Study demonstrates robust antibody response elicited by trivalent protein subunit vaccine against SARS-CoV-2

In a recent study posted to the bioRxiv* preprint server, researchers demonstrated that a trivalent vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) improved the breadth of humoral immune responses in mice.

Study: Trivalent SARS-CoV-2 S1 Subunit Protein Vaccination Induces Broad Humoral Responses in BALB/c Mice. Image Credit: Corona Borealis Studio/Shutterstock


SARS-CoV-2 vaccines have reduced morbidity and mortality due to the coronavirus disease 2019 (COVID-19). Nonetheless, emergent SARS-CoV-2 variants and global vaccination inequities have deprived many low-to-middle-income countries of access to vaccines tailored for variants.

Protein subunit vaccine technology is favorable for equitable global distribution given the relatively low cost of production, thermostability, and high safety profile. There is a growing interest in vaccines that can target multiple variants. A multivalent vaccine is a conventional approach to increase antigen landscape against ever-changing pathogens.

The study and findings

In the present study, researchers assessed the immunogenicity of protein subunit vaccines based on spike S1 of wildtype SARS-CoV-2, Delta, or Omicron variant administered in mice as individual monovalent antigens or a trivalent vaccine incorporating the three antigens. Recombinant S1 subunits of SARS-CoV-2 Wuhan, Delta, and Omicron BA.1 spike proteins were expressed.

Polyclonal anti-spike antibodies recognized the recombinant antigens in a Western blot. Female BALB/c mice were immunized with SARS-CoV-2 Wuhan-S1, Delta-S1, Omicron-S1, or a trivalent vaccine incorporating these antigens. Animals received a homologous booster three weeks later. Serum samples were obtained pre- and post-immunization.

Serum samples were serially diluted to measure immunoglobulin G (IgG) titers against SARS-CoV-2 Wuhan, Delta, and Omicron S1 using enzyme-linked immunosorbent assay (ELISA). All vaccinated mice showed significantly increased geometric mean IgG endpoint titers (EPTs) against Wuhan S1 at week 5 relative to week 3.

Interestingly, mice primed with the Wuhan S1 vaccine showed lower EPTs at week 9 than others. Mice receiving the trivalent vaccine showed higher EPTs at weeks 3, 5, and 7 against Wuhan S1 than those vaccinated with the Omicron S1 vaccine. Mice vaccinated with Delta S1 or trivalent vaccine showed the highest geometric mean EPTs against Delta S1 at week 3.

However, only those vaccinated with Omicron S1 or trivalent vaccine showed higher EPTs against Delta S1 at week 5 relative to week 3. Animals receiving Delta S1, Omicron S1, or trivalent vaccine had significantly higher EPTs against Omicron S1 at week 5 than at week 3. Omicron S1 and trivalent vaccines elicited the highest EPTs against Omicron S1 from week 3 until week 20.

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Competitive immunoassays measuring the inhibition of binding between SARS-CoV-2 spike and angiotensin-converting enzyme 2 (ACE2) correlate well with virus neutralization tests. Such assays are convenient surrogates to quantify the neutralizing ability of vaccinated sera. As such, the authors assessed the neutralizing capacity of vaccine-induced antibodies to inhibit spike-ACE2 binding using spike proteins from different variants.

Sera from vaccinated mice were assessed at weeks 5 and 7. Antibodies inhibiting spike-ACE2 binding were present in all vaccinated mice. Sera from Wuhan S1-vaccinated mice had the lowest median binding inhibition against spikes from SARS-CoV-2 Wuhan, Alpha, Beta, and Delta variants, whereas sera from Delta S1-vaccinated mice exhibited robust inhibition against these variants.

However, Delta S1-immunized sera had a weakened response against Omicron and its sub-lineages. Omicron S1-vaccinated mice displayed moderate to high binding inhibition against all tested spikes and exhibited potent inhibitory activity against Omicron spikes.

Mice immunized with the trivalent vaccine showed enhanced binding inhibition against all tested spikes compared to animals vaccinated with the monovalent counterparts. Moreover, trivalent vaccine recipients had higher median binding inhibition than Omicron S1 recipients against spikes from Wuhan, Alpha, Beta, Delta, and Omicron (BA.1 and BA.2).

Finally, the authors plotted mean values of Wuhan, Delta, and Omicron spike binding IgG EPTs with percent binding inhibition. Delta S1- and trivalent vaccine recipients grouped with the highest mean IgG EPTs and binding inhibition against Wuhan and Delta spikes. Omicron S1- and trivalent vaccine-immunized mice had the highest mean IgG EPTs and binding inhibition.


In summary, the researchers observed robust antibody responses using SARS-CoV-2 spike S1 subunit vaccines in mice. All vaccinated animals displayed strong binding IgG responses against Wuhan, Delta, and Omicron S1. Specifically, the recipients of the trivalent vaccine exhibited cross-reactive antibodies inhibiting ACE2-spike with an increased breadth compared to their monovalent counterparts.

These results indicated that increasing the valency of COVID-19 vaccines might not necessarily attenuate the magnitude of immune responses against individual variants. The findings corroborate the use of the trivalent COVID-19 vaccine incorporating the spike S1 subunit of SARS-CoV-2 Wuhan, Delta, and Omicron variants to increase antigenic coverage and augment immune responses.

*Important notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information

Journal reference:
  • Khan, M. et al. (2022) "Trivalent SARS-CoV-2 S1 Subunit Protein Vaccination Induces Broad Humoral Responses in BALB/c Mice". bioRxiv. doi: 10.1101/2022.12.12.520124.

Posted in: Medical Science News | Medical Research News | Disease/Infection News

Tags: ACE2, Angiotensin, Angiotensin-Converting Enzyme 2, Antibodies, Antibody, Antigen, Assay, Coronavirus, Coronavirus Disease COVID-19, covid-19, ELISA, Enzyme, Homologous, Immunization, Immunoassays, Immunoglobulin, Mortality, Omicron, Protein, Respiratory, SARS, SARS-CoV-2, Severe Acute Respiratory, Severe Acute Respiratory Syndrome, Syndrome, Vaccine, Virus, Western Blot

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Tarun Sai Lomte

Tarun is a writer based in Hyderabad, India. He has a Master’s degree in Biotechnology from the University of Hyderabad and is enthusiastic about scientific research. He enjoys reading research papers and literature reviews and is passionate about writing.

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