Improvement of immune dysregulation and health-related quality of life in individuals with long COVID at 24-months following SARS-CoV-2 infection (preprint)

This study investigated the humoral and cellular immune responses in individuals with long COVID (LC) compared to age and gender matched recovered COVID-19 controls (MC) over 24-months. LC participants showed elevated spike and nucleocapsid IgG levels, higher neutralizing capacity, and increased spike- and nucleocapsid-specific CD4+ T cells, PD-1, and TIM-3 expression on CD4+ and CD8+ T cells at 3- and 8-months, but these differences did not persist at 24-months. Some LC participants had detectable IFN-{beta} and IFN-{gamma} that was attributed to reinfection and antigen re-exposure. Single-cell RNA sequencing at 24-month timepoint revealed similar immune cell proportions and reconstitution of naive T and B cell subsets in LC. No significant differences in exhaustion scores or antigen-specific T cell clones were observed. These findings suggest resolution of immune activation in LC and return to comparable immune responses between LC and MC over time. Improvement in self-reported health-related quality of life at 24-months was also evident in the majority of LC (62%). PTX3, CRP levels and platelet count were associated with improvements in health-related quality of life.

Lasting first impression: Pre-existing immunity restricts mucosal antibody responses during Omicron breakthrough (preprint)

Understanding mucosal antibody responses from SARS-CoV-2 infection and/or vaccination is crucial to develop strategies for longer term immunity, especially against emerging viral variants. We profiled serial paired mucosal and plasma antibodies from: COVID-19 vaccinated only vaccinees (vaccinated, uninfected), COVID-19 recovered vaccinees (convalescent, vaccinated) and individuals with breakthrough Delta or Omicron BA.2 infections (vaccinated, infected). Saliva from COVID-19 recovered vaccinees displayed improved antibody neutralizing activity, Fc{gamma}R engagement and IgA compared to COVID-19 uninfected vaccinees. Furthermore, repeated mRNA vaccination boosted SARS-CoV-2-specific IgG2 and IgG4 responses in both mucosa biofluids (saliva and tears) and plasma. IgG, but not IgA, responses to breakthrough COVID-19 variants were dampened and narrowed by increased pre-existing vaccine-induced immunity to the ancestral strain. Salivary antibodies delayed initiation of boosting following breakthrough COVID-19 infection, especially Omicron BA.2, however, rose rapidly thereafter. Our data highlight how pre-existing immunity shapes mucosal SARS-CoV-2-specific antibody responses and has implications for long-term protection from COVID-19.

Monoclonal antibodies against human coronavirus NL63 spike (preprint)

The COVID-19 pandemic has illustrated the potential for monoclonal antibody therapeutics as prophylactic and therapeutic agents against pandemic viruses. No such therapeutics currently exist for other human coronaviruses. NL63 is a human alphacoronavirus that typically causes the common cold and uses the same receptor, ACE2, as the highly pathogenic SARS-CoV and SARS-CoV-2 pandemic viruses. In a cohort of healthy adults, we characterised humoral responses against the NL63 spike protein. While NL63 spike and receptor binding domain-specific binding antibodies and neutralisation activity could be detected in plasma of all subjects, memory B cells against NL63 spike were variable and relatively low in frequency compared to that against SARS-CoV-2 spike. From these donors, we isolated a panel of antibodies against NL63 spike and characterised their neutralising potential. We identified potent neutralising antibodies that recognised the receptor binding domain (RBD) and other non-RBD epitopes within spike.

