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1.
Preprint in English | Other preprints | ID: ppcovidwho-296014

ABSTRACT

ABSTRACT In response to the need for a safe, efficacious vaccine that elicits vigorous T cell as well as humoral protection against SARS-CoV-2 infection, we have developed a dual-antigen COVID-19 vaccine comprising both the viral spike (S) protein modified to increase cell-surface expression (S-Fusion) and nucleocapsid (N) protein with an Enhanced T-cell Stimulation Domain (N-ETSD) to enhance MHC class I and II presentation and T-cell responses. The antigens are delivered using a human adenovirus serotype 5 (hAd5) platform with E1, E2b, and E3 regions deleted that has been shown previously in cancer vaccine studies to be safe and effective in the presence of pre-existing hAd5 immunity. The findings reported here are focused on human T-cell responses due to the likelihood that such responses will sustain efficacy against emerging variants, a hypothesis supported by our in silico prediction of T-cell epitope HLA binding for both the first-wave SARS-CoV-2 ‘A’ strain and the B.1.351 strain K417N, E484K, and N501Y spike and T201I N variants. We demonstrate the hAd5 S-Fusion + N-ETSD vaccine antigens expressed by previously SARS-CoV-2-infected patient dendritic cells elicit Th1 dominant activation of autologous patient T cells, indicating the vaccine antigens have the potential to elicit immune responses in previously infected patients. For participants in our open-label Phase 1b study of the vaccine ( NCT04591717 ;https://clinicaltrials.gov/ct2/show/NCT04591717 ), the magnitude of Th-1 dominant S- and N-specific T-cell responses after a single prime subcutaneous injection were comparable to T-cell responses from previously infected patients. Furthermore, vaccinated participant T-cell responses to S were similar for A strain S and a series of spike variant peptides, including S variants in the B.1.1.7 and B.1.351 strains. The findings that this dual-antigen vaccine elicits SARS-CoV-2-relevant T-cell responses and that such cell-mediated protection is likely to be sustained against emerging variants supports the testing of this vaccine as a universal booster that would enhance and broaden existing immune protection conferred by currently approved S-based vaccines.

2.
Preprint in English | EuropePMC | ID: ppcovidwho-293414

ABSTRACT

ABSTRACT We assessed if immune responses are enhanced in CD-1 mice by heterologous vaccination with two different nucleic acid-based COVID-19 vaccines: a next-generation human adenovirus serotype 5 (hAd5)-vectored dual-antigen spike (S) and nucleocapsid (N) vaccine (AdS+N) and a self-amplifying and -adjuvanted S RNA vaccine (SASA S) delivered by a nanostructured lipid carrier. The AdS+N vaccine encodes S modified with a fusion motif to increase cell-surface expression. The N antigen is modified with an Enhanced T-cell Stimulation Domain (N-ETSD) to direct N to the endosomal/lysosomal compartment to increase the potential for MHC class I and II stimulation. The S sequence in the SASA S vaccine comprises the D614G mutation, two prolines to stabilize S in the prefusion conformation, and 3 glutamines in the furin cleavage region to increase cross-reactivity across variants. CD-1 mice received vaccination by prime > boost homologous and heterologous combinations. Humoral responses to S were the highest with any regimen including the SASA S vaccine, and IgG against wild type S1 and Delta (B.1.617.2) variant S1 was generated at similar levels. An AdS+N boost of an SASA S prime enhanced both CD4+ and CD8+ T-cell responses to both S wild type and S Delta peptides relative to all other vaccine regimens. Sera from mice receiving SASA S homologous or heterologous vaccination were found to be highly neutralizing of all pseudovirus tested: Wuhan, Delta, and Beta strain pseudoviruses. The findings here support the clinical testing of heterologous vaccination by an SASA S > AdS+N regimen to provide increased protection against COVID-19 and SARS-CoV-2 variants.

3.
MethodsX ; 8: 101586, 2021.
Article in English | MEDLINE | ID: covidwho-1521414

ABSTRACT

The ongoing pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused unprecedented damage to the global economy. Diagnostic testing is a key factor in limiting virus transmission and safeguarding public health. We present the fabrication process of a paper-based device that uses reverse-transcription loop-mediated isothermal amplification (RT-LAMP) to detect SARS-CoV-2 in complex matrices by providing a colorimetric response apparent to the naked eye. Because of LAMP's functionality, this device just requires a simple heat source (e.g., water bath, incubator), it can be deployed in resource-constrained areas and can be used as a supplement to current point-of-care (POC) and community testing procedures. Since the test is based on nucleic acids, the testing platform itself lends to further applications including food safety monitoring, animal diagnostics, etc. simply by changing the specific primers.•We developed a platform capable of on-paper detection of SARS-CoV-2 using colorimetric reporters that produce responses visible to the naked eye.•The platform is easily reconfigurable to target different pathogens by changing the primer sets, and multiplexing is possible by adding additional reaction sites to the device.

