein — a major structural protein of SARS-CoV-2 — functions in virus assembly and genome packaging and exhibits ~90% homology with the N protein of SARS-CoV-1, while M, the most abundant capsid protein of SARS-CoV-2, shares ~91% homology with SARS-CoV-144,45,46,47. Their evolutionary conservation and immunogenicity profiles makes them attractive candidates for the development of a multivalent next-generation SARS-CoV-2 vaccine. Additionally, we used AIP-C5 to enhance antigen-specific CMI based on prior studies showing its ability to boost T-cell responses via autophagy induction31,48,49. Moreover, we evaluated the BAd vector platform for i.n. delivery, due to its efficient transduction at mucosal surfaces and ability to promote enhanced antigen expression and elicit both mucosal and systemic antigen-specific immune responses.
Study #1 demonstrated that inclusion of AIP-C5 improved CMI responses against all three antigens but did not significantly impact humoral immune responses, confirming our earlier findings that inclusion of AIP-C5 with an immunogen enhances immunogen-specific CMI responses31,48. Importantly, the addition of M and N to S did not enhance antibody titers and in some cases, notably in the triple-vector group (HAd-S/C5 + HAd-M/C5 + HAd-N/C5), S-specific VN titers were significantly reduced compared to S-only groups. Mice immunized with vector formulations containing HAd-S/C5 provided complete protection from morbidity and mortality with considerably lower numbers of viral RNA copy numbers or virus antigen-expressing cells in the lungs following the challenge with a Delta variant. Interestingly, groups immunized with HAd-N/C5 or HAd-M/C5 alone showed partial protection. However, co-delivery of M/C5 + N/C5 did not confer survival and was associated with diminished humoral and CMI responses. This outcome was paralleled by elevated levels of IL-10 and IL-4, suggesting a shift toward a Th2 and immunoregulatory response50. IL-10, in particular, suppresses dendritic cell function and Th1 differentiation, while promoting Th2 polarization via IL-4-mechanisms that could explain the reduction in protective efficacy observed in the M/C5 + N/C5 group50,51,52,53. These findings underscore the need for further study of the M + N interaction closely to understand the mechanisms that guide this immunosuppression.
Interestingly, the inclusion of S/C5 appeared to mitigate the suppressive effects observed with M/C5 + N/C5, suggesting a possible immunomodulatory or dominant role of S in restoring a more balanced or protective immune profile. There was approximately a 45% and 41% increase in N- or M-specific CMI responses, respectively, in the group immunized with the triple vector formulation, indicating that N- or M-specific CMI responses in combination with S protein may result in broader protection. Additional experiments will be necessary to further investigate the role of synergy in each component of this multi-valent vaccine.
In Study #2, we explored a heterologous prime-boost strategy using BAd vectors for priming and HAd vectors for boosting. BAd was chosen for priming due to its species specificity and the low likelihood of encountering pre-existing vector immunity in humans, thus avoiding diminished response due to vector-directed immunity as well as to add translational value to our study30,47. Moreover, BAd vectors have shown superior ability to enhance innate immunity54,55, and confer complete protection against influenza virus with a 30-fold less vector dose than HAd vector system56. The BAd vector platform is an excellent i.n. delivery system since it uses α(2,3)-linked and α(2,6)-linked sialic acid-binding proteins as receptors for virus entry34. Additionally, a heterologous prime-boost strategy has been demonstrated in other platforms to enhance both humoral and cellular immunity. Both forms of S proteins utilized in the study elicited similar levels of humoral immune responses. Similar to study #1 high levels of antigen-specific humoral and CMI responses were elicited even at a reduced dose of 1.0 × 107 PFU. The prime-and-boost approach with vector formulation expressing native- or stabilized-form of S protein resulted in higher levels of VN titers against their corresponding SARS-CoV-2 variants. Prime-boost vaccination with S, N, and M containing formulations led to protection against Delta and Omicron challenges, although protection was most robust in groups that included S. Mice immunized with N and M alone showed somewhat low reductions in lung viral titers, consistent with T-cell-mediated protection, but were insufficient for complete viral clearance. Interestingly, the quadruple vector formulation group elicited significantly higher levels of S-, N-, or M-specific CMI responses. Of note, adding N and M antigens to S(Delta)/C5 had an enhancing effect for the group’s VN titers against BA.1 Omicron strain compared to S(Delta)/C5 alone (Fig. 7K). Furthermore, having both S antigens in the same formulation reduced the VN titers compared to the single S antigen against the corresponding strain (Fig. 7J, K). Despite variations in their VN titers, groups immunized with the single, double, triple, or quadruple vector formulation expressing at least one of the S proteins conferred complete protection against challenge with either the Delta (B.1.617.2) or Omicron (B.1.1.529) variant, underscoring the importance of S as a correlate of protection.
