Do anti-nucleocapsid antibodies affect COVID-19 immune reactions?

Coronavirus disease 2019 (COVID-19) first emerged in Wuhan, China, and has since spread worldwide. The rapid transmission rate forced many countries to enact costly and restrictive measures in the hopes of controlling the disease, including mandatory face masks, social distancing laws, and even full lockdowns and stay-at-home orders.

The introduction of vaccines allowed these to be dismantled in many countries, but with new variants continuing to evade both vaccine-induced and natural immunity, more research is required to ensure long-term protection.

Researchers from Northwestern University have been investigating the potential for vaccines and treatments against the nucleocapsid protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

A preprint of the paper can be found on the bioRxiv* preprint server while the study undergoes peer review.

The study

SARS-CoV-2 has four major structural proteins – the spike protein, nucleocapsid protein, membrane protein and envelope protein. The spike protein is key to the pathogenicity of the disease, as following cleavage by a host protein, the S1 subunit binds to angiotensin-converting enzyme 2 (ACE2) to permit viral cell entry, while the S2 subunit mediates membrane fusion.

This is the target of most monoclonal antibody treatments as well as most vaccines, but it is also a highly mutable region that shows significant differences in conformation between different variants. The nucleocapsid protein is not currently targeted by any approved vaccine, and testing for anti-nucleocapsid antibodies tends to be used to test for past infection in vaccinated individuals. There is increased interest in developing vaccines against alternate proteins to reduce the impact of new variants.

Initially, C57BL/6 mice were primed by intramuscular injection with an adenovirus serotype 5 vector expressing the nucleocapsid protein from SARS-CoV-2 at a dose of 10^11 PFU. A 100ug booster of nucleocapsid protein was provided three weeks later. Control mice were immunized with a vector with no nucleocapsid protein and a PBC booster. Nucleocapsid-specific immune response was measured two weeks after the booster, revealing robust responses in CD8 T cells, memory B cells and antibodies. To help confirm the effects, these tests were followed using focus reduction neutralization titres (FRNT) using live virus. Mice that had received a spike-based adenovirus vaccine were used as a control. The sera from mice who received the spike protein-based vaccine successfully prevented infection, but unfortunately there was no antiviral effect from the nucleocapsid based vaccine.

Neutralization is not the only way immune response can help protect against virus-based disease, however, and to investigate if non-spike immune response could help combat COVID-19 through alternate mechanisms the researchers performed a passive immunization study. They transferred 500ul of nucleocapsid-specific sera into otherwise naïve transgenic mice expressing hACE2. Following the transfer, the mice were challenged intranasally with 10^3 PFU of SARS-CoV-2 and quantified the viral load in lungs on day four. The mice who received the injections showed a significantly lower viral load than the control mice.

The conclusion

Further study could attempt to identify the specific mechanism by which anti-nucleocapsid immune response helps protect against the disease, potentially by examining the effects of CD8 T cells or memory B cells on disease progression in isolation, rather than the general immune response.

Nevertheless, the research presented here provides strong evidence that anti-nucleocapsid protein can help protect the host against SARS-CoV-2 infection, as well as showing that neutralizing the nucleocapsid protein does not block infection.

While a previous study suggests that a nucleocapsid-based vaccine was effective to a limited effect, the authors explain this as a difference in dosage – the mice in this study were given a typical physiological dose of 10^3 PFU, whereas the previous study used a much higher dose – 5×10^4.

It is important to examine the effect of the immune response against proteins other than the spike protein, as emerging variants have continued to show significant mutability and changes in conformation that reduce the effect of anti-spike treatments and vaccinations. This information could help future vaccine manufacturers and researchers looking into alternate therapies against COVID-19.

*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.

Written by

Sam Hancock

Sam completed his MSci in Genetics at the University of Nottingham in 2019, fuelled initially by an interest in genetic ageing. As part of his degree, he also investigated the role of rnh genes in originless replication in archaea.