Validation of Saliva as the Clinical Specimen Type for a University-Wide COVID-19 Surveillance Program.

At the beginning of the COVID-19 pandemic, the Georgia Institute of Technology made the decision to keep the university doors open for on-campus attendance. To manage COVID-19 infection rates, internal resources were applied to develop and implement a mass asymptomatic surveillance program. The objective was to identify infections early for proper follow-on verification testing, contact tracing, and quarantine/isolation as needed. Program success depended on frequent and voluntary sample collection from over 40,000 students, faculty, and staff personnel. At that time, the nasopharyngeal (NP) swab, not saliva, was the main accepted sample type for COVID-19 testing. However, due to collection discomfort and the inability to be self-collected, the NP swab was not feasible for voluntary and frequent self-collection. Therefore, saliva was selected as the clinical sample type and validated. A saliva collection kit and a sample processing and analysis workflow were developed. The results of a clinical sample-type comparison study between co-collected and matched NP swabs and saliva samples showed 96.7% positive agreement and 100% negative agreement. During the Fall 2020 and Spring 2021 semesters, 319,988 samples were collected and tested. The program resulted in maintaining a low overall mean positivity rate of 0.78% and 0.54% for the Fall 2020 and Spring 2021 semesters, respectively. For this high-throughput asymptomatic COVID-19 screening application, saliva was an exceptionally good sample type.

Isolation and characterization of IgG3 glycan-targeting antibodies with exceptional cross-reactivity for diverse viral families.

Broadly reactive antibodies that target sequence-diverse antigens are of interest for vaccine design and monoclonal antibody therapeutic development because they can protect against multiple strains of a virus and provide a barrier to evolution of escape mutants. Using LIBRA-seq (linking B cell receptor to antigen specificity through sequencing) data for the B cell repertoire of an individual chronically infected with human immunodeficiency virus type 1 (HIV-1), we identified a lineage of IgG3 antibodies predicted to bind to HIV-1 Envelope (Env) and influenza A Hemagglutinin (HA). Two lineage members, antibodies 2526 and 546, were confirmed to bind to a large panel of diverse antigens, including several strains of HIV-1 Env, influenza HA, coronavirus (CoV) spike, hepatitis C virus (HCV) E protein, Nipah virus (NiV) F protein, and Langya virus (LayV) F protein. We found that both antibodies bind to complex glycans on the antigenic surfaces. Antibody 2526 targets the stem region of influenza HA and the N-terminal domain (NTD) region of SARS-CoV-2 spike. A crystal structure of 2526 Fab bound to mannose revealed the presence of a glycan-binding pocket on the light chain. Antibody 2526 cross-reacted with antigens from multiple pathogens and displayed no signs of autoreactivity. These features distinguish antibody 2526 from previously described glycan-reactive antibodies. Further study of this antibody class may aid in the selection and engineering of broadly reactive antibody therapeutics and can inform the development of effective vaccines with exceptional breadth of pathogen coverage.

Discovery and characterization of a pan-betacoronavirus S2-binding antibody.

The continued emergence of deadly human coronaviruses from animal reservoirs highlights the need for pan-coronavirus interventions for effective pandemic preparedness. Here, using linking B cell receptor to antigen specificity through sequencing (LIBRA-seq), we report a panel of 50 coronavirus antibodies isolated from human B cells. Of these, 54043-5 was shown to bind the S2 subunit of spike proteins from alpha-, beta-, and deltacoronaviruses. A cryoelectron microscopy (cryo-EM) structure of 54043-5 bound to the prefusion S2 subunit of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike defined an epitope at the apex of S2 that is highly conserved among betacoronaviruses. Although non-neutralizing, 54043-5 induced Fc-dependent antiviral responses in vitro, including antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). In murine SARS-CoV-2 challenge studies, protection against disease was observed after introduction of Leu234Ala, Leu235Ala, and Pro329Gly (LALA-PG) substitutions in the Fc region of 54043-5. Together, these data provide new insights into the protective mechanisms of non-neutralizing antibodies and define a broadly conserved epitope within the S2 subunit.

COBRA N2 NA vaccines induce protective immune responses against influenza viral infection.