Similar to Study #1, co-administration of vectors expressing M/C5 and N/C5 resulted in a noticeably lower level of antigen-specific humoral and CMI responses. This was accompanied by significant increases in the number of IL-10- and IL-4-secreting cells in the spleens of immunized groups, suggesting an immunosuppressive effect40. These combined effects underscore the critical role of IL-10 in maintaining an immune balance and preventing excessive Th1-mediated inflammation. Again, we noted that the inclusion of S/C5 in the vaccine formulation appeared to mitigate the immunosuppressive effect, indicating a potential modulatory role of S/C5. These findings highlight the importance of understanding protein-protein interactions in multi-component vaccines and suggest further investigation into mechanisms driving the immunosuppressive or immune-stimulatory response.
Another shortcoming of the first-generation COVID-19 vaccines is their short durability of protective efficacy57. The mRNA-based SARS-CoV-2 vaccine’s protective immunity persisted for three to six months58,59,60, whereas the HAd-26-based COVID-19 vaccine induced protective immunity for at least eight months61,62,63. Waning immunity leads to the emergence of breakthrough infections in vaccinated individuals64. S-specific VN antibodies are the key correlates of protection against SARS-CoV-2 infection; however, CD8+ T-cell response also plays a vital role in controlling viral infection27,28,57.
Overall, these findings support the utility of multi-antigen Ad vector-based vaccines incorporating conserved non-S antigens. The immunological interplay between these antigens — potentially mediated by cytokine profiles and T-cell polarization — requires further study. The durability of protection also remains an open question; a separate exhaustive study will be required to investigate whether our multi-antigen vaccine formulation combined with a prime-and-boost approach using a BAd vector platform and a heterologous delivery system can confer protective immunity for over one year.
Materials and methods
Cell lines
HAd5 E1 expressing human embryonic kidney cells (HEK293)65 were used for growing HAd5 vectors. HEK293 cells expressing Cre recombinase (HEK293Cre)66, bovine-human hybrid clone 2 C (BHH2C)67 was used to transfect and titrate HAd vectors, respectively, as previously described67. BAd3 E1-B expressing bovine-human hybrid clone 5 (BHH/F5), and BHH/F5 expressing endonuclease I-SceI (BHH/F5-ISceI), were used to rescue and grow recombinant BAd3 vectors67. VeroE6 or Vero CCL-81.4 cells (ATCC, USA) were used for growing and titrating SARS-CoV-2 variants. All cells utilized in this study were grown as monolayer cultures in Dulbecco’s Modification of Eagle’s Medium (Life Technologies, Thermo Fisher Scientific, Waltham, MA) supplemented with 10% reconstituted fetal bovine serum (Fetal clone III; Hyclone, Logan, UT) and 50 μg/mL gentamycin.
