Longitudinal analysis of SARS-CoV-2 infection and vaccination in the LA-SPARTA cohort reveals increased risk of infection in vaccinated Hispanic participants.

Introduction: SARS-CoV-2 is the etiologic agent of coronavirus disease 2019 (COVID-19). Questions remain regarding correlates of risk and immune protection against COVID-19. Methods: We prospectively enrolled 200 participants with a high risk of SARS-CoV-2 occupational exposure at a U.S. medical center between December 2020 and April 2022. Participant exposure risks, vaccination/infection status, and symptoms were followed longitudinally at 3, 6, and 12 months, with blood and saliva collection. Serological response to the SARS-CoV-2 spike holoprotein (S), receptor binding domain (RBD) and nucleocapsid proteins (NP) were quantified by ELISA assay. Results: Based on serology, 40 of 200 (20%) participants were infected. Healthcare and non-healthcare occupations had equivalent infection incidence. Only 79.5% of infected participants seroconverted for NP following infection, and 11.5% were unaware they had been infected. The antibody response to S was greater than to RBD. Hispanic ethnicity was associated with 2-fold greater incidence of infection despite vaccination in this cohort. Discussion: Overall, our findings demonstrate: 1) variability in the antibody response to SARS-CoV-2 infection despite similar exposure risk; 2) the concentration of binding antibody to the SARS-CoV-2 S or RBD proteins is not directly correlated with protection against infection in vaccinated individuals; and 3) determinants of infection risk include Hispanic ethnicity despite vaccination and similar occupational exposure.

Recombinant Protein Vaccines Formulated with Enantio-Specific Cationic Lipid R-DOTAP Induce Protective Cellular and Antibody-Mediated Immune Responses in Mice.

Adjuvants are essential components of subunit vaccines added to enhance immune responses to antigens through immunomodulation. Very few adjuvants have been approved for human use by regulatory agencies due to safety concerns. Current subunit vaccine adjuvants approved for human use are very effective in promoting humoral immune responses but are less effective at promoting T-cell immunity. In this study, we evaluated a novel pure enantio-specific cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (R-DOTAP) as an immunomodulator for subunit vaccines capable of inducing both humoral- and cellular-mediated immunity. Using recombinant protein antigens derived from SARS-CoV2 spike or novel computationally optimized broadly reactive influenza antigen (COBRA) proteins, we demonstrated that R-DOTAP nanoparticles promoted strong cellular- and antibody-mediated immune responses in both monovalent and bivalent vaccines. R-DOTAP-based vaccines induced antigen-specific and polyfunctional CD8+ and CD4+ effector T cells and memory T cells, respectively. Antibody responses induced by R-DOTAP showed a balanced Th1/Th2 type immunity, neutralizing activity and protection of mice from challenge with live SARS-CoV2 or influenza viruses. R-DOTAP also facilitated significant dose sparing of the vaccine antigens. These studies demonstrate that R-DOTAP is an excellent immune stimulator for the production of next-generation subunit vaccines containing multiple recombinant proteins.

Functional HIV-1/HCV cross-reactive antibodies isolated from a chronically co-infected donor.

Despite prolific efforts to characterize the antibody response to human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) mono-infections, the response to chronic co-infection with these two ever-evolving viruses is poorly understood. Here, we investigate the antibody repertoire of a chronically HIV-1/HCV co-infected individual using linking B cell receptor to antigen specificity through sequencing (LIBRA-seq). We identify five HIV-1/HCV cross-reactive antibodies demonstrating binding and functional cross-reactivity between HIV-1 and HCV envelope glycoproteins. All five antibodies show exceptional HCV neutralization breadth and effector functions against both HIV-1 and HCV. One antibody, mAb688, also cross-reacts with influenza and coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We examine the development of these antibodies using next-generation sequencing analysis and lineage tracing and find that somatic hypermutation established and enhanced this reactivity. These antibodies provide a potential future direction for therapeutic and vaccine development against current and emerging infectious diseases. More broadly, chronic co-infection represents a complex immunological challenge that can provide insights into the fundamental rules that underly antibody-antigen specificity.

α2,6-Sialylation Is Upregulated in Severe COVID-19, Implicating the Complement Cascade.

