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CD4 T follicular helper (Tfh) cells are important for the generation of durable and specific humoral protection against viral infections. The degree to which SARS-CoV-2 infection generates Tfh cells and stimulates the germinal center (GC) response is an important question as we investigate vaccine induced immunity against COVID-19. Here, we report that SARS-CoV-2 infection in rhesus macaques, either infused with convalescent plasma, normal plasma, or receiving no infusion, resulted in transient accumulation of pro-inflammatory monocytes and proliferating Tfh cells with a Th1 profile in peripheral blood. CD4 helper cell responses skewed predominantly toward a Th1 response in blood, lung, and lymph nodes. SARS-CoV-2 Infection induced GC Tfh cells specific for the SARS-CoV-2 spike and nucleocapsid proteins, and a corresponding early appearance of antiviral serum IgG antibodies. Collectively, the data show induction of GC responses in a rhesus model of mild COVID-19.

Antisense phosphorodiamidate morpholino oligomers (PMOs) were tested for the ability to inhibit gene expression in Escherichia coli. PMOs targeted to either a myc-luciferase reporter gene product or 16S rRNA did not inhibit luciferase expression or growth. However, in a strain with defective lipopolysaccharide (lpxA mutant), which has a leaky outer membrane, PMOs targeted to the myc-luciferase or acyl carrier protein (acpP) mRNA significantly inhibited their targets in a dose-dependent response. A significant improvement was made by covalently joining the peptide (KFF) 3 KC to the end of PMOs. In strains with an intact outer membrane, (KFF) 3 KC-myc PMO inhibited luciferase expression by 63%. A second (KFF) 3 KC-PMO conjugate targeted to lacI mRNA induced ␤-galactosidase in a dose-dependent response. The end of the PMO to which (KFF) 3 KC is attached affected the efficiency of target inhibition but in various ways depending on the PMO. Another peptide-lacI PMO conjugate was synthesized with the cationic peptide CRRRQRRKKR and was found not to induce ␤-galactosidase. We conclude that the outer membrane of E. coli inhibits entry of PMOs and that (KFF) 3 KC-PMO conjugates are transported across both membranes and specifically inhibit expression of their genetic targets.Phosphorodiamidate morpholino oligomers (PMOs) are DNA mimics that inhibit expression of specific mRNA in eukaryotic cells (1,17,22). They are synthesized by using the four natural bases, with a base sequence that is complementary (antisense) to a region of a specific mRNA. They are different than DNA in the chemical structure that links the bases together ( Fig. 1). Ribose has been replaced with a morpholine group, and the phosphodiester is replaced with a phosphorodiamidate. These alterations make the antisense molecule resistant to nucleases (11) and free of charges at physiological pH, yet it retains the molecular architecture required for binding specifically to a complementary strand of nucleic acid (21,22,24). PMOs are currently in phase I and II human clinical trials as therapeutics for restenosis and cancers.Recently, another type of antisense DNA mimic, called peptide nucleic acid (PNA), was shown to inhibit the expression of bacterial genes in vitro and in pure culture (8, 9). However, entry of PNA into Escherichia coli was inefficient because of the outer membrane of this gram-negative bacterium (10). Entry was greatly improved (7) by coupling PNA to cationic peptides that were previously shown to permeabilize the gramnegative outer membrane (18,25).In this report, we show that PMOs inhibit gene expression in E. coli but are limited for cellular uptake by the outer membrane. In addition, uptake across the outer membrane was achieved by coupling PMOs to the peptide (KFF) 3 KC. MATERIALS AND METHODSBacteria and growth conditions. E. coli SM105 [thr-1 araC14 tsx-78 ⌬(galKatt)99 hisG4 rfbD1 rpsL136 sylA5 mtl-1 thi-1] and its isogenic lpxA2(Ts) derivative SM101 were purchased from the E. coli Genetic Stock Center (New Haven, Conn.). E. coli BL...

An 11 base antisense PMO targeted to acpP significantly inhibited viability of a strain of E. coli with a normal, intact outer membrane both in pure culture and in infected mice. Inhibition by PMOs was sequence-specific.