Thu. Jan 20th, 2022

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

Study: Preclinical efficacy, safety and immunogenicity of PHH-1V, a second generation COVID-19 vaccine candidate based on a new recombinant RBD fusion heterodimer of SARS-CoV-2.  Image credit: Tero Vesalainen / ShutterstockStudy: Preclinical efficacy, safety and immunogenicity of PHH-1V, a second generation COVID-19 vaccine candidate based on a new 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 development of more SARS-CoV-2 variants, the lack of homogeneous distribution of the vaccines, and the decline in immunological protection of current vaccines. Therefore, it is important to develop second-generation vaccines that are effective against the SARS-CoV-2 variants and that can be further used as a booster.

Background

SARS-CoV-2 is a new 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, which promotes the penetration of the virus inside the host cell, membrane (M) protein, which is responsible for shaping the virions, nucleocapsid (N) protein, which is involved in genome packaging and envelope protein (E), which is responsible for virion collection and release.

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

The receptor binding domain (RBD), which contains 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 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 have been 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 appropriate safety profile, suitable for people with weakened 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, consisting of a recombinant RBD fusion heterodimer of 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 evaluated 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 ,g, 0.2 .g, 1 .g, 5 .g and 20 Rg RBD fusion heterodimer / dose for safety assay in BALB / c mice. The vaccine was tested at 10 μg and 20 μg fusion heterodimer / dose in K18-hACE2 mice to evaluate efficacy.

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 was 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 weeks old, were divided into four groups to assess the efficacy of the vaccine. All mice were vaccinated and then challenged with SARS-CoV-2. Group A was the control group, group B was infected with SARS-CoV-2 but received placebo, group C was vaccinated with 10 μg / dose 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.

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

RT-qPCR was performed to determine the viral load in airway tissue samples followed by virus titration using TCID50 assay. Finally, histopathological analysis was performed using the upper and lower airways and brain tissue.

Survey results

The results indicated that after the prime-boost immunization with 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 immunized with more than 1 μg of recombinant RBD fusion heterodimer antigen in BALB / c mice. In K18-hACE2 mice, both groups vaccinated with either 10 or 20 µg 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 Rg RBD fusion heterodimer antigen (group F) induced higher neutralizing titers compared to groups C to E. High neutralizing titers were obtained against all SARS-CoV-2 variants (alpha, beta, gamma, delta) from sera obtained from group F.

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

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

Therefore, the current study indicated that the use of the PHH-1V vaccine is safe in mice and leads to the development of RBD-binding and neutralizing antibodies. In addition to safety and efficacy, this second-generation vaccine may also be effective against new 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 message

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

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