Durable reprogramming of neutralising antibody responses following breakthrough Omicron infection (preprint)

SARS-CoV-2 breakthrough infection of vaccinated individuals is increasingly common with the circulation of highly immune evasive and transmissible Omicron variants. Here, we report the dynamics and durability of recalled spike-specific humoral immunity following BA.1 or BA.2 breakthrough infection, with longitudinal sampling up to 8 months post-infection. Both BA.1 and BA.2 infection robustly boosted neutralisation activity against the infecting strain while expanding breadth against other Omicron strains. Cross-reactive memory B cells against both ancestral and Omicron spike were predominantly expanded by infection, with limited recruitment of de novo Omicron-specific B cells or antibodies. Modelling of neutralisation titres predicts that protection from symptomatic reinfection against antigenically similar strains will be remarkably durable, but is undermined by novel emerging strains with further neutralisation escape.

Monoclonal antibody levels and protection from COVID-19 (preprint)

Multiple monoclonal antibodies have been shown to be effective for both prophylaxis and therapy for SARS-CoV-2 infection. Here we aggregate data from randomized controlled trials assessing the use of monoclonal antibodies in preventing symptomatic SARS-CoV-2 infection. We use data on changes in the in vivo concentration of monoclonal antibodies, and the associated protection from COVID-19, over time to model the dose-response relationship of monoclonal antibodies for prophylaxis. We estimate that 50% protection from COVID-19 is achieved with a monoclonal antibody concentration of 939-fold of the in vitro IC50 (95% CI: 135 - 2073). This relationship provides a quantitative tool allowing prediction of the prophylactic efficacy and duration of protection for new monoclonal antibodies administered at different doses and against different SARS-CoV-2 variants. Finally, we compare the relationship between neutralization titer and protection from COVID-19 after either monoclonal antibody treatment or vaccination. We find no evidence for a difference between the 50% protective titer for monoclonal antibodies and vaccination, although vaccination is predicted to be capable of achieving a higher maximum level of protection.

Broad immunity to SARS-CoV-2 variants of concern mediated by a SARS-CoV-2 receptor-binding domain protein vaccine (preprint)

The SARS-CoV-2 global pandemic has fuelled the generation of vaccines at an unprecedented pace and scale. However, many challenges remain, including: the emergence of vaccine-resistant mutant viruses, vaccine stability during storage and transport, waning vaccine-induced immunity, and concerns about infrequent adverse events associated with existing vaccines. Here, we report on a protein subunit vaccine comprising the receptor-binding domain (RBD) of the ancestral SARS-CoV-2 spike protein, dimerised with an immunoglobulin IgG1 Fc domain. These were tested in conjunction with three different adjuvants: a TLR2 agonist R4-Pam2Cys, an NKT cell agonist glycolipid alpha-Galactosylceramide, or MF59 squalene oil-in-water adjuvant. Each formulation drove strong neutralising antibody (nAb) responses and provided durable and highly protective immunity against lower and upper airway infection in mouse models of COVID-19. We have also developed an RBD-human IgG1 Fc vaccine with an RBD sequence of the highly immunoevasive beta variant (N501Y, E484K, K417N). This beta variant RBD vaccine, combined with MF59 adjuvant, induced strong protection in mice against the beta strain as well as the ancestral strain. Furthermore, when used as a third dose booster vaccine following priming with whole spike vaccine, anti-sera from beta-RBD-Fc immunised mice increased titres of nAb against other variants including alpha, delta, delta+, gamma, lambda, mu, and omicron BA.1 and BA.2. These results demonstrated that an RBD-Fc protein subunit/MF59 adjuvanted vaccine can induce high levels of broad nAbs, including when used as a booster following prior immunisation of mice with whole ancestral-strain Spike vaccines. This vaccine platform offers a potential approach to augment some of the currently approved vaccines in the face of emerging variants of concern, and it has now entered a phase I clinical trial.