Influenza neuraminidase (NA) is a promising target for a broadly protective vaccine. In this study, the Computationally Optimized Broadly Reactive Antigen (COBRA) methodology was used to develop N2 NA vaccine candidates. The unique wild type (WT) N2 sequences of human and swine influenza strains isolated between 1957 and 2019 were used to design the COBRA N2-A NA vaccine, while the unique WT N2 sequences of human influenza strains isolated between 2000 and 2019 were used to design the COBRA N2-B NA vaccine. Sera collected from COBRA N2 NA vaccinated mice showed more broadly reactive antibody responses against a broad panel of H×N2 influenza virus strains than sera collected from mice vaccinated with WT N2 NA vaccines. Antibodies elicited by COBRA or WT N2 NA antigens cross react with recent human H3N2 influenza viruses from different clades, while the antibodies elicited by A/Switzerland/9715293/2013 hemagglutinin (HA) reacted with viruses from the same clade. Furthermore, mice vaccinated with COBRA N2-B NA vaccine had lower viral lung titers compared to mock vaccinated mice when challenged with human H3N2 influenza viruses. Thus, the COBRA N2 NA vaccines elicit broadly protective murine anti-NA antibodies against multiple strains across subtypes and the viral loads were significantly decreased in the lungs of the mice in the COBRA N2 NA vaccine groups, compared to the mice in the mock vaccinated group, indicating that the COBRA-based N2 subtype NA vaccines have a potential to be a component in a universal influenza vaccine.

Respiratory viruses: Preventive and therapeutic approaches to diverse pathogens.

Viral respiratory diseases affect millions of individuals worldwide each year. Annual vaccinations are recommended by the World Health Organization for some of them, such as influenza and more recently for the coronavirus disease of 2019 (COVID-19) and respiratory syncytial virus, with the goal of reducing disease severity and limiting transmission. In the context of infection and vaccination, it is of primary importance to evaluate the immune response to pathogens to shed light on the mechanisms of protection.

The response to influenza vaccination is associated with DNA methylation-driven regulation of T cell innate antiviral pathways.

BACKGROUND: The effect of vaccination on the epigenome remains poorly characterized. In previous research, we identified an association between seroprotection against influenza and DNA methylation at sites associated with the RIG-1 signaling pathway, which recognizes viral double-stranded RNA and leads to a type I interferon response. However, these studies did not fully account for confounding factors including age, gender, and BMI, along with changes in cell-type composition. RESULTS: Here, we studied the influenza vaccine response in a longitudinal cohort vaccinated over two consecutive years (2019-2020 and 2020-2021), using peripheral blood mononuclear cells and a targeted DNA methylation approach. To address the effects of multiple factors on the epigenome, we designed a multivariate multiple regression model that included seroprotection levels as quantified by the hemagglutination-inhibition (HAI) assay test. CONCLUSIONS: Our findings indicate that 179 methylation sites can be combined as potential signatures to predict seroprotection. These sites were not only enriched for genes involved in the regulation of the RIG-I signaling pathway, as found previously, but also enriched for other genes associated with innate immunity to viruses and the transcription factor binding sites of BRD4, which is known to impact T cell memory. We propose a model to suggest that the RIG-I pathway and BRD4 could potentially be modulated to improve immunization strategies.

Intranasal administration of octavalent next-generation influenza vaccine elicits protective immune responses against seasonal and pre-pandemic viruses.

