A rationally designed Wuhan-sequence-based COVID-19 vaccine that maintained effectiveness against diverse SARS-CoV-2 variants, including Omicron: What was the key to success?
Jun 2026· Transactions of the Institute of Molecular Biology & Biotechnologies· Vol 10, pp. 62-71· 0 citations
TL;DR
The scientific basis of a plant-produced recombinant COVID-19 cocktail vaccine that demonstrated sustained immunogenicity and cross-variant effectiveness despite being designed from the ancestral Wuhan SARS-CoV-2 sequence is discussed.
Abstract
The continuous emergence of SARS-CoV-2 variants of concern has highlighted important limitations of first-generation COVID-19 vaccines developed against the ancestral Wuhan spike protein. Although these vaccines provided strong protection against severe disease and mortality, the accumulation of mutations in the spike protein, particularly within the receptor-binding domain (RBD), progressively reduced their ability to prevent infection and facilitated immune escape. In contrast, vaccine approaches that combine multiple complementary viral antigens have shown greater potential to maintain broad protective immunity despite ongoing viral evolution and immune escape. In this article, we discuss the scientific basis of a plant-produced recombinant COVID-19 cocktail vaccine that demonstrated sustained immunogenicity and cross-variant effectiveness despite being designed from the ancestral Wuhan SARS-CoV-2 sequence. Based on our experimental studies, we propose that this broad protective potential results from the integration of three key design elements: (i) targeting conserved and functionally constrained epitopes within the RBD, (ii) combining RBD and nucleocapsid (N) proteins in a multi-antigen vaccine formulation, and (iii) applying glycoengineering strategies to optimize antigen structure and immune recognition, thereby broadening the immune response and reducing susceptibility to viral immune escape. We further discuss the advantages of plant molecular farming, particularly transient expression in Nicotiana benthamiana, as a rapid, scalable, and cost-effective platform for recombinant vaccine production. Our studies demonstrated that plant-produced glycosylated and deglycosylated RBD antigens retain functional receptor-binding activity and induce strong neutralizing antibody responses. Moreover, when combined with the N protein, these antigens maintained effectiveness against highly divergent SARS-CoV-2 variants, including Omicron. Importantly, the principles underlying this vaccine design may extend beyond COVID-19. The strategic combination of conserved and immunologically complementary antigens provides a rational framework for the development of broadly protective vaccines against future pandemic threats and other rapidly evolving emerging pathogens. In particular, our findings suggest that integrating conserved and immunologically complementary antigens may represent a universal vaccine design strategy to combat future pandemics caused by rapidly evolving pathogens.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve through mutations in the spike (S) protein, leading to antigenically divergent variants such as the Omicron sublineage JN.1 that markedly reduce vaccine-induced neutralizing antibody activity. These limitations highlight the need for vaccine strategies that can induce multilayered protective immunity rather than relying solely on S protein-targeted immunity. Here, we developed a Wuhan-lineage-based multi-antigen virus-like particle (VLP) vaccine incorporating S, envelope (E), membrane (M), and nucleocapsid (N) proteins using a baculovirus dual-expression system. Mice were primed intramuscularly and boosted via either the intramuscular or intranasal route, and humoral, mucosal, and cellular immune responses were evaluated. Both vaccination regimens induced robust systemic IgG responses, whereas mucosal IgA responses were selectively enhanced following intranasal boosting. Sera from VLP-immunized mice efficiently neutralized the homologous Wuhan-lineage strain but showed no detectable neutralizing activity against the JN.1 variant. Despite this, VLP immunization elicited antigen-specific CD4+ and CD8+ T cell responses in both pulmonary and systemic compartments. In a highly susceptible K18-hACE2 mouse model, VLP immunization completely prevented mortality following challenge with both the ancestral Wuhan-lineage strain and the JN.1 variant and markedly suppressed viral replication in respiratory tissues. Histopathological analysis further showed minimal interstitial lung lesions with organized perivascular lymphocytic infiltration in vaccinated mice, while pulmonary cytokine analysis revealed qualitatively distinct immune response patterns depending on the challenge strain and vaccination status. Our findings show that a Wuhan-lineage-based multi-antigen VLP vaccine can provide cross-protection against an antigenically divergent SARS-CoV-2 variant that is not fully explained by detectable serum neutralizing activity alone, suggesting the importance of integrated immune responses involving humoral, cellular, and local immune mechanisms.