Better understanding of the molecular mechanisms underlying COVID-19 severity is desperately needed in current times. Although hyper-inflammation drives severe COVID-19, precise mechanisms triggering this cascade and what role glycosylation might play therein are unknown. Here we report the first high-throughput glycomic analysis of COVID-19 plasma samples and autopsy tissues. We find that α2,6-sialylation is upregulated in the plasma of patients with severe COVID-19 and in autopsied lung tissue. This glycan motif is enriched on members of the complement cascade (e.g., C5, C9), which show higher levels of sialylation in severe COVID-19. In the lung tissue, we observe increased complement deposition, associated with elevated α2,6-sialylation levels, corresponding to elevated markers of poor prognosis (IL-6) and fibrotic response. We also observe upregulation of the α2,6-sialylation enzyme ST6GAL1 in patients who succumbed to COVID-19. Our work identifies a heretofore undescribed relationship between sialylation and complement in severe COVID-19, potentially informing future therapeutic development.

Antibody affinity and cross-variant neutralization of SARS-CoV-2 Omicron BA.1, BA.2 and BA.3 following third mRNA vaccination.

There is limited knowledge on durability of neutralization capacity and antibody affinity maturation generated following two versus three doses of SARS-CoV-2 mRNA vaccines in naïve versus convalescent individuals (hybrid immunity) against the highly transmissible Omicron BA.1, BA.2 and BA.3 subvariants. Virus neutralization titers against the vaccine-homologous strain (WA1) and Omicron sublineages are measured in a pseudovirus neutralization assay (PsVNA). In addition, antibody binding and antibody affinity against spike proteins from WA1, BA.1, and BA.2 is determined using surface plasmon resonance (SPR). The convalescent individuals who after SARS-CoV-2 infection got vaccinated develop hybrid immunity that shows broader neutralization activity and cross-reactive antibody affinity maturation against the Omicron BA.1 and BA.2 after either second or third vaccination compared with naïve individuals. Neutralization activity correlates with antibody affinity against Omicron subvariants BA.1 and BA.2 spikes. Importantly, at four months post-third vaccination the neutralization activity and antibody affinity against the Omicron subvariants is maintained and trended higher for the individuals with hybrid immunity compared with naïve adults. These findings about hybrid immunity resulting in superior immune kinetics, breadth, and durable high affinity antibodies support the need for booster vaccinations to provide effective protection from emerging SARS-CoV-2 variants like the rapidly spreading Omicron subvariants.

Kinetic of the Antibody Response Following AddaVax-Adjuvanted Immunization with Recombinant Influenza Antigens.

Notwithstanding the current SARS-CoV-2 pandemic, influenza virus infection still represents a global health concern in terms of hospitalizations and possible pandemic threats. The objective of next-generation influenza vaccines is not only to increase the breadth of response but also to improve the elicitation of an effective and robust immune response, especially in high-risk populations. To achieve this second objective, the administration of adjuvanted influenza vaccines has been considered. In this regard, the monitoring and characterization of the antibody response associated with the administration of adjuvanted vaccines has been evaluated in this study in order to shed light on the kinetic, magnitude and subclass usage of antibody secreting cells (ASCs) as well as of circulating antigen-specific serum antibodies. Specifically, we utilized the DBA/2J mouse model to assess the kinetic, magnitude and IgG subclass usage of the antibody response following an intramuscular (IM) or intraperitoneal (IP) immunization regimen with AddaVax-adjuvanted bivalent H1N1 and H3N2 computationally optimized broadly reactive antigen (COBRA) influenza recombinant hemagglutinins (rHAs). While the serological evaluation revealed a homogeneous kinetic of the antibody response, the detection of the ASCs through a FluoroSpot platform revealed a different magnitude, subclass usage and kinetic of the antigen-specific IgG secreting cells peaking at day 5 and day 9 following the IP and IM immunization, respectively.

High Prevalence of Both Previous Infection with SARS-CoV-2 and Persistent Symptoms.