Generation of replication-defective HAd and BAd vectors expressing SARS-CoV-2 S, M, or N
The S, M, and N protein sequences of the SARS-CoV-2 Ancestral strain (Wuhan/WIV04/2019; GenBank: MN908947), S protein sequence of Delta strain (hCoV-19/USA/PHC658/2021; GenBank: OL442162.) and S protein sequence of Omicron 22E strain (hCoV-19/USA/MD-HP20874/2021; GenBank: OP388404.1) with or without AIP-C5 were used for synthesizing codon-optimized gene cassettes for the rodent expression (GenScript Biotech Corporation, Piscataway, NJ).The gene cassette of the S protein sequence of the Omicron 22E strain was modified to stabilize the protein as previously described68. For the synthesis of HAd-5 replication-defective E1- & E3-deleted vectors, the transgene cassette under the control of the CMV promoter and BGH polyadenylation signal was cloned into an E1 transfer plasmid. Vectors were generated in HEK293Cre cells following a Cre-recombinase-mediated recombination protocol69.
For the synthesis of BAd-3 replication-defective E1- & E3-deleted vectors, the transgene cassette under the control of the CMV promoter and BGH polyadenylation signal was generated in BHH/F5-ISceI cells by I-SceI-mediated release of infectious vector genome. HAd or BAd vectors were purified by cesium chloride density gradient ultracentrifugation following an established protocol70 and were titrated in BHH2C or BHH/F5 cells, respectively as described earlier71.
SARS-CoV-2 variants
The following SARS-CoV-2 variants were procured from BEI Resources: hCoV-19/USA-WA1/2020 (Wuhan lineage), NR-52281; hCoV-19/USA/PHC658/2021 (lineage B.1.617.2, Delta), NR-5566; and hCoV-19/USA/MD-HP20874/2021 (lineage B.1.1.529, Omicron), NR-56481. SARS-CoV-2 variants were grown in VeroE6 or Vero CCL-81.4 cells and titrated in the same cell line by tissue culture infectious dose 50 (TCID50) assay.
Immunoblotting
293 or BHH/F5 cells were infected with HAd or BAd vectors, respectively at the multiplicity of infection (MOI) of 2 PFU per cell. Forty-eight h post-infection, cell pellets were harvested in RIPA (Radioimmunoprecipitation assay) buffer and used in immunoblotting as previously described30. The S-specific mouse monoclonal antibody (GeneTex, Zeeland, MI. #GTX632604), N-specific rabbit polyclonal antibody (GeneTex #GTX135357) or M-specific rabbit monoclonal antibody (GeneTex #GTX636246) were used as primary antibodies, and horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG were used as secondary antibodies. To assess equal loading, immunoblotting for β-actin was performed.
Immunogenicity studies in mice
All immunogenicity studies in mice were performed in a BSL-2 facility at Purdue University with the approval from the Institutional Animal Care and Use Committee (IACUC) and the Institutional Biosafety Committee (IBC). Seven-week-old BALB/c mice were purchased from the Jackson Laboratory, Bar Harbor, ME.
Study #1 evaluated the immunogenicity of HAd vectors expressing SARS-CoV-2 Wuhan Hu1 antigens (S, M, and N) with and without AIP-C5. BALB/c mice (3 females + 3 males) were inoculated i.n. with 5 × 10⁷ plaque-forming units (PFU) per vector per mouse. HAd-ΔE1E3 (empty vector), HAd-S, HAd-S/C5, HAd-M, HAd-M/C5, HAd-N, HAd-N/C5, HAd-S + HAd-M, HAd-S/C5 + HAd-M/C5, HAd-S + HAd-N, HAd-S/C5 + HAd-N/C5, HAd-M + HAd-N, HAd-M/C5 + HAd-N/C5, HAd-S + HAd-M + HAd-N, or HAd-S/C5 + HAd-M/C5 + HAd-N/C5. For combination groups, each vector was administered at 5 × 10⁷ PFU, resulting in a total administered dose of 1.0 × 10⁸ (two vectors formulation) or 1.5 × 10⁸ (three vectors formulation) PFU per mouse. Mice were anesthetized using ketamine and xylazine prior to intranasal inoculation and sample collection. At four weeks post-immunization, each group was boosted with the same dose of the same vaccine formulation. A similarly inoculated PBS group was kept as a negative control. Four weeks post-booster, animals were anesthetized to collect blood samples by retro-orbital punctures and lung washes by homogenizing one lung lobe from each animal in 1 mL of 0.5% bovine serum albumin (BSA) in PBS as described previously72 for evaluating humoral immune responses. The second lung lobe was collected and processed using Lymphocyte Separation Medium (Corning, Thermo Fisher Scientific) to collect lung MN cells, which were used along with splenocytes for investigating CMI responses as previously described49.
