key: cord-0913271-j799l55x
authors: Zhang, Ying; Li, Dandan; Zhao, Heng; Wang, Lichun; Liao, Yun; Li, Xueqi; Mou, Tangwei; Li, Qihan
title: The role of multiple SARS-CoV-2 viral antigens in a vaccine-induced integrated immune response
date: 2021-03-22
journal: Vaccine
DOI: 10.1016/j.vaccine.2021.03.067
sha: 35e8f04653441fb2a7a8b6eae38e1a4e5bedebca
doc_id: 913271
cord_uid: j799l55x

nan

The global pandemic caused by the new coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which emerged at the end of 2019, has led to a public health crisis in which more than 30 million people have been infected and 1 million have died [1, 2] . Accelerated vaccine and drug development in different countries and regions (accessed at ClinicalTrials.gov as of Oct. 10, 2020) resulted in the rapid entry of several different types of vaccines into clinical trials [3] . Shortly thereafter, a series of clinical data were obtained, especially immunogenic data [4] [5] [6] .

These studies provide a basis for the development of effective vaccines, but at the same time, they have raised a series of questions regarding the mechanism underlying the immunological response to infection with SARS-CoV-2, the differences in immunogenicity among various types of vaccines and the different immune responses they induce [7] . Our understanding of SARS-CoV-2 is based solely on the limited studies on other members of the coronavirus family [7] . Therefore, the characteristics of the immune response to infection with SARS-CoV-2 should serve as the theoretical basis for the identification of a suitable vaccine antigen. SARS-CoV-2 contains four major structural proteins, namely, spike (S), membrane (M), envelope (E) and nucleocapsid (N). Among these four proteins, S is being used as the leading target antigen in vaccine development because it is responsible for recognition of the host cellular receptor to initiate virus entry. In contrast, N protein packages the viral RNA to form a helical capsid and is essential for virus viability.

Although N proteins are highly immunogenic and are expressed abundantly during infection of many coronaviruses family members [8] , their complicated role in 3 inducing an immune response is unable to judge its significance as a major target antigen for vaccine. Studies of SARS-CoV and avian coronavirus infectious bronchitis virus showed that N antibodies are not neutralizing nor protecting [9] [10] [11] ; however, N antibodies or N-specific T cell epitopes have also been reported to protect animals from infection by mouse hepatitis virus, IBV, SARS-CoV and MERS-CoV [12] [13] [14] [15] . There are also reports showing that although the crystal structure of the SARS-CoV-2 N protein is similar to that of the SARS-CoV N protein, their surface electrostatic potential characteristics are distinct [16] . These findings might suggest a complicated role of N proteins in different coronavirus infections.

With a microneutralization assay using SARS-CoV-2 live virus and an ELISA kit coated with recombinant S protein, N protein and native purified viral antigen, we were able to detect not only varied titers of neutralizing antibodies (1:16-1:256) but also high titers of anti-S, anti-N and anti-whole-virion antibodies (Fig. 1ab) . The presence of these antibodies in the convalescent sera of COVID-19 patients suggests the stimulation of the immune system by various antigen components of SARS-CoV-2 throughout the disease course. This raises the question of whether the antibodies against other viral antigens, especially the N protein as the major nucleocapsid protein, function in the antiviral immune response. The antiviral effect of the anti-N antibody was further observed in a rhesus monkey immunization challenge test using a specially prepared inactivated vaccine containing exposed structural protein components of SARS-CoV-2. In rhesus macaques immunized with two doses of 200, 100 and 20 EUs (ELISA units, the viral antigen concentration determined by ELISA) 4 inactivated vaccine with a 14-day interval, the neutralizing antibody reaction showed a dose-dependent effect of the antigens, and most monkeys were positive on ELISA for the anti-S, anti-N and anti-whole-virion antibodies (Fig. 1cd) . The geometric mean titer (GMT) of the neutralizing antibody was 120 in the 200-EU antigen group (N=4), 32 in the 100-EU antigen group (N=3) and 2 in the 20-EU antigen group (N=3).

Although the GMT of the neutralizing antibody in two animals was less than 1:4, most animals in the three dose groups had high titers of anti-S, anti-N and anti-whole-virion antibodies from ELISA detection. Further challenge test suggested that all animals showed immune protection upon viral challenge via the nasal route 2 weeks after immunization, in which, viral shedding from the nasal cavity, pharynx and fecal content of immunized animals were found decreasing below the detection limit from 10 4 copies/100ul after 48 hours, while those in the positive control were maintained at 10 3-4 copies/100ul during 7-15 days (Fig. 2a) . The detection of viral loads in tissues and organs, especially the respiratory system of animals sacrificed at different time points, also clearly confirmed these findings (Fig. 2b) . These results suggest that, at least in protective immunity studies of inactivated vaccines in rhesus monkeys, the protective efficacy of vaccines does not seem to depend solely on the titer of the neutralizing antibodies but instead might be related to the presence of various types of antibodies in the immunized sera, including anti-N and anti-S antibodies. Based on these findings, we proceeded with a random, double-blinded and control phase 1 clinical trial [17] , in which high titers of anti-S, anti-N and anti-whole-virion antibodies associated with the neutralizing antibody raising in low-, medium-and high-dose groups immunized with 0/14-day procedure and the 0/28-day procedure were observed. Combined with the analysis of neutralizing antibody components in the convalescent sera of COVID-19 patients, these data seem to suggest a theory regarding the viral antigen stimulation of the immune system over the course of an infection with SARS-CoV-2: As we know, the virus infects the human body through the respiratory tract, which means that the binding of the viral S protein RBD to the surface receptor ACE2 on respiratory tract epithelial cells is the first step in initiating the viral infection [18] , and, this binding mediates viral entry into the host cell followed by the separation of the S protein into the S1 and S2 subunits [19] . The S2 subunit mediates the fusion between the viral envelope and the host cell membrane, allowing the virus nucleocapsid to enter the cell without the envelope [20] . This fact suggests that the nucleocapsid antigen (b) Viral loads in respiratory system (trachea, lung), lymph nodes (pulmonary lymph nodes, tracheal lymph nodes and cervical lymph nodes), nervous system (brain, midbrain, cerebellum, cervical spinal cord, thoracic spinal cord, lumbar spinal cord) and other major organs (heart, liver, spleen, kidney and small intestine) of 

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This work was supported by the National Key R&D Program of China (2020YFC0849700 and 2020YFC0860600), the Program of Chinese Academy of Medicine Science (2020HY320001) and the Major Science and Technology Special Projects of Yunnan Province (202003AC100003 and 202003AC100009).

The sponsor of the study played no role in the study design, data and sample collection, data processing, or report writing. The corresponding author had full access to all the data generated by the study and takes full responsibility for the final 7 submission for publication.

The sponsor of the study played no role in the study design, data and sample collection, data processing, or report writing. The corresponding author had full access to all the data generated by the study and takes full responsibility for the final submission for publication.