INTRODUCTION: Universities are unique settings with large populations, congregate housing, and frequent attendance of events in large groups. However, the current prevalence of previous COVID-19 infection in university students, including symptomatic and asymptomatic disease, is unknown. Our goal therefore was to determine the prevalence of previous infection, risk factors for infection, and the prevalence of persistent symptoms following infection among university students. METHODS: This was a cross-sectional study set in a large public university between January 22 and March 22, 2021. We surveyed students about demographics, risk factors, and symptoms, and simultaneously tested their saliva for IgA antibodies to SARS-CoV-2. To estimate the prevalence of previous infection we adjusted our intentional sample of a diverse student population for year in school and age to resemble the composition of the entire student body and adjusted for the imperfect sensitivity and specificity of the antibody test. Univariate and multiple regression analysis was used to identify independent risk factors for infection, and the proportion of students with persistent symptoms following acute infection was determined. RESULTS: A total of 488 students completed the survey, 432 had a valid antibody result, and 428 had both. The estimated prevalence of previous infection for 432 participants with valid antibody results was 41%. Of 145 students in our sample with a positive antibody test, 41.4% denied having a previous positive polymerase chain reaction (PCR) test for SARS-CoV-2 and presumably had an asymptomatic infection; in our adjusted analysis we estimate that approximately 2-thirds of students had asymptomatic infections. Independent risk factors for infection included male sex, having a roommate with a known symptomatic infection, and having two or fewer roommates. More frequent attendance of parties and bars was a univariate risk factor, but not in the multiple regression analysis. Of 122 students reporting a previous symptomatic infection, 14 (11.4%) reported persistent symptoms consistent with postacute COVID-19 a median of 132 days later. CONCLUSIONS AND RELEVANCE: Previous COVID-19 infection, both symptomatic and asymptomatic, was common at a large university. Measures that could prevent resurgence of the infection when students return to campus include mandatory vaccination policies, mass surveillance testing, and testing of sewage for antigen to SARS-CoV-2.

Template-Assisted De Novo Sequencing of SARS-CoV-2 and Influenza Monoclonal Antibodies by Mass Spectrometry.

In this study, we used multiple enzyme digestions, coupled with higher-energy collisional dissociation (HCD) and electron-transfer/higher-energy collision dissociation (EThcD) fragmentation to develop a mass-spectrometric (MS) method for determining the complete protein sequence of monoclonal antibodies (mAbs). The method was refined on an mAb of a known sequence, a SARS-CoV-1 antireceptor binding domain (RBD) spike monoclonal antibody. The data were searched using Supernovo to generate a complete template-assisted de novo sequence for this and two SARS-CoV-2 mAbs of known sequences resulting in correct sequences for the variable regions and correct distinction of Ile and Leu residues. We then used the method on a set of 25 antihemagglutinin (HA) influenza antibodies of unknown sequences and determined high confidence sequences for >99% of the complementarity determining regions (CDRs). The heavy-chain and light-chain genes were cloned and transfected into cells for recombinant expression followed by affinity purification. The recombinant mAbs displayed binding curves matching the original mAbs with specificity to the HA influenza antigen. Our findings indicate that this methodology results in almost complete antibody sequence coverage with high confidence results for CDR regions on diverse mAb sequences.

PARIS and SPARTA: Finding the Achilles' Heel of SARS-CoV-2.

To understand reinfection rates and correlates of protection for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), we established eight different longitudinal cohorts in 2020 under the umbrella of the PARIS (Protection Associated with Rapid Immunity to SARS-CoV-2)/SPARTA (SARS SeroPrevalence And Respiratory Tract Assessment) studies. Here, we describe the PARIS/SPARTA cohorts, the harmonized assays and analysis that are performed across the cohorts, as well as case definitions for SARS-CoV-2 infection and reinfection that have been established by the team of PARIS/SPARTA investigators. IMPORTANCE Determining reinfection rates and correlates of protection against SARS-CoV-2 infection induced by both natural infection and vaccination is of high significance for the prevention and control of coronavirus disease 2019 (COVID-19). Furthermore, understanding reinfections or infection after vaccination and the role immune escape plays in these scenarios will inform the need for updates of the current SARS-CoV-2 vaccines and help update guidelines suitable for the postpandemic world.