Study #2 evaluated the immunogenicity of a heterologous prime-boost vaccine regimen utilizing BAd vector/s priming followed by the boost with HAd vector/s expressing the same antigen/s. BALB/c mice (3 females + 3 males) were inoculated i.n. with 1 × 107 PFU of BAd-∆E1E3 (empty vector), BAd-S/C5(Delta), BAd-S/C5(Omicron/St), BAd-M/C5, BAd-N/C5, BAd-S/C5(Delta) + BAd-S/C5(Omicron/St), BAd-M/C5 + BAd-N/C5, BAd-S/C5(Delta) + BAd-M/C5, BAd-S/C5(Delta) + BAd-N/C5, BAd-S/C5(Delta) + BAd-M/C5 + BAd-N/C5, BAd-S/C5(Omicron/St) + BAd-M/C5, BAd-S/C5(Omicron/St) + BAd-N/C5, BAd-S/C5(Omicron/St) + BAd-M/C5 + BAd-N/C5, or BAd-S/C5(Delta) + BAd-S/C5(Omicron/St) + BAd-M/C5 + BAd-N/C5. At four weeks post-immunizations, each group was boosted with the same dose of the same antigen/s in the HAd vector system [HAd-∆E1E3 (empty vector), HAd-S/C5(Delta), HAd-S/C5(Omicron 22E/St), HAd-M/C5, HAd-N/C5, HAd-S/C5(Delta) + HAd-S/C5(Omicron 22E/St), HAd-M/C5 + HAd-N/C5, HAd-S/C5(Delta) + HAd-M/C5, HAd-S/C5(Delta) + HAd-N/C5, HAd-S/C5(Delta) + HAd-M/C5 + HAd-N/C5, HAd-S/C5(Omicron 22E/St) + HAd-M/C5, HAd-S/C5(Omicron 22E/St) + HAd-N/C5, HAd-S/C5(Omicron 22E/St) + HAd-M/C5 + HAd-N/C5, or HAd-S/C5(Delta) + HAd-S/C5(Omicron 22E/St) + HAd-M/C5 + HAd-N/C5). A similarly inoculated PBS group was kept as a negative control. As in Study #1, four weeks post-booster, animals were anesthetized to collect the blood, lung washes, lung MN cells, and splenocytes. Additionally, MLN cells were also collected for CMI responses.
ELISA to detect SARS-CoV-2 specific antibodies
The development of humoral immune responses was monitored by ELISA as described previously73,74. SARS-CoV-2 S (NR-53937, BEI Resources; Manassas, VA. 2 μg/mL), N (NR-53797, BEI Resources; 2 µg/mL), or M (SCV2-M-050P, E.Enzyme; Gaithersburg, MD. 2 µg/mL) proteins were used to coat 96-well flat-bottom plates (Medisorp and Immulon 2HB, Thermo Fisher Scientific), and incubated overnight at 4 °C. The plates were blocked with 2% BSA in PBS for 2 h. Log diluted serum samples or lung washes were added to the wells and incubated at room temperature for 2 h. Plates were then washed four times with PBS + 0.05% Tween-20, followed by the addition of the horseradish peroxidase-conjugated goat anti-mouse IgG, IgG1, IgG2a, or IgA antibodies (Invitrogen, Thermo Fisher Scientific) at recommended dilutions. Plates were incubated at room temperature in the dark for 2 h and washed four times with PBS-Tween. A BD OptEIA ELISA TMB substrate (Thermo Fisher Scientific) was used for the color development. The reaction was stopped with 2 N sulfuric acid solution, and absorbance was measured at 450 nm using a SpectraMax i3x microplate reader (Molecular Devices, Sunnyvale, CA).