Seung-Ji Kim, Howon Kim, Seung-Eun Son et al.· Vaccine· 0 citations
Vaccination remains the most successful preventative measure against viral infection, but methods to stably deter rapidly-evolving pathogens have remained elusive. Vaccines capable of incorporating and anticipating viral evolution could address current challenges in seasonal vaccination efforts against SARS-CoV-2 and influenza where economic and disease burdens remain high despite decades of combined study. Rare epitope suppression (RES) is an underutilized concept within vaccine design, where humoral epitope targeting can be molded using complex antigen pools. Based in mRNA vaccine technology, ‘wobble vaccines’ represent the novel application of RES to human pathogens designed to anticipate and resist viral evolution. To establish this platform, public SARS-CoV-2 sequencing data was compiled from the first two years of the COVID-19 pandemic to identify high-diversity sites across the receptor binding domain (RBD) of the spike protein. Wobble RBD (WobbRBD) libraries reflecting that entropy were synthesized and incorporated into established self-amplifying (SA) vaccine constructs. Animals immunized with these complex antigen pools showed no obvious adverse effects. By three days-post vaccination, WobbRBD stimulated robust primary immune activation with distinctive characteristics compared to traditional single-strain vaccine modalities. By day 14, germinal centers, class switching, and antibody-secreting cells were induced, creating potent SARS-CoV-2 spike-binding IgG antibodies. Despite similar overall activation profiles, WobbRBD generated significantly increased breadth against SARS-CoV-2 variant spikes in comparison to single-strain controls – even against future-emerging strains. Taken together, wobble vaccines represent a novel method for anticipating and preventing viral escape with promising applications in SARS-CoV-2, influenza, HIV, and beyond.
Peter R. McIlroy, Wendy M. Zinzow-Kramer, M. Ellis et al.· bioRxiv· 0 citations
BACKGROUND
The COVID-19 pandemic caused over seven million deaths, and disease control relied largely on mass immunization campaigns. Although diverse vaccine platforms were used, most formulations targeted the Spike protein because of its central role in virus entry. Persistent viral evolution and global vaccine inequity highlight the need for alternative strategies, particularly for low- and middle-income countries. In this scenario, recombinant protein-based vaccines are attractive due to low cost, established manufacturing pipelines and well-characterized safety profiles. The receptor binding domain (RBD) of Spike is an appealing antigen due to its relatively simple structure, proven immunogenicity - especially in multimerized formats - and because it harbors most neutralizing antibody epitopes. Protein-based vaccines, however, rely on the use of adjuvants to optimize antigen-specific immunity.
METHODS
We used an RBD dimer derived from the ancestral Wuhan strain as a well-characterized protein to evaluate the humoral and cellular immunogenicity of combined AddaS03 and Poly I:C adjuvants in BALB/c mice.
RESULTS
The adjuvant combination preserved the strong humoral responses typically induced by AddaS03 while conferring the Th1-skewing properties of Poly I:C, producing a synergistic effect that exceed those of each adjuvant alone. Specifically, the combination improved antibody affinity, increased the generation of antibody- secreting cells in spleen and bone marrow, and enhanced the induction of polyfunctional, cytokine-producing CD4+ T cells. Additionally, we mapped and characterized three class II T cell epitopes within RBD: peptide 6 (351-YAWNRKRISNCVADYSV-367), peptide 19 (442-SKVGGNYNYLYRLFRK-458) and peptide 20 (449-YNYLYRLFRKSNLKPFE-465).
CONCLUSIONS
Combining AddaS03 and Poly I:C adjuvants is a potent strategy to modulate and enhance immune responses elicited by an RBD-based vaccine.
