The COVID-19 caused by the SARS-CoV-2 coronavirus infection is currently a global pandemic. The most important question is why some individuals produce rapid protective antibodies (Abs) and exhibit null or only mild symptoms, whereas others do not? A second urgent question is if a standard laboratory test can be developed to stratify subjects who would be most likely to develop rapid long-lived B cells that could produce protective Abs after infection or after vaccination? Based on the understanding of efficient Ab responses to other coronavirus, an uncomplicated infection would promote a response to the spike (S-protein) as well as to the viral nucleoprotein (vNP). This proposal will focus on the role of interferon-? (IFN-?) that enhances the B cell Ab response. Our previous studies in autoimmune systemic lupus erythematosus (SLE) patients have shown that high levels of B cell endogenous IFN-? enables development of a B cell repertoire skewed toward recognition of an endogenous ribonuclear protein (RNPs) which we propose then form the immune repertoire that is poised to respond to vNPs. In the case of SARS-CoV-2, the vNP is a positive strand RNA complex with a nuclear protein very similar to the natural repertoire that develops in humans. For the 2003 SARS-CoV, vNP was detected in multiple body fluids during early infection and IgG anti-NP has been shown to be long-lived and elevated in recovered patients. We have shown that type I IFN promotes long-lived plasma B cell development including T-cell mediated development of high affinity antibody to a neo-antigen. A similar mechanism is proposed for development of protective Ab response to the S-protein of SARS-CoV-2. At a mechanistic level, we propose that the high affinity response to S-protein also requires enhanced single stranded RNA-induced toll-like receptor 7 (TLR7) signaling and follicular T-cell cytokines especially type II IFN to propel B cell into long-lived plasma cells (PCs).
Specific Aim 1 will determine if B cells from COVID-19 convalescent subjects with high endogenous IFN-? produce a strong anti-vNP and S-protein response. This will be investigated using fluorescent labeling of antigen strategy to identify vNP and S-protein-specific B cells and to phenotype these B cells by flow cytometry analysis.
Specific Aim 2 will utilize fluorescent and nucleotide barcoded vNP and S-protein to enable analysis by 5' 10X Genomics sequencing to determine the program of type I and type II IFN response pathways that are associated with development of B cells producing protective Abs. These studies are made feasible by our past studies of B cell Ab development and responses from recovered subjects that exhibit high endogenous IFN-? and produce antibodies to vNP or S-proteins. Cloning, sequencing, production and testing of the Ab produced by the vNP and S-protein specific B cells will be carried out using 5' 10X Genomics and cloning of heavy and light chain regions into an expression vector by TWIST Biotechnologies. An outstanding team of investigators from the Birmingham VAMC has been assembled including Dr. John Mountz, with expertise in B cell development and Dr. John Kappes, with expertise in virology. We have recruited scientists with expertise in single-cell analysis and immunology to assist with the project. The studies are highly significant since they will lead to a better understanding and stratifications of subjects who did and did not generate an efficient Ab response of the SARS-CoV-2 virus after infection and to determine the underlying immune landscape required for an efficient response. The studies will be important in the design of vaccines to understand the combined role of the vNP and S-protein of SARS-CoV-2, as well as adjuvant that can promote an efficient interferons and TLR7-mediated B cell response. The studies will also have immediate applications to potential immunomodulatory therapy related to development of the anti-viral response such as a potential mechanism of hydroxychloroquine which can modulate TLR7 signaling and other aspects of viral processing upon entry into cells. Other immediate applications would be to guide development of both innate and adaptive immune responses to boost and enable a long-term B cell immunity.
It is projected that recurrent wintertime outbreaks of SARS-CoV-2 will probably occur after the initial, most severe pandemic wave. To avoid overload of critical care capacities, prolonged or intermittent social distancing may be necessary into 2022 or after. Even in the event of apparent elimination, SARS-CoV-2 surveillance should be maintained since a resurgence in contagion could be possible as late as 2024. Longitudinal serological studies are urgently needed to determine the extent and duration of immunity to SARS-CoV-2. This proposal intends to determine the type of antibodies that will provide the most effective immunity for both immediate and long-term protection to the host. Cutting-edge technologies will be applied to determine the host immune mechanisms that lead to the development of these antibodies. Results generated from the proposed studies should provide an important clue to identify immune phenotypes in protected hosts and also help develop effective vaccination to prevent future infection.