Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Dec;11(1):2359-2370.
doi: 10.1080/22221751.2022.2122580.

A replication-competent smallpox vaccine LC16m8Δ-based COVID-19 vaccine

Akihiko Sakamoto  1 , Hiroaki Osawa  1 , Hinata Hashimoto  1 , Tetsushi Mizuno  1   2 , Ammar A Hasyim  1 , Yu-Ichi Abe  1 , Yuto Okahashi  1 , Ryohei Ogawa  3 , Mitsuhiro Iyori  1 , Hisatoshi Shida  4 , Shigeto Yoshida  1
Affiliations

A replication-competent smallpox vaccine LC16m8Δ-based COVID-19 vaccine

Akihiko Sakamoto et al. Emerg Microbes Infect. 2022 Dec.

Abstract

Viral vectors are a potent vaccine platform for inducing humoral and T-cell immune responses. Among the various viral vectors, replication-competent ones are less commonly used for coronavirus disease 2019 (COVID-19) vaccine development compared with replication-deficient ones. Here, we show the availability of a smallpox vaccine LC16m8Δ (m8Δ) as a replication-competent viral vector for a COVID-19 vaccine. M8Δ is a genetically stable variant of the licensed and highly effective Japanese smallpox vaccine LC16m8. Here, we generated two m8Δ recombinants: one harbouring a gene cassette encoding the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein, named m8Δ-SARS2(P7.5-S)-HA; and one encoding the S protein with a highly polybasic motif at the S1/S2 cleavage site, named m8Δ-SARS2(P7.5-SHN)-HA. M8Δ-SARS2(P7.5-S)-HA induced S-specific antibodies in mice that persisted for at least six weeks after a homologous boost immunization. All eight analysed serum samples displayed neutralizing activity against an S-pseudotyped virus at a level similar to that of serum samples from patients with COVID-19, and more than half (5/8) also had neutralizing activity against the Delta/B.1.617.2 variant of concern. Importantly, most serum samples also neutralized the infectious SARS-CoV-2 Wuhan and Delta/B.1.617.2 strains. In contrast, immunization with m8Δ-SARS2(P7.5-SHN)-HA elicited significantly lower antibody titres, and the induced antibodies had less neutralizing activity. Regarding T-cell immunity, both m8Δ recombinants elicited S-specific multifunctional CD8+ and CD4+ T-cell responses even after just a primary immunization. Thus, m8Δ provides an alternative method for developing a novel COVID-19 vaccine.

Keywords: BALB/c mouse; COVID-19; Delta/B.1.617.2 variant of concern; LC16m8; SARS-CoV-2; cellular immunity; emerging and re-Emerging coronaviruses; humoral immunity; prime-boost immunization.

PubMed Disclaimer

Conflict of interest statement

S.Y., H.S., and M.I. are named inventors on filed patents related to viral-vectored malaria vaccines. H.S. is a named inventor on a filed patent related to m8Δ (WO 2005/054451 A1).