Development of next-generation influenza virus vaccines is crucial to improve protection against circulating and emerging viruses. Current vaccine formulations have to be updated annually due to mutations in seasonal strains and do not offer protection against strains with pandemic potential. Computationally optimized broadly reactive antigen (COBRA) methodology has been utilized by our group to generate broadly reactive immunogens for individual influenza subtypes, which elicit protective immune responses against a broad range of strains over numerous seasons. Octavalent mixtures of COBRA hemagglutinin (HA) (H1, H2, H3, H5, H7, and influenza B virus) plus neuraminidase (NA) (N1 and N2) recombinant proteins mixed with c-di-AMP adjuvant were administered intranasally to naive or pre-immune ferrets in prime-boost fashion. Four weeks after final vaccination, collected sera were analyzed for breadth of antibody response, and the animals were challenged with seasonal or pre-pandemic strains. The octavalent COBRA vaccine elicited antibodies that recognized a broad panel of strains representing different subtypes, and these vaccinated animals were protected against influenza virus challenges. Overall, this study demonstrated that the mixture of eight COBRA HA/NA proteins mixed with an intranasal adjuvant is a promising candidate for a universal influenza vaccine. IMPORTANCE: Influenza is a respiratory virus which infects around a billion people globally every year, with millions experiencing severe illness. Commercial vaccine efficacy varies year to year and can be low due to mismatch of circulating virus strains. Thus, the formulation of current vaccines has to be adapted accordingly every year. The development of a broadly reactive influenza vaccine would lessen the global economic and public health burden caused by the different types of influenza viruses. The significance of our research is producing a promising universal vaccine candidate which provides protection against a wider range of virus strains over a wider range of time.

Pre-existing immunity to influenza aids ferrets in developing stronger and broader H3 vaccine-induced antibody responses.

Influenza seasons occur annually, building immune history for individuals, but the influence of this history on subsequent influenza vaccine protection remains unclear. We extracted data from an animal trial to study its potential impact. The trial involved 80 ferrets, each receiving either one type of infection or a placebo before vaccination. We quantified the vaccine protection by evaluating hemagglutination inhibition (HAI) antibody titer responses. We tested whether hosts with different infection histories exhibited similar level of responses when receiving the same vaccine for all homologous and heterologous outcomes. We observed that different pre-existing immunities were generally beneficial to vaccine induced responses, but varied in magnitude. Without pre-immunity, post-vaccination HAI titers after the 1st dose of the vaccine were less likely to be above 1:40, and a booster shot was needed. Our study suggests that pre-existing immunity may strengthen and extend the homologous and heterologous vaccine responses.

rAAV expressing a COBRA-designed influenza hemagglutinin generates a protective and durable adaptive immune response with a single dose.

Influenza remains a worldwide public health threat. Although seasonal influenza vaccines are currently the best means of preventing severe disease, the standard-of-care vaccines require frequent updating due to antigenic drift and can have low efficacy, particularly in vulnerable populations. Here, we demonstrate that a single administration of a recombinant adenovirus-associated virus (rAAV) vector expressing a computationally optimized broadly reactive antigen (COBRA)-derived influenza H1 hemagglutinin (HA) induces strongly neutralizing and broadly protective antibodies in naïve mice and ferrets with pre-existing influenza immunity. Following a lethal viral challenge, the rAAV-COBRA vaccine allowed for significantly reduced viral loads in the upper and lower respiratory tracts and complete protection from morbidity and mortality that lasted for at least 5 months post-vaccination. We observed no signs of antibody waning during this study. CpG motif enrichment of the antigen can act as an internal adjuvant to further enhance the immune responses to allow for lower vaccine dosages with the induction of unique interferon-producing CD4+ and CD8+ T cells specific to HA head and stem peptide sequences. Our studies highlight the utility of rAAV as an effective platform to improve seasonal influenza vaccines. IMPORTANCE: Developing an improved seasonal influenza vaccine remains an ambitious goal of researchers and clinicians alike. With influenza routinely causing severe epidemics with the potential to rise to pandemic levels, it is critical to create an effective, broadly protective, and durable vaccine to improve public health worldwide. As a potential solution, we created a rAAV viral vector expressing a COBRA-optimized influenza hemagglutinin antigen with modestly enriched CpG motifs to evoke a robust and long-lasting immune response after a single intramuscular dose without needing boosts or adjuvants. Importantly, the rAAV vaccine boosted antibody breadth to future strains in ferrets with pre-existing influenza immunity. Together, our data support further investigation into the utility of viral vectors as a potential avenue to improve our seasonal influenza vaccines.

A Computationally Optimized Broadly Reactive Hemagglutinin and Neuraminidase Vaccine Boosts Antibody-Secreting Cells and Induces a Robust Serological Response, Preventing Lung Damage in a Pre-Immune Model.

