Immune response enhancement with GLS-5310 DNA primary vaccine against SARS-CoV-2 followed by administration of an mRNA vaccine heterologous boost.

Heterologous boost regimens are being increasingly considered against SARS-CoV-2. We report results for the 32 of 45 participants in the Phase 1 CoV2-001 clinical trial (Kim et al., Int J Iinfect Dis 2023, 128:112-120) who elected to receive an EUA-approved SARS-CoV-2 mRNA vaccine 6 to 8 months following a two-dose primary vaccination with the GLS-5310 bi-cistronic DNA vaccine given intradermally and followed by application of suction using the GeneDerm device. Receipt of EUA-approved mRNA vaccines after GLS-5310 vaccination was well-tolerated, with no reported adverse events. Immune responses were enhanced such that binding antibody titers, neutralizing antibody titers, and T-cell responses increased 1,187-fold, 110-fold, and 2.9-fold, respectively. This paper is the first description of the immune responses following heterologous vaccination with a DNA primary series and mRNA boost.

Safety and immunogenicity of the bi-cistronic GLS-5310 COVID-19 DNA vaccine delivered with the GeneDerm suction device.

OBJECTIVES: The CoV2-001 phase I randomized trial evaluated the safety and immunogenicity of the GLS-5310 bi-cistronic DNA vaccine through 48 weeks of follow-up. DESIGN: A total of 45 vaccine-naïve participants were recruited between December 31, 2020, and March 30, 2021. GLS-5310, encoding for the SARS-CoV-2 spike and open reading frame 3a (ORF3a) proteins, was administered intradermally at 0.6 mg or 1.2 mg per dose, followed by application of the GeneDerm suction device as part of a two-dose regimen spaced either 8 or 12 weeks between vaccinations. RESULTS: GLS-5310 was well tolerated with no serious adverse events reported. Antibody and T cell responses were dose-independent. Anti-spike antibodies were induced in 95.5% of participants with an average geometric mean titer of ∼480 four weeks after vaccination and declined minimally through 48 weeks. Neutralizing antibodies were induced in 55.5% of participants with post-vaccination geometric mean titer of 28.4. T cell responses were induced in 97.8% of participants, averaging 716 site forming units/106 cells four weeks after vaccination, increasing to 1248 at week 24, and remaining greater than 1000 through 48 weeks. CONCLUSION: GLS-5310 administered with the GeneDerm suction device was well tolerated and induced high levels of binding antibodies and T-cell responses. Antibody responses were similar to other DNA vaccines, whereas T cell responses were many-fold greater than DNA and non-DNA vaccines.

Siglec-9 Restrains Antibody-Dependent Natural Killer Cell Cytotoxicity against SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection alters the immunological profiles of natural killer (NK) cells. However, whether NK antiviral functions are impaired during severe coronavirus disease 2019 (COVID-19) and what host factors modulate these functions remain unclear. We found that NK cells from hospitalized COVID-19 patients degranulate less against SARS-CoV-2 antigen-expressing cells (in direct cytolytic and antibody-dependent cell cytotoxicity [ADCC] assays) than NK cells from mild COVID-19 patients or negative controls. The lower NK degranulation was associated with higher plasma levels of SARS-CoV-2 nucleocapsid antigen. Phenotypic and functional analyses showed that NK cells expressing the glyco-immune checkpoint Siglec-9 elicited higher ADCC than Siglec-9- NK cells. Consistently, Siglec-9+ NK cells exhibit an activated and mature phenotype with higher expression of CD16 (FcγRIII; mediator of ADCC), CD57 (maturation marker), and NKG2C (activating receptor), along with lower expression of the inhibitory receptor NKG2A, than Siglec-9- CD56dim NK cells. These data are consistent with the concept that the NK cell subpopulation expressing Siglec-9 is highly activated and cytotoxic. However, the Siglec-9 molecule itself is an inhibitory receptor that restrains NK cytotoxicity during cancer and other viral infections. Indeed, blocking Siglec-9 significantly enhanced the ADCC-mediated NK degranulation and lysis of SARS-CoV-2-antigen-positive target cells. These data support a model in which the Siglec-9+ CD56dim NK subpopulation is cytotoxic even while it is restrained by the inhibitory effects of Siglec-9. Alleviating the Siglec-9-mediated restriction on NK cytotoxicity may further improve NK immune surveillance and presents an opportunity to develop novel immunotherapeutic tools against SARS-CoV-2 infected cells. IMPORTANCE One mechanism that cancer cells use to evade natural killer cell immune surveillance is by expressing high levels of sialoglycans, which bind to Siglec-9, a glyco-immune checkpoint molecule on NK cells. This binding inhibits NK cell cytotoxicity. Several viruses, such as hepatitis B virus (HBV) and HIV, also use a similar mechanism to evade NK surveillance. We found that NK cells from SARS-CoV-2-hospitalized patients are less able to function against cells expressing SARS-CoV-2 Spike protein than NK cells from SARS-CoV-2 mild patients or uninfected controls. We also found that the cytotoxicity of the Siglec-9+ NK subpopulation is indeed restrained by the inhibitory nature of the Siglec-9 molecule and that blocking Siglec-9 can enhance the ability of NK cells to target cells expressing SARS-CoV-2 antigens. Our results suggest that a targetable glyco-immune checkpoint mechanism, Siglec-9/sialoglycan interaction, may contribute to the ability of SARS-CoV-2 to evade NK immune surveillance.