Neutralising antibodies predict protection from severe COVID-19 (preprint)

Background: Vaccine protection from COVID-19 has been shown to decline with time-since-vaccination and against SARS-CoV-2 variants. Protection against severe COVID-19 is higher than against symptomatic infection, and also appears relatively preserved over time and against variants. Although Protection protection from symptomatic SARS-CoV-2 infection has been shown to be strongly correlated with neutralising antibody titres, however, this relationship has been is less well described for severe COVID-19. Protection against severe COVID-19 is higher than against symptomatic infection, and also appears relatively preserved over time and against variants. Here we analyse whether neutralising antibody titre remains predictive of protection against severe COVID-19 in the face of waning neutralising antibody levels and emerging variants. Methods: We extracted data from 15 studies reporting on protection against a range of SARS-CoV-2 clinical endpoints ("any infection", "symptomatic infection" and "severe COVID-19"). We then estimated the concurrent neutralising antibody titres using existing parameters on vaccine potency, neutralising antibody decay, and loss of recognition of variants and investigated the relationship between neutralising antibody titre and vaccine effectiveness against severe COVID-19. Findings: Predicted neutralising antibody titres are strongly correlated with vaccine effectiveness against symptomatic and severe COVID-19 (Spearman rho = .94 and 0.63 respectively, p

Correlates of protection, thresholds of protection, and immunobridging in SARS-CoV-2 infection (preprint)

Several studies show neutralizing antibody levels are an important correlate of immune protection from COVID-19 and have estimated the relationship between neutralizing antibodies and protection. However, a number of these studies appear to yield quite different estimates of the level of neutralizing antibodies required for protection. Here we show that after normalization of antibody titers current studies converge on a consistent relationship between antibody levels and protection from COVID-19.

Dynamics of immune recall following SARS-CoV-2 vaccination or breakthrough infection (preprint)

Vaccination against SARS-CoV-2 results in protection from acquisition of infection as well as improved clinical outcomes even if infection occurs, likely reflecting a combination of residual vaccine-elicited immunity and the recall of immunological memory. Here, we define the early kinetics of spike-specific humoral and T cell immunity after vaccination of seropositive individuals, and after breakthrough infection in vaccinated individuals. Intensive and early longitudinal sampling reveals the timing and magnitude of recall, with the phenotypic activation of B cells preceding an increase in neutralizing antibody titres. In breakthrough infections, the delayed kinetics of humoral immune recall provides a mechanism for the lack of early control of viral replication but likely underpins accelerated viral clearance and the protective effects of vaccination against severe COVID-19.

Tear antibodies to SARS-CoV-2: implications for transmission (preprint)

ObjectivesSARS-CoV-2 can be transmitted by aerosols and the ocular surface may be an important route of transmission. Little is known about protective antibody responses to SARS-CoV-2 in tears after infection or vaccination. We analysed SARS-CoV-2 specific IgG and IgA responses in human tears after either COVID-19 infection or vaccination. MethodsWe recruited 16 subjects with COVID-19 infection an average of 7 months previously and 15 subjects before and 2 weeks after Comirnaty (Pfizer-BioNtech) vaccination. Plasma, saliva and basal tears were collected. Pre-pandemic plasma, saliva and basal tears from 11 individuals were included as healthy controls. Antibody responses to 5 SARS-CoV-2 antigens were measured via multiplex. ResultsIgG antibodies to Spike and Nucleoprotein were detected in tears, saliva and plasma from subjects with prior SARS-CoV-2 infection in comparison to uninfected controls. While RBD-specific antibodies were detected in plasma, minimal RBD-specific antibodies were detected in tears and saliva. In contrast, high levels of IgG antibodies to Spike and RBD, but not Nucleoprotein, were induced in tears, saliva and plasma of subjects receiving 2 doses of the Comirnaty vaccine. Increased levels of IgA1 and IgA2 antibodies to SARS-CoV-2 antigens were detected in plasma following infection or vaccination, but were unchanged in tears and saliva. ConclusionBoth infection and vaccination induce SARS-CoV-2-specific IgG antibodies in tears. RBD-specific IgG antibodies in tears were induced by vaccination but were not present 7 months post-infection. This suggests neutralising antibodies may be low in the tears late following infection.