The hemagglutinin (HA) and neuraminidase (NA) surface proteins are the primary and secondary immune targets for most influenza vaccines. In this study, H2, H5, H7, N1, and N2 antigens designed by the computationally optimized broadly reactive antigen (COBRA) methodology were incorporated into an adjuvant-formulated vaccine to assess the protective efficacy and immune response against A/Hong Kong/125/2017 H7N9 virus challenge in pre-immune mice. The elicited antibodies bound to H2, H5, H7, N1, and N2 wild-type antigens; cH6/1 antigens; and cH7/3 antigens, with hemagglutinin inhibition (HAI) activity against broad panels of the H2Nx, H5Nx, and H7Nx influenza strains. Mice vaccinated with the pentavalent COBRA HA/NA vaccine showed little to no weight loss, no clinical signs of diseases, and were protected from mortality when challenged with the lethal H7N9 virus. Virus titers in the lungs of vaccinated mice were lower and cleared more rapidly than in mock-vaccinated mice. Some vaccinated mice showed no detectable lung injury or inflammation. Antibody-secreting cells were significantly increased in COBRA-vaccinated mice, with higher total Ig and H7-specific ASC. Thus, the combination of H2, H5, H7, N1, and N2 COBRA antigens presents a potential for the formulation of a universal influenza virus vaccine.

Polymeric cGAMP microparticles affect the immunogenicity of a broadly active influenza mRNA lipid nanoparticle vaccine.

Influenza outbreaks are a major burden worldwide annually. While seasonal vaccines do provide protection against infection, they are limited in that they need to be updated every year to account for the constantly mutating virus. Recently, lipid nanoparticles (LNPs) encapsulating mRNA have seen major success as a vaccine platform for SARS-CoV-2. Herein, we applied LNPs to deliver an mRNA encoding a computationally optimized broadly active (COBRA) influenza immunogen. These COBRA mRNA LNPs induced a broadly active neutralizing antibody response and protection after lethal influenza challenge. To further increase the immunogenicity of the COBRA mRNA LNPs, we combined them with acetalated dextran microparticles encapsulating a STING agonist. Contrary to recent findings, the STING agonist decreased the immunogenicity of the COBRA mRNA LNPs which was likely due to a decrease in mRNA translation as shown in vitro. Overall, this work aids in future selection of adjuvants to use with mRNA LNP vaccines.

Immune imprinting in early life shapes cross-reactivity to influenza B virus haemagglutinin.

Influenza exposures early in life are believed to shape future susceptibility to influenza infections by imprinting immunological biases that affect cross-reactivity to future influenza viruses. However, direct serological evidence linked to susceptibility is limited. Here we analysed haemagglutination-inhibition titres in 1,451 cross-sectional samples collected between 1992 and 2020, from individuals born between 1917 and 2008, against influenza B virus (IBV) isolates from 1940 to 2021. We included testing of 'future' isolates that circulated after sample collection. We show that immunological biases are conferred by early life IBV infection and result in lineage-specific cross-reactivity of a birth cohort towards future IBV isolates. This translates into differential estimates of susceptibility between birth cohorts towards the B/Yamagata and B/Victoria lineages, predicting lineage-specific birth-cohort distributions of observed medically attended IBV infections. Our data suggest that immunological measurements of imprinting could be important in modelling and predicting virus epidemiology.

Longitudinal assessment of human antibody binding to hemagglutinin elicited by split-inactivated influenza vaccination over six consecutive seasons.