Figures

Figure 1.
Figure 1.
Construction of the m8Δ-based vaccines. (A) Schematic of the vaccines. Characteristic polybasic sequences are listed above the S1/S2 cleavage sites, indicated by scissors. Arrows indicate the primers used for validation by PCR. m8Δ-S, m8Δ-SARS2(P7.5-S)-HA; m8Δ-SHN, m8Δ-SARS2(P7.5-SHN)-HA; SP, signal peptide; G, VSV G protein-derived membrane anchor; L, left arm; R, right arm. (B) Validation of the recombinant viruses by conducting PCR with the indicated primer pairs. The indicated viral DNA was used as the template. bp, base pair. (C) Loss of HA expression in the recombinant viruses. Three days after RK13 cells were inoculated with the indicated recombinant viruses at a dose of 50–100 plaque-forming units per 7.5×105 cells, they were used in a haemadsorption assay. Data are representative from two independent experiments with similar results. Scale bars, 1 mm.
Figure 2.
Figure 2.
In vitro expression of the S protein. (A) Immunoblotting of HEK293T cells that were inoculated with the indicated recombinant viruses at an MOI of 1 or mock infected one day before the analysis. Antibody targets are shown on the left side of the panels. The cleaved form frequency was calculated via densitometry from three independent experiments and is shown as the mean±SEM. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. m8Δ-S, m8Δ-SARS2(P7.5-S)-HA; m8Δ-SHN, m8Δ-SARS2(P7.5-SHN)-HA. (B) Immunofluorescence assays of HEK293T cells that were inoculated with the indicated viruses at an MOI of 0.1 one day before the analysis. SARS-CoV-2 S2 (green) and nuclei (blue) were visualized on the non-permeabilized formalin-fixed cells. Data are representative of three (A) or at least two (B) independent experiments. Scale bars, 10 µm.
Figure 3.
Figure 3.
Antibody production induced by immunization with the m8Δ-based vaccines. (A) Experimental outline. (B) Time course of the antibody levels after primary immunization with the indicated virus. The antibody levels were measured by ELISA using diluted serum samples (1:200). The dilution factor of 200 corresponded to the 50% effective concentration of the serum samples obtained six weeks after the boost immunization. Symbols and bars represent the means and SEM, respectively. A414, absorbance at 414 nm; m8Δ-S, m8Δ-SARS2(P7.5-S)-HA; m8Δ-SHN, m8Δ-SARS2(P7.5-SHN)-HA. (C) Endpoint titres of the indicated antibodies six weeks after the primary (×1) or boost (×2) immunization. Titres of <100 are listed as not determined (ND). The proportions of samples with measurable titres are shown in parentheses. Each symbol represents an individual mouse. Horizontal lines represent geometric means. Data were pooled from three independent experiments using four or more mice per experimental group. Data at each time point in (B) were analysed by Tukey test. The statistical significance of the difference from the m8Δ×2 group is indicated by * near the individual symbols. The log-transformed data in (C) were analysed by a Welch t-test. ***P < 0.001, **P < 0.01, *P < 0.05.
Figure 4.
Figure 4.
Neutralizing activity of the serum after immunization with the m8Δ-based vaccines. (A) Neutralization of the Wuhan-Hu-1 pseudovirus by the serum samples in a concentration-dependent manner, as determined by luciferase assay. The pseudovirus was incubated with the serum samples obtained six weeks after the boost (×2) immunization with the indicated virus. m8Δ-S, m8Δ-SARS2(P7.5-S)-HA; m8Δ-SHN, m8Δ-SARS2(P7.5-SHN)-HA. (B) Neutralizing activities of the serum samples obtained six weeks after the primary (×1) or boost immunization with the indicated virus. The proportions of samples with measurable neutralizing activity are shown in parentheses. NT50, 50% neutralizing concentration; ND, not determined. (C) Neutralizing activities of the serum samples described in (B) obtained six weeks after the boost immunization with m8Δ-S, as measured using the indicated pseudovirus. (D) Neutralization of the Wuhan SARS-CoV-2 by the serum samples described in (A) in a concentration-dependent manner, as determined by crystal violet assay. Serum samples lacking neutralizing activity against the Wuhan-Hu-1 pseudovirus were excluded. A570, absorbance at 570 nm. (E) Neutralizing activities of the serum samples described in (D). (F) Neutralizing activities of the serum samples described in (C) against the indicated SARS-CoV-2. Serum samples displaying neutralizing activity against the corresponding pseudovirus were analysed. Symbols and bars in (A,D) represent the means and SEM, respectively. Each symbol in (B,C,E,F) represents an individual mouse or patient. The same serum samples are linked in (C,F). Horizontal lines in (B,C,E,F) represent the geometric means. Data were pooled from three independent experiments using four or more mice per experimental group. The log-transformed data in (B,C,E,F) were analysed by an unpaired (B,E) or paired Welch t-test (C,F). *P < 0.05. NS, not statistically significant.