Immunological dysfunction persists for 8 months following initial mild-moderate SARS-CoV-2 infection (preprint)

A proportion of patients surviving acute COVID-19 infection develop post-COVID syndrome (long COVID) encompassing physical and neuropsychiatric symptoms lasting longer than 12 weeks. Here we studied a prospective cohort of individuals with long COVID (the ADAPT study) compared to age/gender matched subjects without long COVID, healthy donors and individuals infected with other non-SARS CoV2 human coronaviruses (the ADAPT-C study). We found an elevated diffuse serum inflammatory cytokine profile in symptomatic long COVID subjects that was maintained at 8 months post-infection and was not observed in asymptomatic COVID-19 survivors. This inflammatory profile consisted of 15 cytokines that positively correlated; revealing an apparent diffuse, potentially coordinated, low level up regulation of a spectrum of immune and inflammatory mediators. In addition, we found an absence of subsets of un-activated naive T and B cells in peripheral blood of long COVID subjects, that did not reconstitute over time. In contrast, individual serum cytokines from the interferon I and III classes, T cell activation markers and plasma ACE2, while elevated in the serum of people previously infected with SARS-CoV-2 were not further elevated in subjects with long COVID symptoms. This work defines immunological parameters associated with long COVID and suggests future opportunities to prevention and treatment.

What level of neutralising antibody protects from COVID-19? . (preprint)

Both previous infection and vaccination have been shown to provide potent protection from COVID-19. However, there are concerns that waning immunity and viral variation may lead to a loss of protection over time. Predictive models of immune protection are urgently needed to identify immune correlates of protection to assist in the future deployment of vaccines. To address this, we modelled the relationship between in vitro neutralisation levels and observed protection from SARS-CoV-2 infection using data from seven current vaccines as well as convalescent cohorts. Here we show that neutralisation level is highly predictive of immune protection. The 50% protective neutralisation level was estimated to be approximately 20% of the average convalescent level (95% CI = 14-28%). The estimated neutralisation level required for 50% protection from severe infection was significantly lower (3% of the mean convalescent level (CI = 0.7-13%, p = 0.0004). Given the relationship between in vitro neutralization titer and protection, we then used this to investigate how waning immunity and antigenic variation might affect vaccine efficacy. We found that the decay of neutralising titre in vaccinated subjects over the first 3-4 months after vaccination was at least as rapid as the decay observed in convalescent subjects. Modelling the decay of neutralisation titre over the first 250 days after immunisation predicts a significant loss in protection from SARS-CoV-2 infection will occur, although protection from severe disease should be largely retained. Neutralisation titres against some SARS-CoV-2 variants of concern are reduced compared to the vaccine strain and our model predicts the relationship between neutralisation and efficacy against viral variants. Our analyses provide an evidence-based prediction of SARS-CoV-2 immune protection that will assist in developing vaccine strategies to control the future trajectory of the pandemic.

Adaptive immunity to human coronaviruses is widespread but low in magnitude (preprint)

Endemic human coronaviruses (hCoV) circulate worldwide but cause minimal mortality. Although seroconversion to hCoV is near ubiquitous during childhood, little is known about hCoV-specific T cell memory in adults. We quantified CD4 T cell and antibody responses to hCoV spike antigens in 42 SARS-CoV-2 uninfected individuals. T cell responses were widespread within conventional memory and cTFH compartments but did not correlate with IgG titres. SARS-CoV-2 cross-reactive T cells were observed in 48% of participants and correlated with HKU1 memory. hCoV-specific T cells exhibited a CCR6+ central memory phenotype in the blood, but were enriched for frequency and CXCR3 expression in human lung draining lymph nodes. Overall, hCoV-specific humoral and cellular memory are independently maintained, with a shared phenotype existing among coronavirus-specific CD4 T cells. This understanding of endemic coronavirus immunity provides insight into the homeostatic maintenance of immune responses that are likely to be critical components of protection against SARS-CoV-2.