Participants between the ages of 10-86 years old were vaccinated with split-inactivated influenza vaccine (Fluzone®) in six consecutive influenza seasons from 2016-2017 to 2021-2022. Vaccine effectiveness varies from season to season as a result of both host immune responses as well as evolutionary changes in the influenza virus surface glycoproteins that provide challenges to vaccine manufacturers to produce more effective annual vaccines. Next generation influenza vaccines are in development and may provide protective immune responses against a broader number of influenza viruses and reduce the need for annual vaccination. An improved understanding how current influenza vaccines are influenced by human host immune responses in people of different ages and co-morbidities is necessary for designing the next-generation of 'universal' or broadly-protective influenza vaccines. Overall, pre-existing immune responses to previous influenza virus exposures, either by past infections or vaccinations, is a critical factor influencing host responses to seasonal influenza vaccination. Participants vaccinated in consecutive seasons had reduced serum hemagglutination-inhibition (HAI) activity against strains included in the vaccine compared to participants that had not been vaccinated in the preceding 1-2 years prior to entering this study. The magnitude and breadth of these antibody responses were also modulated by the age of the participant. Elderly participants over 65 years of age, in general, had lower pre-existing HAI titers each season prior to vaccination with lower post-vaccination titers compared to children or young adults under the age of 35. The administration of higher doses (HD) of the split-inactivated vaccine enhanced the antibody titers in the elderly. This report showcases 6 consecutive years of antibody HAI activity in human subjects receiving seasonal split-inactivated influenza vaccine.

Inactivated influenza virus vaccines expressing COBRA hemagglutinin elicited broadly reactive, long-lived protective antibodies.

The influenza viruses cause seasonal respiratory illness that affect millions of people globally every year. Prophylactic vaccines are the recommended method to prevent the breakout of influenza epidemics. One of the current commercial influenza vaccines consists of inactivated viruses that are selected months prior to the start of a new influenza season. In many seasons, the vaccine effectiveness (VE) of these vaccines can be relatively low. Therefore, there is an urgent need to develop an improved, more universal influenza vaccine (UIV) that can provide broad protection against various drifted strains in all age groups. To meet this need, the computationally optimized broadly reactive antigen (COBRA) methodology was developed to design a hemagglutinin (HA) molecule as a new influenza vaccine. In this study, COBRA HA-based inactivated influenza viruses (IIV) expressing the COBRA HA from H1 or H3 influenza viruses were developed and characterized for the elicitation of immediate and long-term protective immunity in both immunologically naïve or influenza pre-immune animal models. These results were compared to animals vaccinated with IIV vaccines expressing wild-type H1 or H3 HA proteins (WT-IIV). The COBRA-IIV elicited long-lasting broadly reactive antibodies that had hemagglutination-inhibition (HAI) activity against drifted influenza variants.

COBRA HA and NA vaccination elicits long-live protective immune responses against pre-pandemic H2, H5, and H7 influenza virus subtypes.

Highly pathogenic avian influenza (HPAI) viruses remain a major threat to both the poultry industry and human public health, and these viruses continue to spread worldwide. In this study, mice were vaccinated with COBRA H2, H5, and H7 hemagglutinin (HA) and two neuraminidase (NA) proteins, N1 and N2. Vaccinated mice were fully protected against lethal challenge with H5N6 influenza virus. Sera collected after vaccination showed cross-reactive IgG antibodies against a panel of wild-type H2, H5, and H7 HA proteins, and N1 and N2 NA proteins. Mice with pre-existing immunity to H1N1 and H3N2 influenza viruses that were subsequently vaccinated with COBRA HA/NA vaccines had enhanced anti-HA stem antibodies compared to vaccinated mice without pre-existing immunity. In addition, sera collected after vaccination had hemagglutinin inhibitory activity against a panel of H2Nx, H5Nx, and H7Nx influenza viruses. These protective antibodies were maintained up for up to 4 months after vaccination.

Multi-COBRA hemagglutinin formulated with cGAMP microparticles elicits protective immune responses against influenza viruses.