4.
Front Immunol ; 12: 729837, 2021.
Article in English | MEDLINE | ID: covidwho-1450810

ABSTRACT

We have developed a dual-antigen COVID-19 vaccine incorporating genes for a modified SARS-CoV-2 spike protein (S-Fusion) and the viral nucleocapsid (N) protein with an Enhanced T-cell Stimulation Domain (N-ETSD) to increase the potential for MHC class II responses. The vaccine antigens are delivered by a human adenovirus serotype 5 platform, hAd5 [E1-, E2b-, E3-], previously demonstrated to be effective in the presence of Ad immunity. Vaccination of rhesus macaques with the hAd5 S-Fusion + N-ETSD vaccine by subcutaneous prime injection followed by two oral boosts elicited neutralizing anti-S IgG and T helper cell 1-biased T-cell responses to both S and N that protected the upper and lower respiratory tracts from high titer (1 x 106 TCID50) SARS-CoV-2 challenge. Notably, viral replication was inhibited within 24 hours of challenge in both lung and nasal passages, becoming undetectable within 7 days post-challenge.


Subject(s)
COVID-19 Vaccines/immunology , COVID-19/prevention & control , Coronavirus Nucleocapsid Proteins/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Adenoviruses, Human/genetics , Adenoviruses, Human/immunology , Adenoviruses, Human/metabolism , Administration, Oral , Animals , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , COVID-19 Vaccines/administration & dosage , Cytokines/blood , Immunization, Secondary/methods , Immunoglobulin G/blood , Lung/virology , Macaca mulatta , Nose/virology , Phosphoproteins/immunology , Protein Domains/immunology , T-Lymphocytes, Helper-Inducer/immunology , Vaccination , Virus Replication/immunology
5.
Biosens Bioelectron X ; 9: 100076, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1356145

ABSTRACT

Herein, we describe the development of a paper-based device to detect nucleic acids of pathogens of interest in complex samples using loop-mediated isothermal amplification (LAMP) by producing a colorimetric response visible to the human eye. To demonstrate the utility of this device in emerging public health emergencies, we developed and optimized our device to detect SARS-CoV-2 in human saliva without preprocessing. The resulting device was capable of detecting the virus within 60 min and had an analytical sensitivity of 97% and a specificity of 100% with a limit of detection of 200 genomic copies/µL of patient saliva using image analysis. The device consists of a configurable number of reaction zones constructed of Grade 222 chromatography paper separated by 20 mil polystyrene spacers attached to a Melinex® backing via an ARclean® double-sided adhesive. The resulting device is easily configurable to detect multiple targets and has the potential to detect a variety of pathogens simply by changing the LAMP primer sets.

6.
Sci Rep ; 11(1): 14917, 2021 07 21.
Article in English | MEDLINE | ID: covidwho-1320238

ABSTRACT

We have developed a COVID-19 vaccine, hAd5 S-Fusion + N-ETSD, that expresses SARS-CoV-2 spike (S) and nucleocapsid (N) proteins with modifications to increase immune responses delivered using a human adenovirus serotype 5 (hAd5) platform. Here, we demonstrate subcutaneous (SC) prime and SC boost vaccination of CD-1 mice with this dual-antigen vaccine elicits T-helper cell 1 (Th1) biased T-cell and humoral responses to both S and N that are greater than those seen with hAd5 S wild type delivering only unmodified S. We then compared SC to intranasal (IN) prime vaccination with SC or IN boosts and show that an IN prime with an IN boost is as effective at generating Th1 biased humoral responses as the other combinations tested, but an SC prime with an IN or SC boost elicits greater T cell responses. Finally, we used a combined SC plus IN (SC + IN) prime with or without a boost and found the SC + IN prime alone to be as effective in generating humoral and T-cell responses as the SC + IN prime with a boost. The finding that SC + IN prime-only delivery has the potential to provide broad immunity-including mucosal immunity-against SARS-CoV-2 supports further testing of this vaccine and delivery approach in animal models of viral challenge.


Subject(s)
COVID-19 Vaccines/administration & dosage , COVID-19/immunology , COVID-19/prevention & control , SARS-CoV-2/immunology , Adenoviridae/genetics , Administration, Intranasal , Animals , Antibodies, Neutralizing , Antibodies, Viral/biosynthesis , Antibodies, Viral/immunology , COVID-19 Vaccines/immunology , Female , Genetic Vectors , Hypodermoclysis , Immunity, Cellular/immunology , Immunity, Mucosal/immunology , Immunization, Secondary , Mice , Mice, Inbred Strains , Vaccination/methods
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