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Principal Investigator: John D Mountz
Organization: BIRMINGHAM VA MEDICAL CENTER
Fiscal Year: 2021
Funding agency: Veterans Affairs
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-β (IFN-β) 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-β 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-β 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-β 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.
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