Sex disparities in influenza: A multiscale network analysis

Summary Sex differences in the pathogenesis of infectious diseases because of differential immune responses between females and males have been well-documented for multiple pathogens. However, the molecular mechanism underlying the observed sex differences in influenza virus infection remains poorly understood. In this study, we used a network-based approach to characterize the blood transcriptome collected over the course of infection with influenza A virus from female and male ferrets to dissect sex-biased gene expression. We identified significant differences in the temporal dynamics and regulation of immune responses between females and males. Our results elucidate sex-differentiated pathways involved in the unfolded protein response (UPR), lipid metabolism, and inflammatory responses, including a female-biased IRE1/XBP1 activation and male-biased crosstalk between metabolic reprogramming and IL-1 and AP-1 pathways. Overall, our study provides molecular insights into sex differences in transcriptional regulation of immune responses and contributes to a better understanding of sex biases in influenza pathogenesis. Graphical Highlights • Regulation of immune responses between females and males is significantly different• Rapid activation of UPR in females triggers potent immune and inflammatory responses• Male-specific regulatory pattern in the AP1 pathway indicate a bias in immune response Biological sciences;Immunology;Virology;Systems biology

The Effect of Waning on Antibody Levels and Memory B Cell Recall following SARS-CoV-2 Infection or Vaccination.

In order to longitudinally track SARS-CoV-2 antibody levels after vaccination or infection, we assessed anti-RBD antibody levels in over 1000 people and found no significant decrease in antibody levels during the first 14 months after infection in unvaccinated participants, however, a significant waning of antibody levels was observed following vaccination. Participants who were pre-immune to SARS-CoV-2 prior to vaccination seroconverted to higher antibody levels, which were maintained at higher levels than in previously infected, unvaccinated participants. Older participants exhibited lower level of antibodies after vaccination, but a higher level after infection than younger people. The rate of antibody waning was not affected by pre-immunity or age. Participants who received a third dose of an mRNA vaccine not only increased their antibody levels ~14-fold, but also had ~3 times more antibodies compared to when they received their primary vaccine series. PBMC-derived memory B cells from 13 participants who lost all circulating antibodies were differentiated into antibody secreting cells (ASCs). There was a significant recall of memory B cell ASCs in the absence of serum antibodies in 5-8 of the 10 vaccinated participants, but not in any of the 3 infected participants, suggesting a strong connection between antibody levels and the effectiveness of memory B cell recall.

SARS-CoV-2 and Influenza A Virus Coinfections in Ferrets.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and seasonal influenza viruses are cocirculating in the human population. However, only a few cases of viral coinfection with these two viruses have been documented in humans with some people having severe disease and others mild disease. To examine this phenomenon, ferrets were coinfected with SARS-CoV-2 and human seasonal influenza A viruses (IAVs; H1N1 or H3N2) and were compared to animals that received each virus alone. Ferrets were either immunologically naive to both viruses or vaccinated with the 2019 to 2020 split-inactivated influenza virus vaccine. Coinfected naive ferrets lost significantly more body weight than ferrets infected with each virus alone and had more severe inflammation in both the nose and lungs compared to that of ferrets that were single infected with each virus. Coinfected, naive animals had predominantly higher IAV titers than SARS-CoV-2 titers, and IAVs were efficiently transmitted by direct contact to the cohoused ferrets. Comparatively, SARS-CoV-2 failed to transmit to the ferrets that cohoused with coinfected ferrets by direct contact. Moreover, vaccination significantly reduced IAV titers and shortened the viral shedding but did not completely block direct contact transmission of the influenza virus. Notably, vaccination significantly ameliorated influenza-associated disease by protecting vaccinated animals from severe morbidity after IAV single infection or IAV and SARS-CoV-2 coinfection, suggesting that seasonal influenza virus vaccination is pivotal to prevent severe disease induced by IAV and SARS-CoV-2 coinfection during the COVID-19 pandemic. IMPORTANCE Influenza A viruses cause severe morbidity and mortality during each influenza virus season. The emergence of SARS-CoV-2 infection in the human population offers the opportunity to potential coinfections of both viruses. The development of useful animal models to assess the pathogenesis, transmission, and viral evolution of these viruses as they coinfect a host is of critical importance for the development of vaccines and therapeutics. The ability to prevent the most severe effects of viral coinfections can be studied using effect coinfection ferret models described in this report.

Clinical validation of a multiplex PCR-based detection assay using saliva or nasopharyngeal samples for SARS-Cov-2, influenza A and B.

