T-cell recognition of cancer neoantigens is important for effective immune checkpoint blockade therapy, and an increasing interest exists in developing personalized tumor neoantigen vaccines. Previous studies utilizing RNA and long-peptide neoantigen vaccines in pre-clinical and early phase clinical studies have shown immune responses predominantly driven by MHC class II CD4 + T cells. Here, we report on a pre-clinical study utilizing a DNA vaccine platform to target tumor neoantigens. We showed that optimized strings of tumor neoantigens, when delivered by potent electroporation-mediated DNA delivery, were immunogenic and generated predominantly MHC class I-restricted, CD8 + T-cell responses. High MHC class I affinity was associated specifically with immunogenic CD8 + T-cell epitopes. These DNA neoantigen vaccines induced a therapeutic antitumor response in vivo, and neoantigen-specific T cells expanded from immunized mice directly killed tumor cells ex vivo. These data illustrate a unique advantage of this DNA platform to drive CD8 + T-cell immunity for neoantigen immunotherapy.

T-cell immunity is likely to play a role in protection against SARS-CoV-2 by helping generate neutralizing antibodies. We longitudinally studied CD4 T-cell responses to the M, N, and S structural proteins of SARS-CoV-2 in 26 convalescent individuals. Within the first two months following symptom onset, a majority of individuals (81%) mounted at least one CD4 T-cell response, and 48% of individuals mounted detectable SARS-CoV-2-specific circulating T follicular helper cells (cTfh, defined as CXCR5+PD1+ CD4 T cells). SARS-CoV-2-specific cTfh responses across all three protein specificities correlated with antibody neutralization with the strongest correlation observed for S protein-specific responses. When examined over time, cTfh responses, particularly to the M protein, increased in convalescence, and robust cTfh responses with magnitudes greater than 5% were detected at the second convalescent visit, a median of 38 days post-symptom onset. CD4 T-cell responses declined but persisted at low magnitudes three months and six months after symptom onset. These data deepen our understanding of antigen-specific cTfh responses in SARS-CoV-2 infection, suggesting that in addition to S protein, M and N protein-specific cTfh may also assist in the development of neutralizing antibodies and that cTfh response formation may be delayed in SARS-CoV-2 infection.

Antibody-based immune therapies targeting the T cell checkpoint molecules CTLA-4 and PD-1 have impacted cancer therapy. However, this immune therapy requires complex manufacturing and frequent dosing, limiting the global use of this treatment. Here we focused on the development of a DNA-encoded monoclonal antibody (DMAb) approach for delivery of anti-CTLA-4 monoclonal antibodies in vivo. With this technology, engineered and formulated DMAb plasmids encoding IgG inserts were directly injected into muscle and delivered intracellularly by electroporation, leading to in vivo expression and secretion of the encoded IgG. DMAb expression from a single dose can continue for several months without the need for repeated administration. Delivery of an optimized DMAb encoding anti-mouse CTLA-4 IgG resulted in high serum levels of the antibody as well as tumor regression in Sa1N and CT26 tumor models. DNA-delivery of the anti-human CTLA-4 antibodies ipilimumab and tremelimumab in mice achieved potent peak levels of approximately 85μg/mL and 58μg/mL, respectively. These DMAb exhibited prolonged expression, with maintenance of serum levels at or above 15μg/mL for over a year. Anti-human CTLA-4 DMAbs produced in vivo bound to human CTLA-4 protein expressed on stimulated human PBMC and induced T cell activation in a functional assay ex vivo. In summary, direct in vivo expression of DMAb encoding checkpoint inhibitors serve as a novel tool for immunotherapy that could significantly improve availability and provide broader access to such therapies.

C oronavirus disease (COVID-19) is typically diagnosed by reverse transcription PCR (RT-PCR) amplifi cation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA from nasopharyngeal fl uids (1). RT-PCR yields cycle threshold (C t ) values that are inversely correlated with viral loads (2) and thus provide an estimate of the number of SARS-CoV-2 RNA copies in the sample. Serologic assays complement COVID-19 diagnosis by documenting past infections. In most persons, binding and neutralizing antibodies develop within 1-3 weeks after onset of symptoms (3), and titers correlate with disease severity (4).Initial serosurveys identifi ed antibodies in nearly 100% of persons with RT-PCR-confi rmed SARS-CoV-2 infection (5). However, more recent studies have shown that seroconversion rates are surprisingly variable (6-10). For example, a multicenter study from Israel reported that 5% of participants remained seronegative despite a positive test result on a nasal swab specimen (6). In contrast, a seroprevalence study from New York found that 20% of persons with a positive RT-PCR test result did not seroconvert (8). Another study from Germany reported that 85% of confi rmed infected COVID-19 contacts failed to develop antibodies (9). To examine the reasons for these differences, we investigated the relationship between seroconversion and demographic, clinical, and laboratory data in a convenience sample of convalescent persons recruited at the University of Alabama at Birmingham (Birmingham, Alabama, USA) in 2020. The StudyWe studied 72 persons, all of whom had a previous positive RT-PCR test but were symptom-free for >3 weeks before blood was collected for testing (Table ). Only 2 persons (3%) reported no symptoms, whereas 13 (18%) persons reported mild disease, 48 (67%) reported moderate disease, and 9 (12%) reported severe disease (Appendix Table 1, https://wwwnc.cdc.gov/ EID/article/27/9/21-1024-App1.pdf).We tested plasma samples (n = 144) collected at enrollment and follow-up visits for antibodies to the spike protein by using a validated ELISA (Appendix). Only 46 of the 72 participants had detectable IgG responses, IgA responses, or both (Table ); reciprocal endpoint titers ranged from 182 to >312,500 (Appendix Table 2). Analysis of the same samples for receptor-binding domain (RBD) and nucleocapsid (N)

Highlights d Convalescent plasma clinically benefited a severely ill immunodeficient patient d The transfer of high-titer neutralizing antibodies led to rapid clinical recovery d Neutralizing activity was low in most convalescent plasmas but high in recipients d Neutralizing activity should be tested in both plasma donors and recipients