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Next-generation SARS-CoV-2 subunit vaccine candidate elicits powerful immunity in preclinical trials

While over a score of vaccines against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been rolled out in different countries, the emergence of new variants with partial resistance to antibodies elicited by earlier variants poses an ongoing challenge to the effort to control the spread of the virus. New research – by scientists at the University of Chicago, Johns Hopkins School of Medicine and the University of Iowa in the USA – describes a novel vaccine candidate that has achieved impressive immunologic results in mouse studies.

A pre-print version of the study is available on the bioRxiv* server, while the article undergoes peer review.

Vaccine platform concept

The new vaccine prototype uses a co-polymer comprising both the carbohydrate molecule, mannose, and a toll-like receptor 7 (TLR7) agonist in the form of an imidazoquinoline molecule. This has been shown to be a potent adjuvant for egg albumin and malarial parasite proteins.

Represented as p(Man-TLR7), this copolymer is combined with the SARS-CoV-2 antigen, either the whole spike (stabilized in its prefusion form) or just the receptor-binding domain (RBD), on a single macromolecular platform.

The antigen-conjugated form will release the antigen intact once it enters the DC. The DCs will take up and present the antigen and activate effector immune cells.

The conjugated spike and RBD antigens bound the ACE2 receptor, though at half the levels of the native antigens, perhaps because of steric hindrance or because some of the surface lysine residues are modified.

Advantages

The advantages of this platform are the ability of mannose to bind to C-type lectins, a type of sugar molecule that targets dendritic cells (DCs). These cells are specialized to present antigens to immune cells for antibody production and the destruction of the infected cell.

With this molecule, not only are DCs activated to present the viral antigen to the effector cells, but the presence of the TLR7 agonist acts as a powerful booster of the antigen response.

The conjugated antigen-activated bone marrow-derived DCs (BMDCs) produce cytokines IL-12p70, IL-6, and TNFα, which stimulate the immune system, unlike the native antigens.

Robust humoral and cellular protection in adult mice

Secondly, these induced a robust neutralizing antibody response in adult mice vaccinated with two doses of the antigen.

The antibody titers were higher than with the native RBD and more than those found in convalescent serum. However, they were less than with RBD+, the commonly used alum adjuvant AS04+ monophosphoryl lipid A (MPLA). Alum is a benchmark depot-forming adjuvant.

The immune T cell response was Th1-skewed, with high IgG2b levels compared to IgG1, and anti-RBD IgA levels were also noted. IgG2b antibodies have powerful antiviral activity, while RBD-specific IgA can neutralize the virus rapidly.

Plasma containing these antibodies was capable of preventing SARS-CoV-2 infection of cells in culture, unlike antibodies from mice immunized with native RBD.

Alum-adsorbed Spike-p(Man-TLR7) antigen

Both Spike-p(Man-TLR7) and the AS04 alum-MPLA-adjuvanted RBD formulation showed comparable in vitro neutralization results. Indeed, all adjuvanted formulations of the spike, with or without alum, exceeded the required threshold for virus neutralization titer (VNT) set by the USA’s regulatory body, the Food and Drug Administration (FDA).

Similar responses were seen with the adjuvanted Spike-p(Man-TLR7) antigen following vaccination. Even higher responses were obtained when alum was added, with or without MPLA.

However, the best Th1-skewed response was seen with the Spike-p(Man-TLR7) antigen. This antigen also induced high anti-spike IgA antibodies, though even higher titers were elicited by spike-AS03 alum-MPLA antigen.

Both these formulations also led to elevated numbers of antibody-secreting cells (ASCs) producing spike-specific antibodies.

Robust antibody response in elderly mice

The antigen also appeared to induce high anti-spike IgG antibodies in elderly mice, just as in adult mice, from four weeks after the first dose to 12 or more weeks afterward. Again, this reaction becomes more robust when alum is added to the formulation.

Again, the spike-Spike-p(Man-TLR7) vaccine-elicited antigen-specific germinal center B cells and expansion of T follicular helper (Tfh) cells in the spleen and the draining lymph nodes.

Advantages of alum addition

The alum-Spike-p(Man-TLR7) antigen led to antibodies that recognized even more RBD epitopes, including the ACE2-binding site than those elicited by spike-alum-MPLA formulations alone.

Adsorbing Spike-p(Man-TLR7) to the depot-forming adjuvant alum, amplified the broadly neutralizing humoral responses to levels matching those in mice vaccinated with formulations based off of clinically-approved adjuvants.”

B cell and Tfh cell responses were also enhanced when alum was incorporated into the formulation.

Th1 cytokines, specifically IFNγ and IL-2, were produced in the spleen after vaccination with Spike-p(Man-TLR7), with or without alum, on re-exposure to the spike protein. Both CD4+ and CD8+ T cells were activated by the resulting Th1 cytokines induced by these antigens.

Th2 cytokines were higher when the alum-containing formulation was used. However, the response remained strongly biased towards a Th1-functional T cell response, irrespective of the presence of alum.

What are the implications?

These findings show that durable neutralizing responses can be achieved using this adjuvanted spike or RBD formulation for vaccination, unlike with natural infection. The larger antigen is preferred, firstly because it is less likely that the copolymeric adjuvant will completely hide the neutralizing epitopes, and secondly because it elicits a broader range of neutralizing epitopes.

The additional use of alum, which is the gold standard for vaccine adjuvants, boosted B cell expansion in the lymph nodes but not the spleen, indicating that alum keeps the vaccines near the vaccination site. Still, without it, systemic spread of the antigen tends to occur.

This could further enhance the efficacy of this platform, as the antibody titer, neutralizing ability, and breadth of epitope recognition, are alike boosted by the addition of alum.

However, alum may shift the T cell responses towards a Th2 bias, which is undesirable. Further studies are probably warranted to evaluate the utility of alum in this formulation.

The new study is noteworthy for many reasons. Firstly, it describes a candidate vaccine that protects against infection with SARS-CoV-2, and thus can prevent severe COVID-19. It will elicit high neutralizing antibody titers to block virus-host cell interactions and cell entry by the virus.

In addition, it will elicit high levels of cytotoxic and memory T cell responses that will promote viral clearance and durable immunity. This cellular immune response is biased towards T helper cell type 1 (Th1) rather than Th2, which promotes antibody-dependent enhancement of disease (ADE) of the lungs, as opposed to better antiviral responses with Th1 responses.

Lastly, the vaccine effectively induces immune responses in those above 65 years, even with the weaker vaccine efficacy often seen in this age group.

The platform is so elegantly simple in its design that any antigen containing an amine group may be reversibly conjugated to the p(Man-TLR7). It is therefore versatile enough to easily produce new vaccine variants as viral strains mutate. It can also be applied across species.

Not just the TLR7, but instead the TLR8 agonist, can be used, as both are required for strong cellular immune responses.

However, the need for intracellular processing to release the antigen from the polymer may limit its utility in the case of smaller antigens, as fewer epitopes are present. This increases the probability that the neutralizing epitope may be blocked following conjugation with the copolymer adjuvant.

Notwithstanding, this study adds a potentially important new tool to the field of subunit vaccine development.

*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.

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