The COVID-19 pandemic has resulted in significant diversion of human and material resources to COVID-19 diagnostics, to the extent that influenza viruses and co-infection in COVID-19 patients remains undocumented and pose serious public-health consequences. We optimized and validated a highly sensitive RT-PCR based multiplex-assay for the detection of SARS-CoV-2, influenza A and B viruses in a single-test. This study evaluated clinical specimens (n = 1411), 1019 saliva and 392 nasopharyngeal swab (NPS), tested using two-assays: FDA-EUA approved SARS-CoV-2 assay that targets N and ORF1ab gene, and the PKamp-RT-PCR based assay that targets SARS-CoV-2, influenza viruses A and B. Of the 1019 saliva samples, 17.0% (174/1019) tested positive for SARS-CoV-2 using either assay. The detection rate for SARS-CoV-2 was higher with the multiplex assay compared to SARS-specific assay [91.9% (160/174) vs. 87.9% (153/174)], respectively. Of the 392 NPS samples, 10.4% (41/392) tested positive for SARS-CoV-2 using either assay. The detection rate for SARS-CoV-2 was higher with the multiplex assay compared to SARS-specific assay [97.5% (40/41) vs. 92.1% (39/41)], respectively. This study presents clinical validation of a multiplex-PCR assay for testing SARS-CoV-2, influenza A and B viruses, using NPS and saliva samples, and demonstrates the feasibility of implementing the assay without disrupting the existing laboratory workflow.

Antibody affinity maturation and cross-variant activity following SARS-CoV-2 mRNA vaccination: Impact of prior exposure and sex.

BACKGROUND: Limited knowledge exists regarding antibody affinity maturation following mRNA vaccination in naïve vs. COVID-19 recovered individuals and potential sex differences. METHODS: We elucidated post-vaccination antibody profiles of 69 naïve and 17 COVID-19 convalescent adults using pseudovirus neutralization assay (PsVNA) covering SARS-CoV-2 WA-1, variants of concern (VOCs) and variants of interest (VOIs). Surface Plasmon Resonance (SPR) was used to measure antibody affinity against prefusion spike and receptor binding domain (RBD) and RBD mutants. FINDINGS: Higher neutralizing antibodies were observed in convalescent vs. naïve adults against, WA-1, VOCs, and VOIs. Antibody binding to RBD and RBD mutants showed lower binding of post-vaccination sera from naïve compared with convalescent individuals. Moreover, we observed early antibody affinity maturation in convalescent individuals after one vaccine dose and higher antibody affinity after two doses compared with the naïve group. Among the naïve participants, antibody affinity against the SARS-CoV-2 prefusion spike was significantly higher for males than females even though there were no difference in neutralization titers between sexes. INTERPRETATION: This study demonstrates the impact of prior infection on vaccine-induced antibody affinity maturation and difference in antibody affinity between males and females. Further studies are needed to determine whether antibody affinity may contribute to correlates of protection against SARS-CoV-2 and its variants. FUNDING: The antibody characterization work described in this manuscript was supported by FDA's Medical Countermeasures Initiative (MCMi) grant #OCET 2021-1565 to S.K and intramural FDA-CBER COVID-19 supplemental funds. The SPARTA program was supported by the National Institute of Allergy and Infectious Diseases (NIAID), U.S. National Institutes of Health (NIH), Department of Health and Human Services contract 75N93019C00052, and the University of Georgia (US) grant UGA-001. T.M.R is also supported by the Georgia Research Alliance (US) grant GRA-001. The CTRU was supported by the National Center for Advancing Translational Sciences of the National Institutes of Health under Award Number UL1TR002378.

Influenza vaccination in the elderly boosts antibodies against conserved viral proteins and egg-produced glycans.