João Pedro da Silva Nunes, Isabela P Daher, Armando da Silva Pires et al.· Vaccine· 0 citations
The first-generation Spike-based COVID-19 vaccines reduced the risk of hospitalization, serious illness, and death from SARS-CoV-2 infections. However, waning immunity failed to prevent immune escape, resulting in multiple variants of concern (VOCs) and prolonging the pandemic. We hypothesize that next-generation CoV vaccines incorporating highly conserved SARS-CoV-2 T cell antigens would confer potent, broad, long-lasting cross-protective immunity against multiple VOCs. In this study, we identified 10 non-Spike antigens common and highly conserved across 8.7 million SARS-CoV-2 strains, prior VOCs, SARS-CoV-1, MERS-CoV, seasonal human cold CoVs, and animal CoVs. Seven antigens were preferentially recognized by CD8
+
and CD4
+
T cells from unvaccinated asymptomatic COVID-19 patients, irrespective of VOC. Three conserved antigens (NSP2, NSP14, Nucleoprotein) administered to golden Syrian hamsters: (
i
) induced high frequencies of lung-resident polyfunctional CXCR5
+
CD4
+
T
FH
cells, GzmB
+
CD4
+
/CD8
+
cytotoxic T cells, and CD69
+
IFN-γ
+
TNFα
+
CD4
+
/CD8
+
effector T cells; (
ii
) reduced morbidity, viral load, lung pathology, and COVID-19-like symptoms from various VOCs, including the pathogenic B.1.617.2 Delta and the recent KP.3 Omicron variants; (
iii
) improved protection conferred by spike-alone mRNA; and (
iv
) conferred protection that lasted for more than 1 year. This multi-antigen CoV vaccine could provide potent, broad-spectrum, and durable cross-protective immunity against current and future variants of concern in the clinic.
Swayam Prakash, N. Dhanushkodi, Afshana Quadiri et al.· npj Vaccines· 0 citations
In the present study, by building on the previous development of a DC-SIGN-targeting virus-like structure (VLS) vaccine platform and a comprehensive characterization of SARS-CoV-2 structural biology, particularly insights into the role of the nucleocapsid (N) protein in eliciting cytotoxic T lymphocyte (CTL) responses during infection, we designed a SARS-CoV-2 virion-mimetic structural vaccine that encapsulates an mRNA encoding the spike S1 antigen complexed with N protein complexes, with S1 proteins loaded on its surface. This characterized virion-mimetic structural vaccine not only induces the production of high-efficiency antibodies against both the spike and N proteins but also elicits robust S1-specific and N-specific CTL responses in animal models. Furthermore, the generated antibodies exhibit cross-reactive neutralizing activity against multiple SARS-CoV-2 variants and provide protective immunity against challenge with mutant viruses in immunized hosts. This SARS-CoV-2 virion-mimetic structure effectively recapitulates natural infection pathways, comprehensively activating the innate immune system and thereby creating an optimal microenvironment for eliciting potent and broad-spectrum adaptive immune responses.
Jingjing Zhang, Fen Zeng, Yanmei Li et al.· Scientific Reports· 0 citations
Porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) is one of the major pathogens threatening the global swine industry and causes enormous economic losses annually. Vaccination remains one of the most important strategies for the prevention and control of PRRSV infection. In this study, a novel genotype 2 NADC30‐like PRRSV strain, designated AHFY, was isolated and characterized from eastern China. Phylogenetic analysis revealed that strain AHFY belonged to sublineage 1.8. An attenuated strain, AHFY‐F140, was obtained through 140 serial passages in MARC‐145 cells. Genomic sequence comparison between AHFY‐F140 and the parental strain AHFY‐F1 identified two discontinuous amino acid (aa) deletions in the nonstructural protein 2 (NSP2) gene, spanning residues 355–364 and 391–583, which were first detected at passage 90 and persisted through passage 140. The viral titer gradually increased with passaging, rising from 104.3 TCID50/mL for the parental strain AHFY‐F1 to 108.7 TCID50/mL for AHFY‐F140. A pathogenicity evaluation was conducted in 4‐week‐old PRRSV‐free New American Line piglets using AHFY‐F1 and AHFY‐F140. Piglets inoculated with AHFY‐F1 developed persistent fever and obvious respiratory clinical signs, accompanied by severe and typical histopathological lung lesions. In contrast, piglets inoculated with AHFY‐F140 showed no fever or other clinical signs. PRRSV‐specific antibodies became positive at 11 days postinoculation (dpi) in AHFY‐F140‐inoculated piglets, which was later than the 7 dpi observed in AHFY‐F1‐inoculated piglets. Furthermore, immunization with AHFY‐F140 provided robust protection against subsequent challenge with either the parental strain AHFY‐F1 or the highly pathogenic PRRSV (HP‐PRRSV) strain JXA1 (lineage 8). Collectively, these findings demonstrate that the attenuated strain AHFY‐F140 represents a promising vaccine candidate against both NADC30‐like and JXA1‐like PRRSV infections.
Heng-jiao Zhang, Jiakai Zhao, Chengxin Zhang et al.· Transboundary and Emerging D...· 0 citations