ELISpot assay
The INF-γ and IL-2 ELISpot assays were performed as described earlier75. Splenocytes, MLN cells, or lung MN cells were stimulated with a dominant CD8+ CTL epitope of S (GPKKSTNL), N (LALLLLDRL), or N (RTLSYYKL)76. The peptides were synthesized commercially (GenScript Biotech Corporation, Piscataway, NJ). The stimulated cells were processed INF-γ or IL-2 to assess CMI responses, and for IL-10 and IL-4 to evaluate immune suppression by ELISpot assay. The number of spot-forming units (SFU) was enumerated using the AID iSpot Advanced Imaging Device (Autoimmun Diagnostika GmbH, Strassberg, Germany).
VN assay
The VN titers in serum samples of immunized animals were determined by microneutralization assay as described earlier49. For Study #1, VN titers were determined against three SARS-CoV-2 variants: USA-WA1/2020 (Wuhan), hCoV-19/USA/PHC658/2021 (Lineage B.1.617.2; Delta), and hCoV-19/USA/GR484A/2021 (Lineage B.1.1.529; Omicron). Similarly, for Study #2, VN titers were determined against three SARS-CoV-2 variants: hCoV-19/USA/PHC658/2021 (Lineage B.1.617.2; Delta), and hCoV-19/USA/GR484A/2021 (Lineage B.1.1.529; Omicron). Briefly, 2.5 ×104 VeroE6 cells were seeded per well in a 96 microwell plate. Heat-inactivated serum samples were diluted 2-fold, mixed with 100 TCID50 of the virus in triplicates, and incubated at 37 °C for 1 h. The medium was then replaced with the serum-virus mixture and incubated for 72 h in a 5% CO2 incubator at 37 °C. The plates were inspected for the development of cytopathic effect (CPE). The VN titer was determined as the reciprocal of the highest serum dilution where at least 2 out of 3 wells showed CPE.
Challenge studies of immunized animals
Animal origin and arrival
K18-hACE2 transgenic mice [B6.Cg-Tg(K18-ACE2)2Prlmn/J] aged four weeks were procured from The Jackson Laboratory. The mice were housed at the Centralized Biological Laboratory (CBL) at Pennsylvania State University, and food and water ad libitum were provided. They underwent a 5-day acclimatization period before the commencement of the experiment.
Vaccination of the animals
Study #1: The mice were randomly segregated into two nine groups (2 females and 3 males/group) and were similarly immunized i.n. with only HAd vectors containing AIP-C5 as described for Study #1 immunogenicity.
Study #2: The mice were randomly segregated into two batches, each comprising 75 mice. They were further subdivided into 15 groups (2 females and 3 males/group). 15 groups of each batch were similarly immunized i.n. with BAd and HAd vectors (prime-boost strategy) as described for Study #2 immunogenicity.
Animal transfer to an animal biosafety level 3 laboratory (ABSL3)
Three weeks after the booster vaccination, the animals were relocated to the Eva J. Pell ABSL-3 Laboratory for Advanced Biological Research at Pennsylvania State University, maintaining their original batches and groupings. They were granted ad libitum access to food and water.
SARS-CoV-2 challenge and follow-up
Study #1: All groups were i.n. challenged under anesthesia with 104 TCID50 of SARS-CoV-2 Delta (Lineage B.1.617.2) Animals were monitored daily for morbidity and mortality for 14 days post-challenge. Similarly, other set of challenged groups were used for monitoring lung virus titers at 5 days post-challenge by RT-PCR.
Study #2: The 15 groups of the first batch were i.n. challenged under anesthesia with hCoV-19/USA/PHC658/2021 (Lineage B.1.617.2, Delta) and the other 15 groups of the second batch were challenged with hCoV-19/USA/MD-HP20874/2021 (Lineage B.1.1.529, Omicron) at a dosage of 105 TCID50. Five days post-challenge, all animals were euthanized using the CO2 euthanasia method. For virus load quantification, the left lung was promptly stored at -80 °C.
