The magnitude of the COVID-19 pandemic underscores the urgency for a safe and effective vaccine. Many vaccine candidates focus on the Spike protein, as it is targeted by neutralizing antibodies and plays a key role in viral entry. Here we investigate the diversity seen in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequences and compare it to the sequence on which most vaccine candidates are based. Using 18,514 sequences, we perform phylogenetic, population genetics, and structural bioinformatics analyses. We find limited diversity across SARS-CoV-2 genomes: Only 11 sites show polymorphisms in >5% of sequences; yet two mutations, including the D614G mutation in Spike, have already become consensus. Because SARS-CoV-2 is being transmitted more rapidly than it evolves, the viral population is becoming more homogeneous, with a median of seven nucleotide substitutions between genomes. There is evidence of purifying selection but little evidence of diversifying selection, with substitution rates comparable across structural versus nonstructural genes. Finally, the Wuhan-Hu-1 reference sequence for the Spike protein, which is the basis for different vaccine candidates, matches optimized vaccine inserts, being identical to an ancestral sequence and one mutation away from the consensus. While the rapid spread of the D614G mutation warrants further study, our results indicate that drift and bottleneck events can explain the minimal diversity found among SARS-CoV-2 sequences. These findings suggest that a single vaccine candidate should be efficacious against currently circulating lineages.

Infantile Pompe disease progresses to a lethal cardiomyopathy in absence of effective treatment. Enzyme replacement therapy (ERT) with recombinant human acid α-glucosidase (GAA) has been effective in most patients with Pompe disease, but efficacy was reduced by high titer antibody responses. Immunomodulatory gene therapy with a low dose adeno-associated virus (AAV) vector (2×1010 particles) containing a liver-specific regulatory cassette significantly lowered IgG, IgG1, and IgE antibodies to GAA in Pompe disease mice, when compared with mock-treated mice (p<0.05). AAV-LSPhGAApA had the same effect on GAA-antibody production whether it was given prior to, following, or simultaneously with the initial GAA injection. Mice given AAV-LSPhGAApA had significantly less decrease in body temperature (p<0.001) and lower anaphylactic scores (p<0.01) following the GAA challenge. Mouse mast cell protease-1 followed the pattern associated with hypersensitivity reactions (p<0.05). Regulatory T cells (Treg) were demonstrated to play a role in the tolerance induced by gene therapy as depletion of Treg led to an increase in GAA-specific IgG (p<0.001). Treg depleted mice were challenged with GAA and had significantly stronger allergic reactions than mice given gene therapy without subsequent Treg depletion (temperature: p<0.01; symptoms: p<0.05). Ubiquitous GAA expression failed to prevent antibody formation. Thus, immunomodulatory gene therapy could provide adjunctive therapy in lysosomal storage disorders treated by enzyme replacement.

Background IgE-mediated allergic reactions to cashews and other nuts can trigger life-threatening anaphylaxis. Proactive therapies to decrease reaction severity do not exist. Objectives We aimed to determine the efficacy of pepsin-digested cashew proteins used as immunotherapy in a murine model of cashew allergy. Methods Mice were sensitized to cashew and then underwent challenges with digested or native cashew allergens to assess the allergenicity of the protein preparations. Using native or pepsinized cashew proteins, mice underwent oral or intraperitoneal sensitization protocols to determine the immunogenic properties of the protein preparations. Finally, cashew-sensitized mice underwent an immunotherapy protocol with native or pepsinized cashew proteins and subsequent provocation challenges. Results Pepsinized cashew proteins elicited weaker allergic reactions than native cashew proteins but importantly retained the ability to stimulate cellular proliferation and cytokine production. Mice sensitized with pepsinized proteins reacted on challenge with native allergens, demonstrating that pepsinized allergens retain immunogenicity in vivo. Immunotherapy with pepsinized cashew allergens significantly decreased allergic symptoms and body temperature decrease relative to placebo after challenge with native and pepsinized proteins. Immunologic changes were comparable after immunotherapy with native or pepsinized allergens: TH2-type cytokine secretion from splenocytes was decreased, whereas specific IgG1 and IgG2a levels were increased. Conclusions Pepsinized cashew proteins are effective in treating cashew allergy in mice and appear to work through the same mechanisms as native protein immunotherapy.