Plasma ACE2 activity is persistently elevated following SARS-CoV-2 infection: implications for COVID-19 pathogenesis and consequences (preprint)

COVID-19 causes persistent endothelial inflammation, lung and cardiovascular complications. SARS-CoV-2 utilises the catalytic site of full-length membrane-bound angiotensin converting enzyme 2 (ACE2) for cell entry causing downregulation of tissue ACE2. We reported downregulation of cardiac ACE2 is associated with increased plasma ACE2 activity. In this prospective observational study in recovered COVID-19 patients, we hypothesised that SARS-CoV-2 infection would be associated with shedding of ACE2 from cell membranes and increased plasma ACE2 activity. MethodsWe measured plasma ACE2 catalytic activity using a validated, sensitive quenched fluorescent substrate-based assay in a cohort of Australians aged [≥]18 years (n=66) who had recovered from mild, moderate or severe SARS-CoV-2 infection (positive result by PCR testing) and age and gender matched uninfected controls (n=70). Serial samples were available in 23 recovered SARS-CoV-2 patients. ResultsPlasma ACE2 activity at a median of 35 days post-infection [interquartile range 30-38 days] was 97-fold higher in recovered SARS-CoV-2 patients compared to controls (5.8 [2-11.3] vs. 0.06 [0.02-2.2] pmol/min/ml, p<0.0001). There was a significant difference in plasma ACE2 activity according to disease severity (p=0.033), with severe COVID-19 associated with higher ACE2 activity compared to mild disease (p=0.027). Men (n=39) who were SARS-CoV-2 positive had higher median plasma ACE2 levels compared to women (n=27) (p<0.0001). We next analysed whether an elevated plasma ACE2 activity level persisted following SARS-CoV-2 infection in subjects with blood samples at 63 [56-65] and 114 [111-125] days post infection. Plasma ACE2 activity remained persistently elevated in almost all subjects, with no significant differences between timepoints in post-hoc comparisons (p>0.05). DiscussionThis is the first description that plasma ACE2 activity is elevated after COVID-19 infection, and the first with longitudinal data indicating plasma ACE2 activity remains elevated out to a median of 114 days post-infection. Larger studies are now needed to determine if persistent elevated plasma ACE2 activity identifies people at risk of prolonged illness following COVID-19.

rSWeeP: A R/Bioconductor package deal with SWeeP sequences representation (preprint)

The rSWeeP package is an R implementation of the SWeeP model, designed to handle Big Data. rSweeP meets to the growing demand for efficient methods of heuristic representation in the field of Bioinformatics, on platforms accessible to the entire scientific community. We explored the implementation of rSWeeP using a dataset containing 31,386 viral proteomes, performing phylogenetic and principal component analysis. As a case study we analyze the viral strains closest to the SARS-CoV, responsible for the current pandemic of COVID-19, confirming that rSWeeP can accurately classify organisms taxonomically. rSWeeP package is freely available at https://bioconductor.org/packages/ release/bioc/html/rSWeeP.html.

Susceptibility of domestic swine to experimental infection with SARS-CoV-2 (preprint)

SARS-CoV-2, the agent responsible for COVID-19 has been shown to infect a number of species. The role of domestic livestock and the risk associated for humans in close contact remains unknown for many production animals. Determination of the susceptibility of pigs to SARS-CoV-2 is critical towards a One Health approach to manage the potential risk of zoonotic transmission. Here, we show pigs are susceptible to SARS-CoV-2 following oronasal inoculation. Viral RNA was detected in group oral fluids and nasal wash from at least two animals while live virus was isolated from a pig. Further, antibodies could be detected in two animals at 11 and 13 days post infection, while oral fluid samples at 6 days post inoculation indicated the presence of secreted antibodies. These data highlight the need for additional livestock assessment to determine the potential role domestic animals may contribute towards the SARS-CoV-2 pandemic.