Lung virus load quantification by TCID50 assay and RT-PCR
The left lung lobe from each animal was thawed and homogenized using the QIAGEN Tissue Lyser II at 50 Hz for 3 min, followed by centrifugation at 3000 ×g for 3 min. The supernatant was utilized for virus titration by TCID50 assay in VeroE6 cells, with results expressed as log TCID50/mL. Alternatively, viral RNA from the lung was extracted and utilized to quantify the SARS-CoV-2 N gene (Primetime One Step 2X, Integrated DNA Technologies) as previously described77, and the outcomes were expressed as log10RNA copies/mL.
Immunohistochemistry
Briefly, the paraffin-embedded 5 µm thick tissue sections were deparaffinized in xylene and rehydrated in ethanol. Following 10 min presoaking of the rehydrated sections in Tris-buffered saline (TBS), sections were subjected to heat-mediated antigen retrieval. Briefly, the slides were placed in 10 mM Tris 1 mM EDTA buffer and heated at 90 °C for 10 min, following a cool-down period of 15 min. The antigen retrieved sections were blocked at 4 °C overnight using inactivated goat serum (Abcam Cambridge, UK. catalog#ab7481) (diluted 25uL/mL TBS), followed by a TBS wash. SARS-CoV-2 N was stained using the primary Anti-SARS-CoV-2 N protein antibody (6F10, Abcam catalog# ab288116) for 1 h. Following three washes with TBS, sections were further incubated with a secondary goat anti-mouse IgG H&L (Alexa Fluor 647, Abcam catalog# ab150115) for 35 min. The sections were washed three times with TBS and mounted in ProLong Gold antifade mountant with DNA stain DAPI (Thermo Fisher Scientific, catalog # P36931). Negative controls were performed, omitting the primary antibody staining. Following 24 h of curing at room temperature, the sections were imaged using Echo Revolve Fluorescent Microscope (ECHO A BICO Company, San Diego, CA).
Statistical analyses
All statistical analyses were performed using GraphPad Prism 10. Statistical significance was established at p < 0.05. and was generally assessed by two-tailed unpaired Student’s t-tests unless indicated otherwise in figure legends. Nonparametric analyses, were employed when appropriate, as specified in figure legends. In all graphs, lines represent mean values unless otherwise stated. Error bars represent the standard deviations for parametric data or interquartile ranges for nonparametric data.
Data availability
The data supporting the findings of this study are available from the corresponding author upon request. This includes the raw data underlying key figures and any additional datasets generated or analyzed during the study.
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Acknowledgements
This work was supported by Public Health Service grant AI158177 from the National Institute of Allergy and Infectious Diseases. The following reagent was produced under HHSN272201400008C and obtained through BEI Resources, NIAID, NIH: Spike Glycoprotein Receptor Binding Domain (RBD) from SARS-Related Coronavirus 2, Nucleocapsid protein from SARS-Related Coronavirus 2. USA-WA1/2020 virus, hCoV-19/England/204820464/2020 (B.1.1.7), hCoV-19/USA/PHC658/2021 (Lineage B.1.617.2), and hCoV-19/USA/MD-HP20874/2021 (Lineage B.1.1.529).
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M.A., E.E.S., A.E., S.K.C., W.-C.W., M.S.T.M., V.G., N.W., P.J., A.G., S.R., L.L., M.S.N., and R.N. performed the experiments. M.A., E.E.S., A.E., and S.K.C. wrote the first version of the manuscript. M.A., E.E.S., A.E., and S.K.C. prepared all figures. S.V.K. and S.K.M. designed the study, and revised the manuscript. N.W. performed immunohistology evaluations. All authors reviewed the manuscript.
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Alhashimi, M., Sayedahmed, E.E., Elkashif, A. et al. A multi-antigen-based SARS-CoV-2 vaccine provides higher immune responses and protection against SARS-CoV-2 variants. npj Vaccines 10, 159 (2025). https://doi.org/10.1038/s41541-025-01198-7
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DOI: https://doi.org/10.1038/s41541-025-01198-7