Harnessing Recent Advances in Synthetic DNA and Electroporation Technologies for Rapid Vaccine Development Against COVID-19 and Other Emerging Infectious Diseases.

DNA vaccines are considered as a third-generation vaccination approach in which antigenic materials are encoded as DNA plasmids for direct in vivo production to elicit adaptive immunity. As compared to other platforms, DNA vaccination is considered to have a strong safety profile, as DNA plasmids neither replicate nor elicit vector-directed immune responses in hosts. While earlier work found the immune responses induced by DNA vaccines to be sub-optimal in larger mammals and humans, recent developments in key synthetic DNA and electroporation delivery technologies have now allowed DNA vaccines to elicit significantly more potent and consistent responses in several clinical studies. This paper will review findings from the recent clinical and preclinical studies on DNA vaccines targeting emerging infectious diseases (EID) including COVID-19 caused by the SARS-CoV-2 virus, and the technological advancements pivotal to the improved responses-including the use of the advanced delivery technology, DNA-encoded cytokine/mucosal adjuvants, and innovative concepts in immunogen design. With continuous advancement over the past three decades, the DNA approach is now poised to develop vaccines against COVID-19, as well as other EIDs.

Immune profile and responses of a novel dengue DNA vaccine encoding an EDIII-NS1 consensus design based on Indo-African sequences.

The ongoing COVID-19 pandemic highlights the need to tackle viral variants, expand the number of antigens, and assess diverse delivery systems for vaccines against emerging viruses. In the present study, a DNA vaccine candidate was generated by combining in tandem envelope protein domain III (EDIII) of dengue virus serotypes 1-4 and a dengue virus (DENV)-2 non-structural protein 1 (NS1) protein-coding region. Each domain was designed as a serotype-specific consensus coding sequence derived from different genotypes based on the whole genome sequencing of clinical isolates in India and complemented with data from Africa. This sequence was further optimized for protein expression. In silico structural analysis of the EDIII consensus sequence revealed that epitopes are structurally conserved and immunogenic. The vaccination of mice with this construct induced pan-serotype neutralizing antibodies and antigen-specific T cell responses. Assaying intracellular interferon (IFN)-γ staining, immunoglobulin IgG2(a/c)/IgG1 ratios, and immune gene profiling suggests a strong Th1-dominant immune response. Finally, the passive transfer of immune sera protected AG129 mice challenged with a virulent, non-mouse-adapted DENV-2 strain. Our findings collectively suggest an alternative strategy for dengue vaccine design by offering a novel vaccine candidate with a possible broad-spectrum protection and a successful clinical translation either as a stand alone or in a mix and match strategy.

Techniques for Developing and Assessing Immune Responses Induced by Synthetic DNA Vaccines for Emerging Infectious Diseases.

Vaccines are one of mankind's greatest medical advances, and their use has drastically reduced and in some cases eliminated (e.g., smallpox) disease and death caused by infectious agents. Traditional vaccine modalities including live-attenuated pathogen vaccines, wholly inactivated pathogen vaccines, and protein-based pathogen subunit vaccines have successfully been used to create efficacious vaccines against measles, mumps, rubella, polio, and yellow fever. These traditional vaccine modalities, however, take many months to years to develop and have thus proven less effective for use in creating vaccines to emerging or reemerging infectious diseases (EIDs) including influenza, Human immunodeficiency virus (HIV), dengue virus (DENV), chikungunya virus (CHIKV), West Nile virus (WNV), Middle East respiratory syndrome (MERS), and the severe acute respiratory syndrome coronaviruses 1 and 2 (SARS-CoV and SARS-CoV-2). As factors such as climate change and increased globalization continue to increase the pace of EID development, newer vaccine modalities are required to develop vaccines that can prevent or attenuate EID outbreaks throughout the world. One such modality, DNA vaccines, has been studied for over 30 years and has numerous qualities that make them ideal for meeting the challenge of EIDs including; (1) DNA vaccine candidates can be designed within hours of publishing of a pathogens genetic sequence; (2) they can be manufactured cheaply and rapidly in large quantities; (3) they are thermostable and have reduced requirement for a cold-chain during distribution, and (4) they have a remarkable safety record in the clinic. Optimizations made in plasmid design as well as in DNA vaccine delivery have greatly improved the immunogenicity of these vaccines. Here we describe the process of making a DNA vaccine to an EID pathogen and describe methods used for assessing the immunogenicity and protective efficacy of DNA vaccines in small animal models.

