*Important notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

A new study posted in the preprint server bioRxiv* aimed to analyze the qualitative and quantitative impact of spike (S)-induced humoral immunity along with its antibody-dependent cellular cytotoxicity (ADCC) capability in individuals who received a vaccination, recovered from an infection, or both.

Background

Infection and vaccination using any prevalent globally used coronavirus disease 2019 (COVID-19) vaccines can induce humoral immunity against the severe acute respiratory syndrome 2 (SARS-CoV-2 spike) (S) protein. Anti-S antibodies (Ab) can target several regions within the protein. However, most of the focus is on those that neutralize cell-free virions. They bind with the receptor binding domain (RBD) or the N-terminal domain (NTD) in a few cases.

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Neutralizing antibodies can block or prevent the binding of SARS-CoV-2 and the entry receptor angiotensin converting enzyme-2 (ACE-2), as well as prevent post-binding events that are essential for virus entry. They are considered to play an important role in reducing SARS-CoV-2 transmission and, thereby, important for predicting COVID-19 vaccine efficacy.

However, neutralizing Abs has several limitations, including a limited number of neutralizing epitopes. SARS-CoV-2 variants of concern can escape the neutralizing activity of Abs induced by the ancestral S antigen after three years of evolution in the human population. This results in the reduction of the efficacy of vaccines within a few months of their introduction. Moreover, SARS-CoV-2 can undergo direct cell-cell transmission post-infection, which further reduces the efficacy of neutralizing Ab.

To reduce the spread of the virus, Abs that recognize viral antigens on the surface of infected cells are required instead of those neutralizing cell-free virions. They recruit effector cells, such as natural killer (NK) cells, that can kill infected cells through antibody-dependent cellular cytotoxicity (ADCC), which helps control cell-associated viruses.

Previous research has indicated that infection with SARS-CoV-2 induces Abs capable of supporting ADCC. Studies have also reported that Ab, capable of ADCC, prevents disease in animals even without neutralizing activity. Therefore, maximizing and inducing this activity through vaccination can be very important.

Moreover, expression of SARS-CoV-2 S also takes place on the infected cell surface, where it can be efficiently bound by Abs. This makes S an effective target for Fc receptor (FcR)-mediated NK cell activation, thereby making a single vaccine antigen capable of inducing Abs that can target both cell-associated and cell-free viruses. Additionally, studies have reported that infection with SARS-CoV-2 before vaccination with S encoding vaccine can provide greater protection than infection or vaccination alone.

About the study

The study involved the collection of peripheral blood samples from participants after infection or vaccination. Enrollment of most participants took place after the first COVID-19 wave, where the ancestral Wuhan-Hu-1 SARS-CoV-2 caused infections. Information on comorbidity or any undergoing medical treatments was collected from the participants.

Recombinant lentiviral vector expression systems introduced Wuhan-Hu-1, Delta (B.1.617.2), or Omicron (BA.1) S sequences into the human lung fibroblast MRC-5 cell line. Thereafter, antibody-dependent cell-mediated killing assays, virus neutralization assay, and serological testing was carried out.

Depletions of specific Abs from sera were carried out using magnetic bead conjugated proteins based on either the entire S1, entire S2, NT, or RBD domain of SARS-CoV-2 Wuhan-Hu-1 S. ELISA was carried out to determine the individual overlapping peptides that spanned the Wuhan-Hu-1 S sequence.

Study findings

The results indicated that S-specific ADCC responses were weaker among participants who were previously infected and vaccinated but were not infected than those with hybrid immunity. Greater than 10 percent lysis was observed among those with hybrid immunity. Anti-S Abs produced by a hybrid combination of infection followed by vaccination was observed to mediate S-specific Ab-dependent NK cell activity (ADNKA) against infected or transduced cells. Moreover, hybrid immunity was reported to boost S-specific ADNKA to levels similar to the potent multi-antigen ADNKA response of individuals who recovered from mild infection but not those who recovered from severe infection.

ADNKA and neutralization were observed to show positive correlations between infected people and those who received the vaccine. However, the relationship between the two was quite different. Infection was observed to show higher levels of ADNKA as compared to vaccination for any neutralization level. ADCC was reported to be correlated with increased anti-full length S (FLS) IgG and IgG3 Ab levels in both the vaccine and hybrid cohorts. Vaccines were reported to elicit a much weaker ADCC than the hybrid cohort.

The levels of anti-FLS Abs increased after the first vaccination but remained unchanged after the second vaccination for the hybrid cohort. On the contrary, the Ab levels increased significantly after the second vaccination for the vaccine cohort but remained quite low. Moreover, selective increases in anti-S1 IgG Ab levels were reported after vaccination, while higher levels of anti-S2 IgG Abs were reported after infection.

Anti-S1 IgG3 Ab levels were reported to be low after infection but increased after the first vaccination. Anti-S2 IgG3 Abs were higher after infection, and vaccination further improved their levels. Anti-S2 IgG3 Abs levels were reported to be higher r than anti-S1 IgG3 Abs for people with hybrid immunity. Additionally, Ab depletion indicated that a combination of Abs targeting S1 and S2 is important for robust ADNKA. ADNKA induced by hybrid immunity was observed to be dependent on the presence of Abs reactive to both the S1 and S2 regions.

Furthermore, Ab reactivity for three regions along S was reported to be enriched for the hybrid cohort. These regions were contained within the C-terminal domain (CTD) 1 and CTD2 of S1 as well as a region in S2 that was located upstream of the heptad repeat 2 (HR2) sequence in the connector domain (CD). These regions were reported not to show any variation between the Delta or Omicron variants. Moreover, increasing anti-IgG Ab levels specific for each of the three regions was reported to increase ADCC. Finally, overexpression of S, thereby targeting a broader range of epitopes, resulted in only a small decline in ADNKA against Omicron-infected cells.

Conclusion

The current study indicates that hybrid immunity can elicit higher ADCC compared to infection or vaccination alone. Moreover, hybrid immunity can generate an Ab response which retains activity against variant strains to a greater extent than neutralization.

Therefore, strong ADCC can play a role in protection through hybrid immunity, help in the identification of the role of S2-targeted Abs, as well as suggest that inducing Abs that are targeted broadly across spikes can be effective in determining newer and more robust vaccine strategies.

*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

Suchandrima Bhowmik

Suchandrima has a Bachelor of Science (B.Sc.) degree in Microbiology and a Master of Science (M.Sc.) degree in Microbiology from the University of Calcutta, India. The study of health and diseases was always very important to her. In addition to Microbiology, she also gained extensive knowledge in Biochemistry, Immunology, Medical Microbiology, Metabolism, and Biotechnology as part of her master's degree.