Seasonal influenza vaccination elicits a diminished adaptive immune response in the elderly, and the mechanisms of immunosenescence are not fully understood. Using Ig-Seq, we found a marked increase with age in the prevalence of cross-reactive (CR) serum antibodies that recognize both the H1N1 (vaccine-H1) and H3N2 (vaccine-H3) components of an egg-produced split influenza vaccine. CR antibodies accounted for 73% ± 18% of the serum vaccine responses in a cohort of elderly donors, 65% ± 15% in late middle-aged donors, and only 13% ± 5% in persons under 35 years of age. The antibody response to non-HA antigens was boosted by vaccination. Recombinant expression of 19 vaccine-H1+H3 CR serum monoclonal antibodies (s-mAbs) revealed that they predominantly bound to non-HA influenza proteins. A sizable fraction of vaccine-H1+H3 CR s-mAbs recognized with high affinity the sulfated glycans, in particular sulfated type 2 N-acetyllactosamine (Galß1-4GalNAcß), which is found on egg-produced proteins and thus unlikely to contribute to protection against influenza infection in humans. Antibodies against sulfated glycans in egg-produced vaccine had been identified in animals but were not previously characterized in humans. Collectively, our results provide a quantitative basis for how repeated exposure to split influenza vaccine correlates with unintended focusing of serum antibody responses to non-HA antigens that may result in suboptimal immunity against influenza.

SARS-CoV-2 mRNA Vaccines Elicit Different Responses in Immunologically Naïve and Pre-Immune Humans.

As the COVID-19 pandemic continues, the authorization of vaccines for emergency use has been crucial in slowing down the rate of infection and transmission of the SARS-CoV-2 virus that causes COVID-19. In order to investigate the longitudinal serological responses to SARS-CoV-2 natural infection and vaccination, a large-scale, multi-year serosurveillance program entitled SPARTA (SARS SeroPrevalence and Respiratory Tract Assessment) was initiated at 4 locations in the U.S. The serological assay presented here measuring IgG binding to the SARS-CoV-2 receptor binding domain (RBD) detected antibodies elicited by SARS-CoV-2 infection or vaccination with a 95.5% sensitivity and a 95.9% specificity. We used this assay to screen more than 3100 participants and selected 20 previously infected pre-immune and 32 immunologically naïve participants to analyze their antibody binding to RBD and viral neutralization (VN) responses following vaccination with two doses of either the Pfizer-BioNTech BNT162b2 or the Moderna mRNA-1273 vaccine. Vaccination not only elicited a more robust immune reaction than natural infection, but the level of neutralizing and anti-RBD antibody binding after vaccination is also significantly higher in pre-immune participants compared to immunologically naïve participants (p<0.0033). Furthermore, the administration of the second vaccination did not further increase the neutralizing or binding antibody levels in pre-immune participants (p=0.69). However, ~46% of the immunologically naïve participants required both vaccinations to seroconvert.

High-Throughput Next-Generation Sequencing Respiratory Viral Panel: A Diagnostic and Epidemiologic Tool for SARS-CoV-2 and Other Viruses.

Two serious public health challenges have emerged in the current COVID-19 pandemic namely, deficits in SARS-CoV-2 variant monitoring and neglect of other co-circulating respiratory viruses. Additionally, accurate assessment of the evolution, extent, and dynamics of the outbreak is required to understand the transmission of the virus. To address these challenges, we evaluated 533 samples using a high-throughput next-generation sequencing (NGS) respiratory viral panel (RVP) that includes 40 viral pathogens. The performance metrics revealed a PPA, NPA, and accuracy of 95.98%, 85.96%, and 94.4%, respectively. The clade for pangolin lineage B that contains certain distant variants, including P4715L in ORF1ab, Q57H in ORF3a, and S84L in ORF8 covarying with the D614G spike protein mutation, were the most prevalent early in the pandemic in Georgia, USA. The isolates from the same county formed paraphyletic groups, indicating virus transmission between counties. The study demonstrates the clinical and public health utility of the NGS-RVP to identify novel variants that can provide actionable information to prevent or mitigate emerging viral threats and models that provide insights into viral transmission patterns and predict transmission/resurgence of regional outbreaks as well as providing critical information on co-circulating respiratory viruses that might be independent factors contributing to the global disease burden.

Immunisation of ferrets and mice with recombinant SARS-CoV-2 spike protein formulated with Advax-SM adjuvant protects against COVID-19 infection.

