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Expanding a previous study of the immune response to SARS-CoV-2 in 10 New Jersey long-term care facilities (LTCFs) during the first wave of the pandemic, this study characterized the neutralizing antibody (NAb) response to infection and vaccination among residents and staff. Sera from the original study were tested using the semi-quantitative enzyme-linked immunosorbent cPass neutralization-antibody detection assay. Almost all residents (97.8%) and staff (98.1%) who were positive for IgG S antibody to the spike protein were positive for NAb. In non-vaccinated subjects with a history of infection (positive polymerase chain reaction (PCR) or antigen test), the distribution of mean intervals from infection to serology date was not significantly different for S antibody positives versus negatives. More than 80% of both were positive at 10 months. Similarly, the mean NAb titer for residents and staff was not associated with interval from PCR/antigen positive to serology date, F = 0.1.01, Pr F = 0.4269 and F = 0.77, Pr F = 0.6548 respectively. Titers remained high as the interval reached 10 months. In vaccinees who had no history of infection, the NAb titer was near the test maximum when the serum was drawn seven or more days after the second vaccine dose. In staff the mean NAb titer increased significantly as the vaccine number increased from one to two doses, F = 11.69, Pr F 0.0001. NAb titers to SARS-CoV-2 in residents and staff of LTCFs were consistently high 10 months after infection and after two doses of vaccine. Ongoing study is needed to determine whether this antibody provides protection as the virus continues to mutate.

Early in the pandemic, New Jersey (NJ) long-term care facilities (LTCFs) witnessed severe COVID-19 illness. With limited surveillance to characterize the scope of infection, we estimated the prevalence of antibody to the SARS-CoV-2 nucleocapsid protein among residents and staff, to describe the epidemiology, and to measure antibody distribution by prior PCR/antigen status and symptomatology. 10 NJ LTCFs of 20 solicited with diverse geography and bed-capacities were visited between October 2020 and March 2021. A single serum was tested for total N-antibody (ELISA) by the state laboratory. Residents' demographics and clinical history were transcribed from the patient record. For staff, this information was solicited directly from employees, supplemented by prior PCR/antigen results from facilities. 62% of 332 residents and 46% of 661 staff tested N-antibody positive. In a multivariable logistic regression in residents, odds ratios for older age and admission prior before March 1, 2020 were significant. Among the staff, odds ratios for older age, ethnic-racial group, nursing-related job, and COVID-19 symptoms were significantly associated with N-antibody positivity. In a sub-analysis in five better record-keeping LTCFs, 90% of residents and 85% of staff with positive PCR/antigen results were seropositive for N-antibody, yet 25% of residents and 22% of staff were N-antibody positive but PCR/antigen and symptoms negative. The high rate of clinically unsuspected infections likely contributed to the spread. These findings argue for robust surveillance, regular screening of asymptomatic individuals, and vaccinating both residents and staff to abate the pandemic. The data also provide guidance to prevent future outbreaks.

Porcine organs are rapidly rejected after transplantation into primate recipients due to the presence of preexisting immunoglobulins that bind to terminal galactose alpha1,3 galactose residues (alpha-galactosyl) present on porcine glycoproteins and glycolipids. Currently available immunosuppressive reagents have been largely ineffective at controlling the synthesis of these anti-Gal antibodies. Nonantigenic hapten polymers have been shown to be effective materials for blocking humoral immune responses in various model systems. We have developed a series of alpha-galactosyl glycoconjugate polymers and tested their ability to block anti-Gal antibody binding in vitro and in vivo. A galactose alpha1,3 galactose beta 1,4 GlcNAc trisaccharide free acid (TRFA) with a hexanoic acid spacer, containing five methylene groups and a carboxylic acid, was produced and coupled to a variety of polymeric backbones including dextran, branched poly(ethylene glycol) (PEG), and poly-L-lysine. The ability of monomeric TRFA and the alpha-galactosyl conjugates to block anti-Gal IgG and IgM binding was determined using a competition ELISA assay on defined HSA-Gal glycoconjugates and porcine microvascular endothelial cell substrates. We show that branched PEG carriers, with a TRFA sugar attached to each branch, exhibit enhanced antibody blocking ability compared to TRFA, but at higher target antigen densities these simple PEG conjugates are no more effective then an equivalent amount of TRFA in blocking anti-Gal IgM antibody interactions. In contrast, polymers of the branched PEG conjugates and linear conjugates made using dextran and poly-L-lysine were 2000 to 70000-fold more effective inhibitors of anti-Gal antibodies. In a study using nonhuman primates, a single dose infusion of polymeric PEG or dextran glycoconjugates dramatically reduced the level of circulating anti-Gal antibodies in cynomologus monkeys for at least 72 h. Glycoconjugates similar to these might be useful both to block anti-Gal interactions in vivo and to specifically control the induced anti-Gal immune response.

Unlike natural antibodies, single-chain Fv (sFv) proteins normally lack asparagine-linked glycosylation. Many designed immunoconjugates and other therapeutics currently employ the advantageous conjugation chemistry or targeting properties provided by the glycoprotein oligosaccharide domain. sFv proteins with engineered N-glycan designs were evaluated in Pichia pastoris for glycosylation efficiency, expression level, oligosaccharide chain length and composition, and affinity. In contrast to nearly all natural glycoproteins, the engineered attachment of N-glycans conveniently near the polypeptide C-terminus was found to produce the optimal results. Furthermore, the percentage modification and chain length of the attached mannose chains were controllable by the use of tandem and overlapping Asn-X-Thr tripeptide sites. The glycosylated sFv mannose chains could be effectively conjugated to polyethylene glycol and the resulting conjugate displayed a 10-fold increased circulating life in mice. The potential to control polymer:sFv or drug:sFv molar ratios by site-specific conjugation may substantially improve the therapeutic efficacy of these minimal antigen-binding molecules.