Figure 5.
Figure 5.
Cellular immune responses to the m8Δ-based vaccines as measured by an ELISpot assay. Splenocytes were obtained six weeks after the primary (×1) or boost (×2) immunization with the indicated virus. IFN-γ spot-forming units (SFU) were measured after in vitro stimulation with or without the indicated peptide. Each symbol represents an individual mouse. Horizontal lines represent the geometric means. Data were pooled from two or three independent experiments using four or more mice per experimental group. m8Δ-S, m8Δ-SARS2(P7.5-S)-HA; m8Δ-SHN, m8Δ-SARS2(P7.5-SHN)-HA. The log-transformed data from after the primary or boost immunization were analysed individually by a Tukey test. Combinations without mark are not statistically different. ***P < 0.001, **P < 0.01, *P < 0.05.
Figure 6.
Figure 6.
Cytokine profiles of the CD8+ memory T cells. (A) Frequencies and absolute numbers of the CD8+ effector memory T (TEM) cells reactive to the S peptide mix, as measured by intracellular cytokine staining. Splenocytes were obtained six weeks after the primary (×1) or boost (×2) immunization with the indicated virus. The gating strategy applied to isolate the CD8+ TEM cells is shown in Supplementary Figure 5. Cytokine-producing cells were identified as shown in Supplementary Figure 6(A). Each symbol represents an individual mouse. Horizontal lines represent the means (upper panels) or geometric means (lower panels). Data were pooled from two independent experiments using four or more mice per experimental group. m8Δ-S, m8Δ-SARS2(P7.5-S)-HA; m8Δ-SHN, m8Δ-SARS2(P7.5-SHN)-HA. (B) Percentages of the S peptide mix-reactive CD8+ TEM cells described in (A) that expressed the indicated number of cytokines among IFN-γ, TNF-α, and IL-2. The data shown in (A, upper panels) and the log-transformed data shown in (A, lower panels) after the primary and boost immunization were analysed individually by a Tukey test. Combinations without mark are not statistically different. ***P < 0.001, **P < 0.01, *P < 0.05.
Figure 7.
Figure 7.
Cytokine profiles of the CD4+ memory T cells. (A) Frequencies and absolute numbers of the CD4+ effector memory T (TEM) cells reactive to the S peptide mix, as measured by intracellular cytokine staining. Splenocytes were obtained six weeks after the primary (×1) or boost (×2) immunization with the indicated virus. The gating strategy applied to isolate the CD4+ TEM cells is shown in Supplementary Figure 5. The cytokine-producing cells were identified as shown in Supplementary Figure 6(B). Each symbol represents an individual mouse. Horizontal lines represent the means (upper panels) or geometric means (lower panels). Data were pooled from two independent experiments using four or more mice per experimental group. m8Δ-S, m8Δ-SARS2(P7.5-S)-HA; m8Δ-SHN, m8Δ-SARS2(P7.5-SHN)-HA. (B) Percentages of the S peptide mix-reactive CD4+ TEM cells described in (A) that expressed the indicated number of cytokines among IFN-γ, TNF-α, and IL-2. The data shown in (A, upper panels) and the log-transformed data shown in (A, lower panels) after the primary and boost immunization were analysed individually by a Tukey test. Combinations without mark are not statistically different. ***P < 0.001, **P < 0.01, *P < 0.05.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Sadarangani M, Marchant A, Kollmann TR.. Immunological mechanisms of vaccine-induced protection against COVID-19 in humans. Nat Rev Immunol. 2021 Aug;21(8):475–484. - PMC - PubMed
    1. Tregoning JS, Flight KE, Higham SL, et al. . Progress of the COVID-19 vaccine effort: viruses, vaccines and variants versus efficacy, effectiveness and escape. Nat Rev Immunol. 2021 Oct;21(10):626–636. - PMC - PubMed
    1. Jackson CB, Farzan M, Chen B, et al. . Mechanisms of SARS-CoV-2 entry into cells. Nat Rev Mol Cell Biol. 2022 Jan;23(1):3–20. - PMC - PubMed
    1. Chavda VP, Bezbaruah R, Athalye M, et al. . Replicating viral vector-based vaccines for COVID-19: potential avenue in vaccination arena. Viruses. 2022 Apr 6;14(4):759. - PMC - PubMed
    1. Pegu A, O'Connell SE, Schmidt SD, et al. . Durability of mRNA-1273 vaccine-induced antibodies against SARS-CoV-2 variants. Science. 2021 Sep 17;373(6561):1372–1377. - PMC - PubMed

Supplementary concepts

Grants and funding

This work was supported by the Japan Society for the Promotion of Science (JSPS) under Grant [number 21K06545] and Sumitomo Mitsui Trust Bank under Grant.