The development of a safe and effective vaccine is a key requirement to overcoming the COVID-19 pandemic. Recombinant proteins represent the most reliable and safe vaccine approach but generally require a suitable adjuvant for robust and durable immunity. We used the SARS-CoV-2 genomic sequence and in silico structural modelling to design a recombinant spike protein vaccine (Covax-19™). A synthetic gene encoding the spike extracellular domain (ECD) was inserted into a baculovirus backbone to express the protein in insect cell cultures. The spike ECD was formulated with Advax-SM adjuvant and first tested for immunogenicity in C57BL/6 and BALB/c mice. Covax-19 vaccine induced high spike protein binding antibody levels that neutralised the original lineage B.1.319 virus from which the vaccine spike protein was derived, as well as the variant B.1.1.7 lineage virus. Covax-19 vaccine also induced a high frequency of spike-specific CD4 + and CD8 + memory T-cells with a dominant Th1 phenotype associated with the ability to kill spike-labelled target cells in vivo. Ferrets immunised with Covax-19 vaccine intramuscularly twice 2 weeks apart made spike receptor binding domain (RBD) IgG and were protected against an intranasal challenge with SARS-CoV-2 virus given two weeks after the last immunisation. Notably, ferrets that received the two higher doses of Covax-19 vaccine had no detectable virus in their lungs or in nasal washes at day 3 post-challenge, suggesting that in addition to lung protection, Covax-19 vaccine may have the potential to reduce virus transmission. This data supports advancement of Covax-19 vaccine into human clinical trials.

Affinity Tag Coating Enables Reliable Detection of Antigen-Specific B Cells in Immunospot Assays.

Assessment of humoral immunity to SARS-CoV-2 and other infectious agents is typically restricted to detecting antigen-specific antibodies in the serum. Rarely does immune monitoring entail assessment of the memory B-cell compartment itself, although it is these cells that engage in secondary antibody responses capable of mediating immune protection when pre-existing antibodies fail to prevent re-infection. There are few techniques that are capable of detecting rare antigen-specific B cells while also providing information regarding their relative abundance, class/subclass usage and functional affinity. In theory, the ELISPOT/FluoroSpot (collectively ImmunoSpot) assay platform is ideally suited for antigen-specific B-cell assessments since it provides this information at single-cell resolution for individual antibody-secreting cells (ASC). Here, we tested the hypothesis that antigen-coating efficiency could be universally improved across a diverse set of viral antigens if the standard direct (non-specific, low affinity) antigen absorption to the membrane was substituted by high-affinity capture. Specifically, we report an enhancement in assay sensitivity and a reduction in required protein concentrations through the capture of recombinant proteins via their encoded hexahistidine (6XHis) affinity tag. Affinity tag antigen coating enabled detection of SARS-CoV-2 Spike receptor binding domain (RBD)-reactive ASC, and also significantly improved assay performance using additional control antigens. Collectively, establishment of a universal antigen-coating approach streamlines characterization of the memory B-cell compartment after SARS-CoV-2 infection or COVID-19 vaccinations, and facilitates high-throughput immune-monitoring efforts of large donor cohorts in general.

Clinical Validation of a Sensitive Test for Saliva Collected in Healthcare and Community Settings with Pooling Utility for Severe Acute Respiratory Syndrome Coronavirus 2 Mass Surveillance.

The clinical performance of saliva compared with nasopharyngeal swabs (NPSs) has shown conflicting results in healthcare and community settings. In the present study, a total of 429 matched NPS and saliva sample pairs, collected in either healthcare or community setting, were evaluated. Phase-1 (protocol U) tested 240 matched NPS and saliva sample pairs; phase 2 (SalivaAll protocol) tested 189 matched NPS and saliva sample pairs, with an additional sample homogenization step before RNA extraction. A total of 85 saliva samples were evaluated with both protocols. In phase-1, 28.3% (68/240) samples tested positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from saliva, NPS, or both. The detection rate from saliva was lower compared with that from NPS samples (50.0% versus 89.7%). In phase-2, 50.2% (95/189) samples tested positive for SARS-CoV-2 from saliva, NPS, or both. The detection rate from saliva was higher compared with that from NPS samples (97.8% versus 78.9%). Of the 85 saliva samples evaluated with both protocols, the detection rate was 100% for samples tested with SalivaAll, and 36.7% with protocol U. The limit of detection with SalivaAll protocol was 20 to 60 copies/mL. The pooled testing approach demonstrated a 95% positive and 100% negative percentage agreement. This protocol for saliva samples results in higher sensitivity compared with NPS samples and breaks the barrier to using pooled saliva for SARS-CoV-2 testing.