In humans, seasonal influenza viruses cause epidemics. Avian influenza viruses are of particular concern because they can infect multiple species and lead to unpredictable and severe disease. Therefore, there is an urgent need for a universal influenza vaccine that provides protection against all influenza strains. The cyclic GMP-AMP (cGAMP) is a promising adjuvant for subunit vaccines, which promotes type I interferons' production through the stimulator of interferon genes (STING) pathway. The encapsulation of cGAMP in acetalated dextran (Ace-DEX) microparticles (MPs) enhances its intracellular delivery. In this study, the Computationally Optimized Broadly Reactive Antigen (COBRA) methodology was used to generate H1, H3, and H5 vaccine candidates. Monovalent and multivalent COBRA HA vaccines formulated with cGAMP Ace-DEX MPs were evaluated in mice for protective antibody responses. cGAMP MPs adjuvanted COBRA HA vaccines elicited robust antigen-specific antibodies following vaccination. Compared with COBRA HA vaccine groups with no adjuvant or blank MPs, the cGAMP MPs enhanced HAI activity elicited by COBRA HA vaccines. The HAI activity was not significantly different between cGAMP MPs adjuvanted monovalent or multivalent COBRA HA vaccines. The cGAMP MPs adjuvanted COBRA vaccine groups had higher antigen-specific IgG2a-binding titers than the COBRA vaccine groups with no adjuvant or blank MPs. The COBRA vaccines formulated with cGAMP MPs mitigated diseases caused by influenza viral challenge and decreased pulmonary viral titers in mice. Therefore, the formulation of COBRA vaccines plus cGAMP MPs is a promising universal influenza vaccine that elicits protective immune responses against human seasonal and pre-pandemic strains. IMPORTANCE: Influenza viruses cause severe respiratory disease, particularly in the very young and the elderly. Next-generation influenza vaccines are needed to protect against new influenza variants. This report used a promising adjuvant, cyclic GMP-AMP (cGAMP), to enhance the elicited antibodies by an improved influenza hemagglutinin candidate and protect against influenza virus infection. Overall, adding adjuvants to influenza vaccines is an effective method to improve vaccines.

The response to influenza vaccination is associated with DNA methylation-driven regulation of T cell innate antiviral pathways.

Background: The effect of vaccination on the epigenome remains poorly characterized. In previous research, we identified an association between seroprotection against influenza and DNA methylation at sites associated with the RIG-1 signaling pathway, which recognizes viral double-stranded RNA and leads to a type I interferon response. However, these studies did not fully account for confounding factors including age, gender, and BMI, along with changes in cell type composition. Results: Here, we studied the influenza vaccine response in a longitudinal cohort vaccinated over two consecutive years (2019-2020 and 2020-2021), using peripheral blood mononuclear cells and a targeted DNA methylation approach. To address the effects of multiple factors on the epigenome, we designed a multivariate multiple regression model that included seroprotection levels as quantified by the hemagglutination-inhibition (HAI) assay test. Conclusions: Our findings indicate that 179 methylation sites can be combined as potential signatures to predict seroprotection. These sites were not only enriched for genes involved in the regulation of the RIG-I signaling pathway, as found previously, but also enriched for other genes associated with innate immunity to viruses and the transcription factor binding sites of BRD4, which is known to impact T cell memory. We propose a model to suggest that the RIG-I pathway and BRD4 could potentially be modulated to improve immunization strategies.

Mastoparan-7 adjuvanted COBRA H1 and H3 hemagglutinin influenza vaccines.

Adjuvants enhance, prolong, and modulate immune responses by vaccine antigens to maximize protective immunity and enable more effective immunization in the young and elderly. Most adjuvants are formulated with injectable vaccines. However, an intranasal route of vaccination may induce mucosal and systemic immune responses for enhancing protective immunity in individuals and be easier to administer compared to injectable vaccines. In this study, a next generation of broadly-reactive influenza hemagglutinin (HA) vaccines were developed using the Computationally Optimized Broadly Reactive Antigen (COBRA) methodology. These HA vaccines were formulated with Mastoparan 7 (M7-NH2) mast cell degranulating peptide adjuvant and administered intranasally to determine vaccine-induced seroconversion of antibodies against a panel of influenza viruses and protection following infection with H1N1 and H3N2 viruses in mice. Mice vaccinated intranasally with M7-NH2-adjuvanted COBRA HA vaccines had high HAIs against a panel of H1N1 and H3N2 influenza viruses and were protected against both morbidity and mortality, with reduced viral lung titers, following challenge with an H1N1 influenza virus. Additionally, M7-NH2 adjuvanted COBRA HA vaccines induced Th2 skewed immune responses with robust IgG and isotype antibodies in the serum and mucosal lung lavages. Overall, this intranasally delivered M7-NH2 -adjuvanted COBRA HA vaccine provides effective protection against drifted H1N1 and H3N2 viruses.