Novel suction-based in vivo cutaneous DNA transfection platform.

This work reports a suction-based cutaneous delivery method for in vivo DNA transfection. Following intradermal Mantoux injection of plasmid DNA in a rat model, a moderate negative pressure is applied to the injection site, a technique similar to Chinese báguàn and Middle Eastern hijama cupping therapies. Strong GFP expression was demonstrated with pEGFP-N1 plasmids where fluorescence was observed as early as 1 hour after dosing. Modeling indicates a strong correlation between focal strain/stress and expression patterns. The absence of visible and/or histological tissue injury contrasts with current in vivo transfection systems such as electroporation. Specific utility was demonstrated with a synthetic SARS-CoV-2 DNA vaccine, which generated host humoral immune response in rats with notable antibody production. This method enables an easy-to-use, cost-effective, and highly scalable platform for both laboratorial transfection needs and clinical applications for nucleic acid­based therapeutics and vaccines.

Preexisting vs. de novo antibodies against SARS-CoV-2 in individuals without or with virus infection: impact on antibody therapy, vaccine research and serological testing.

The causative agent of the ongoing pandemic in the world is SARS-CoV-2. The research on SARS-CoV-2 has progressed with lightning speed on various fronts, including clinical research and treatment, virology, epidemiology, drug development, and vaccine research. Recent studies reported that sera from healthy individuals, who were confirmed negative for SARS-CoV-2 by RT-PCR method, tested positive for antibodies against spike and nucleocapsid proteins of SARS-CoV-2. Further, such antibodies also exhibited neutralizing activity against the virus. These observations have prompted us to prepare a commentary on this topic. While the preexisting antibodies are likely to protect against SARS-CoV-2 infection, they may also complicate serological testing results. Another unknown is the influence of preexisting antibodies on immune responses in individuals receiving vaccines against  SARS-CoV-2. The commentary identifies the potential limitations with the serological tests based on spike and nucleocapsid proteins as these tests may overestimate the seroprevalence due to cross-reactive antibodies. The inclusion of tests specific to SARS-CoV-2 (such as RBD of spike protein) could overcome these limitations.

Intradermal-delivered DNA vaccine induces durable immunity mediating a reduction in viral load in a rhesus macaque SARS-CoV-2 challenge model.

Coronavirus disease 2019 (COVID-19), caused by the SARS-CoV-2 virus, has had a dramatic global impact on public health and social and economic infrastructures. Here, we assess the immunogenicity and anamnestic protective efficacy in rhesus macaques of an intradermal (i.d.)-delivered SARS-CoV-2 spike DNA vaccine, INO-4800, currently being evaluated in clinical trials. Vaccination with INO-4800 induced T cell responses and induced spike antigen and RBD binding antibodies with ADCP and ADCD activity. Sera from the animals neutralized both the D614 and G614 SARS-CoV-2 pseudotype viruses. Several months after vaccination, animals were challenged with SARS-CoV-2 resulting in rapid recall of anti-SARS-CoV-2 spike protein T cell and neutralizing antibody responses. These responses were associated with lower viral loads in the lung. These studies support the immune impact of INO-4800 for inducing both humoral and cellular arms of the adaptive immune system, which are likely important for providing durable protection against COVID-19 disease.

Identification of Novel Neutralizing Monoclonal Antibodies against SARS-CoV-2 Spike Glycoprotein.

Coronavirus disease 2019 (COVID-19) is caused by the newly emerged human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Due to the highly contagious nature of SARS-CoV-2, it has infected more than 137 million individuals and caused more than 2.9 million deaths globally as of April 13, 2021. There is an urgent need to develop effective novel therapeutic strategies to treat or prevent this infection. Toward this goal, we focused on the development of monoclonal antibodies (mAbs) directed against the SARS-CoV-2 spike glycoprotein (SARS-CoV-2 Spike) present on the surface of virus particles as well as virus-infected cells. We isolated anti-SARS-CoV-2 Spike mAbs from animals immunized with a DNA vaccine. We then selected a highly potent set of mAbs against SARS-CoV-2 Spike protein and evaluated each candidate for their expression, target binding affinity, and neutralization potential using complementary ACE2-blocking and pseudovirus neutralization assays. We identified a total of 10 antibodies, which specifically and strongly bound to SARS-CoV-2 Spike, blocked the receptor binding domain (RBD) and angiotensin-converting enzyme 2 (ACE2) interaction, and neutralized SARS-CoV-2. Furthermore, the glycomic profile of the antibodies suggested that they have high Fc-mediated effector functions. These antibodies should be further investigated for elucidating the neutralizing epitopes on Spike for the design of next-generation vaccines and for their potential in diagnostic as well as therapeutic utilities against SARS-CoV-2.

