Efficacy and safety of an RBD-based recombinant protein COVID-19 vaccine

The emergence of the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led the scientific community and pharmaceutical industry to focus on the development of vaccines to combat the health emergency.

Study: Preclinical efficacy, safety, and immunogenicity of PHH-1V, a second-generation COVID-19 vaccine candidate based on a novel recombinant RBD fusion heterodimer of SARS-CoV-2. Image Credit: Tero Vesalainen/Shutterstock

Several vaccines are currently available against COVID-19, and more than 7.55 billion doses have been administered globally.

However, COVID-19 cases continue to emerge due to the evolution of multiple SARS-CoV-2 variants, the lack of homogeneous distribution of the vaccines, and the decline in immunological protection by the current vaccines. Therefore, it is important to develop second-generation vaccines that are effective against the SARS-CoV-2 variants and can be further used as a booster.

Background

SARS-CoV-2 is a novel beta coronavirus belonging to the subfamily Coronovirinae within the family Coronaviridae. The genome of SARS-CoV-2 encodes at least four structural proteins: spike (S) glycoprotein that promotes entry of the virus inside the host cell, membrane (M) protein that is responsible for shaping the virions, nucleocapsid (N) protein that is involved in genome packaging and envelope (E) protein that is responsible for virion assembly and release.

The S glycoprotein is the primary target of viral neutralizing antibodies and is the main candidate for vaccine development since it binds to the host receptor angiotensin-converting enzyme 2 (ACE2). The S glycoprotein consists of two domains S1 and S2 that allow binding of the viral particle and promotes cellular entry by fusion with the host cell membrane.

The receptor-binding domain (RBD) containing a highly immunogenic receptor binding motif (RBM) that interacts with ACE2 and neutralizes antibodies is located in the S1 domain. Therefore, most of the key mutations occur in the RBM, leading to the emergence of variants.

Two proline substitutions in the original S protein sequence (S-2P) of MERS-CoV, SARS-CoV, and HKU1 coronavirus are involved in maintaining the antigenic conformation. The mRNA-based vaccines and adenoviral vaccines are developed based on this S-2P design.

Although adjuvanted protein-based vaccines are considered an important type of vaccine, their development has lagged due to the need to optimize the manufacturing process for each antigen. Two of the most common subunit vaccines are the Novavax vaccine candidate and the Sanofi-GSK vaccine candidate.

Recombinant protein vaccines have several advantages: no risk of genome integration, an adequate safety profile, suitable for people with compromised immune systems, high productivity, and good stability.

A new study published in the pre-print server bioRxiv* developed a protein-based subunit vaccine, PHH-1V, that consisted of a recombinant RBD fusion heterodimer of the B.1.1.7 (alpha) and B.1.351 (beta) variants of SARS-CoV-2 produced in Chinese Hamster Ovary (CHO) cells with an oil-based adjuvant equivalent to MF59C.1. The study assessed the safety and efficacy of the PHH-1V vaccine in transgenic mice and characterized the RBD fusion heterodimer antigen and its immunogenicity.

About the study

The study involved the production of the viral antigen and its purification. The purified RBD fusion heterodimer was formulated with an oil in water adjuvant. The PHH-1V vaccine was tested at different concentrations: 0.04 µg, 0.2 µg, 1 µg, 5 µg, and 20 µg of RBD fusion heterodimer/dose for safety assay in BALB/c mice. The vaccine was tested at 10 µg and 20 µg of fusion heterodimer/dose in K18-hACE2 mice for efficacy assessment.

Seventy-two of the five-week-old BALB/c mice involved in the study were divided into six groups for safety and immunogenicity assays. Group A was the control, group B was immunized with 0.04 μg recombinant protein RBD fusion heterodimer/dose, group C was 0.2 μg dose, group D were immunized with 1 μg dose, group E was immunized with 5 μg dose, and group F immunized with 20 μg dose.  

62 K18- humanized ACE2 (hACE2) mice which were four/five-week-old were divided into four groups for assessment of vaccine efficacy. All the mice were vaccinated and, after that, challenged with SARS-CoV-2. Group A was the control group, group B was infected with SARS-CoV-2 but received the placebo, group C was vaccinated with 10 μg/dose of recombinant protein RBD fusion heterodimer and infected with SARS-CoV-2, and group D was vaccinated with 20 μg/dose and infected with SARS-CoV-2.

Furthermore, analysis of serum binding SARS-CoV-2 specific antibodies and SARS-CoV-2 neutralizing antibodies was carried out. Following this, intracellular cytokine staining (ICS) along with mouse cytokine assay and IFN-ɣ and IL-4 ELISpot assays.

RT-qPCR was done to determine the viral load in respiratory tissue samples, followed by virus titration using TCID50 assay. Finally, histopathological analysis was carried out using the upper and lower respiratory tract and brain tissues.

Study findings

The results indicated that after the prime-boost immunization with the PHH-1V, higher titers of RBD antibodies were observed in all groups except the control. However, no significant difference in IgG response was observed among the groups that were immunized with more than 1 µg of recombinant RBD fusion heterodimer antigen in BALB/c mice. Also, in K18-hACE2 mice, both the groups vaccinated with either 10 or 20 µg of recombinant heterodimer showed similar antibody titers.

Prime-boost immunization of groups C to F induced higher neutralizing antibody titers against the S protein of the alpha variant. In contrast, no neutralizing antibody response was observed in group B. However, the mean neutralizing antibody titers observed in groups C to E remained the same. Vaccination with 20 µg of RBD fusion heterodimer antigen (group F) induced higher neutralizing titers as compared to groups C to E. High neutralizing titers were obtained against all the SARS-CoV-2 variants (alpha, beta, gamma, delta) from the sera obtained from group F.

The results also indicated activation of CD4+ and CD8+ T cells expressing IFN-γ and IL-2 upon stimulation with an RBD peptide pool in group F. Additionally, higher levels of IL-2, IL-5, and TNF-α were observed in group E. In comparison, higher levels of IL-5 and TNF-α was observed in group F. Also, a balanced Th1/Th2 was observed in group F as compared to group E.

The results showed that the PHH-1V vaccine candidate was safe in mice since it did not cause any clinical symptoms or bodyweight loss in either immunized BALB/c or K18-hACE2 mice. However, mild lesions were observed in a few mice due to local innate immune response. Additionally, immunization with either 10 or 20 µg of PHH-1V was found to reduce the viral load detected by RT-qPCR in the mice's lungs, nasal turbinate, and brain.

Therefore, the current study indicated that using the PHH-1V vaccine is safe in mice and leads to the development of RBD-binding and neutralizing antibodies. Also, besides safety and efficacy, this second-generation vaccine could be effective against emerging SARS-CoV-2 variants. In this regard, the PHH-1V vaccine candidate has shown promising preclinical data and is currently being evaluated in a Phase I/IIa clinical trial.

*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