ABSTRACT
Given the rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and the recent implementation of SARS-CoV-2 vaccination, we have much to learn about the duration of immune protection and the interface between the immune responses to infection and to vaccination. To address these questions, we monitored immune responses to SARS-CoV-2 infection in convalescent individuals over seven months and following mRNA vaccination. Spike Receptor-Binding-Domain (RBD)-specific circulating antibodies and plasma neutralizing activity generally decreased over time, whereas RBD-specific memory B cells persisted. Additionally, using antibody depletion techniques, we showed that the neutralizing activity of plasma specifically resides in the anti-RBD antibodies. More vigorous antibody and B cell responses to vaccination were observed in previously infected subjects relative to uninfected comparators, presumably due to immune priming by infection. SARS-CoV-2 infection also led to increased numbers of double negative B memory cells, which are described as a dysfunctional B cell subset. This effect was reversed by SARS-CoV-2 vaccination, providing a potential mechanistic explanation for the vaccination-induced reduction in symptoms in patients with "Long-COVID".
ABSTRACT
Development of effective vaccines against Coronavirus Disease 2019 (COVID-19) is a global imperative. Rapid immunization of the world human population against a widespread, continually evolving, and highly pathogenic virus is an unprecedented challenge, and many different vaccine approaches are being pursued to meet this task. Engineered filamentous bacteriophage (phage) have unique potential in vaccine development due to their inherent immunogenicity, genetic plasticity, stability, cost-effectiveness for large-scale production, and proven safety profile in humans. Herein we report the design, development, and initial evaluation of targeted phage-based vaccination approaches against Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) by using dual ligand peptide-targeted phage and adeno-associated virus/phage (AAVP) particles. Towards a unique phage- and AAVP-based dual-display candidate approach, we first performed structure-guided antigen design to select six solvent-exposed epitopes of the SARS-CoV-2 spike (S) protein for display on the recombinant major capsid coat protein pVIII. Targeted phage particles carrying one of these epitopes induced a strong and specific humoral response. In an initial experimental approach, when these targeted phage particles were further genetically engineered to simultaneously display a ligand peptide (CAKSMGDIVC) on the minor capsid protein pIII, which enables receptor-mediated transport of phage particles from the lung epithelium into the systemic circulation (termed "dual-display"), they enhanced a systemic and specific spike (S) protein-specific antibody response upon aerosolization into the lungs of mice. In a second line of investigation, we engineered targeted AAVP particles to deliver the entire S protein gene under the control of a constitutive cytomegalovirus (CMV) promoter, which induced tissue-specific transgene expression stimulating a systemic S protein-specific antibody response. As proof-of-concept preclinical experiments, we show that targeted phage- and AAVP-based particles serve as robust yet versatile enabling platforms for ligand-directed immunization and promptly yield COVID-19 vaccine prototypes for further translational development. SignificanceThe ongoing COVID-19 global pandemic has accounted for over 2.5 million deaths and an unprecedented impact on the health of mankind worldwide. Over the past several months, while a few COVID-19 vaccines have received Emergency Use Authorization and are currently being administered to the entire human population, the demand for prompt global immunization has created enormous logistical challenges--including but not limited to supply, access, and distribution--that justify and reinforce the research for additional strategic alternatives. Phage are viruses that only infect bacteria and have been safely administered to humans as antibiotics for decades. As experimental proof-of-concept, we demonstrated that aerosol pulmonary vaccination with lung-targeted phage particles that display short epitopes of the S protein on the capsid as well as preclinical vaccination with targeted AAVP particles carrying the S protein gene elicit a systemic and specific immune response against SARS-CoV-2 in immunocompetent mice. Given that targeted phage- and AAVP-based viral particles are sturdy yet simple to genetically engineer, cost-effective for rapid large-scale production in clinical grade, and relatively stable at room temperature, such unique attributes might perhaps become additional tools towards COVID-19 vaccine design and development for immediate and future unmet needs.
ABSTRACT
BackgroundSARS-CoV-2, the virus that causes COVID-19, has rapidly spread globally beginning in late 2019. Early areas impacted by this pandemic in the US include Essex County, New Jersey. Beyond understanding the prevalence of active infections and deaths, it is important to understand the true burden of infection in the community, as indicated by seroprevalence of antibodies directed to the virus. Understanding the spectrum of disease is key to the effectiveness of primary prevention and control measures and the design of interventions against transmission of infection. MethodsWe utilized venue-based-sampling (VBS), implemented by a community partner, to sample members of the community in Essex County. In VBS the venues are randomized as a proxy for randomizing the attendees of the venues. We asked standard demographic questions, questions about symptoms and PCR testing and previous antibody testing. Participants provide a blood sample collected by finger stick with the Neoteryx Mitra Collection device. Samples were tested using a novel ELISA based approached developed by our team. ResultsFrom September 15, 2020 to December 22, 2020, we conducted 92 randomly selected sampling events where we approached 1349 individuals for screening. Of these, 924 consented and had complete data for analysis. Only 6.5% of the sample reported any COVID-19 like symptoms while 45.9% had sought out a COVID-19 test. In total 13 (1.4%) participants received a positive SARS-CoV-2 PCR test result. While 33 participants (2.6%) sought a SARS-CoV-2 antibody test, only 0.5% of the sample reported a positive antibody result. Testing in this study identified 83 (9.0%) participants positive for SARS-CoV-2 antibodies. ConclusionWe recruited a large sample of the population of Essex County, New Jersey using VBS, electronic surveys, novel sample collection and lab methods. Our findings suggest that the burden of SARS-Cov-2 is slightly more than six times than that suggested by PCR testing. This burden is higher than most estimates obtained through studies of remnant blood samples from hospitals (4.2%), samples from staff at a public-school system (2.9%), and residents of a California county recruited with targeted Facebook ads (1.5%). (9-11) Moreover, with only 6.5% of the sample reporting any COVID-19-like symptoms, our finding suggests that the number of asymptomatic persons may be close to 1.5 times greater than anyone reporting symptoms.