A novel mouse AAV6 hACE2 transduction model of wild-type SARS-CoV-2 infection studied using synDNA immunogens.

More than 100 million people have been infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Common laboratory mice are not susceptible to wild-type SARS-CoV-2 infection, challenging the development and testing of effective interventions. Here, we describe the development and testing of a mouse model for SARS-CoV-2 infection based on transduction of the respiratory tract of laboratory mice with an adeno-associated virus vector (AAV6) expressing human ACE-2 (AAV6.2FF-hACE2). We validated this model using a previously described synthetic DNA vaccine plasmid, INO-4800 (pS). Intranasal instillation of AAV6.2FF-hACE2 resulted in robust hACE2 expression in the respiratory tract. pS induced robust cellular and humoral responses. Vaccinated animals were challenged with 105 TCID50 SARS-CoV-2 (hCoV-19/Canada/ON-VIDO-01/2020) and euthanized four days post-challenge to assess viral load. One immunization resulted in 50% protection and two immunizations were completely protective. Overall, the AAV6.2FF-hACE2 mouse transduction model represents an easily accessible, genetically diverse mouse model for wild-type SARS-CoV-2 infection and preclinical evaluation of potential interventions.

Intradermal delivery of a synthetic DNA vaccine protects macaques from Middle East respiratory syndrome coronavirus.

Emerging coronaviruses from zoonotic reservoirs, including severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), have been associated with human-to-human transmission and significant morbidity and mortality. Here, we study both intradermal and intramuscular 2-dose delivery regimens of an advanced synthetic DNA vaccine candidate encoding a full-length MERS-CoV spike (S) protein, which induced potent binding and neutralizing antibodies as well as cellular immune responses in rhesus macaques. In a MERS-CoV challenge, all immunized rhesus macaques exhibited reduced clinical symptoms, lowered viral lung load, and decreased severity of pathological signs of disease compared with controls. Intradermal vaccination was dose sparing and more effective in this model at protecting animals from disease. The data support the further study of this vaccine for preventing MERS-CoV infection and transmission, including investigation of such vaccines and simplified delivery routes against emerging coronaviruses.

Landscape of humoral immune responses against SARS-CoV-2 in patients with COVID-19 disease and the value of antibody testing.

A new pandemic is ongoing in several parts of the world. The agent responsible is the newly emerged severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The symptoms associated with this virus are known as the coronavirus disease-2019 (COVID-19). In this review, we summarize the published data on virus specific antibodies in hospitalized patients with COVID-19 disease, patients recovered from the disease and the individuals who are asymptomatic with SARS-CoV-2 infections. The review highlights the following: i) an adjunct role of antibody tests in the diagnosis of COVID-19 in combination with RT-PCR; ii) status of antibodies from COVID-19 convalescent patients to select donors for plasma therapy; iii) the potential confounding effects of other coronaviruses, measles, mumps and rubella in antibody testing due to homology of certain viral genes; and iv) the role of antibody testing for conducting surveillance in populations, incidence estimation, contact tracing and epidemiologic studies.

SARS-CoV-2 Assays To Detect Functional Antibody Responses That Block ACE2 Recognition in Vaccinated Animals and Infected Patients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic of COVID-19, resulting in cases of mild to severe respiratory distress and significant mortality. The global outbreak of this novel coronavirus has now infected >20 million people worldwide, with >5 million cases in the United States (11 August 2020). The development of diagnostic and research tools to determine infection and vaccine efficacy is critically needed. We have developed multiple serologic assays using newly designed SARS-CoV-2 reagents for detecting the presence of receptor-binding antibodies in sera. The first assay is surface plasmon resonance (SPR) based and can quantitate both antibody binding to the SARS-CoV-2 spike protein and blocking to the Angiotensin-converting enzyme 2 (ACE2) receptor in a single experiment. The second assay is enzyme-linked immunosorbent assay (ELISA) based and can measure competition and blocking of the ACE2 receptor to the SARS-CoV-2 spike protein with antispike antibodies. The assay is highly versatile, and we demonstrate the broad utility of the assay by measuring antibody functionality of sera from small animals and nonhuman primates immunized with an experimental SARS-CoV-2 vaccine. In addition, we employ the assay to measure receptor blocking of sera from SARS-CoV-2-infected patients. The assay is shown to correlate with pseudovirus neutralization titers. This type of rapid, surrogate neutralization diagnostic can be employed widely to help study SARS-CoV-2 infection and assess the efficacy of vaccines.