Conserved longitudinal alterations of anti-S-protein IgG subclasses in disease progression in initial ancestral Wuhan and vaccine breakthrough Delta infections.

Introduction: COVID-19 has a wide disease spectrum ranging from asymptomatic to severe. While humoral immune responses are critical in preventing infection, the immune mechanisms leading to severe disease, and the identification of biomarkers of disease progression and/or resolution of the infection remains to be determined. Methods: Plasma samples were obtained from infections during the initial wave of ancestral wildtype SARS-CoV-2 and from vaccine breakthrough infections during the wave of Delta variant, up to six months post infection. The spike-specific antibody profiles were compared across different severity groups and timepoints. Results: We found an association between spike-specific IgM, IgA and IgG and disease severity in unvaccinated infected individuals. In addition to strong IgG1 and IgG3 response, patients with severe disease develop a robust IgG2 and IgG4 response. A comparison of the ratio of IgG1 and IgG3 to IgG2 and IgG4 showed that disease progression is associated with a smaller ratio in both the initial wave of WT and the vaccine breakthrough Delta infections. Time-course analysis revealed that smaller (IgG1 and IgG3)/(IgG2 and IgG4) ratio is associated with disease progression, while the reverse associates with clinical recovery. Discussion: While each IgG subclass is associated with disease severity, the balance within the four IgG subclasses may affect disease outcome. Acute disease progression or infection resolution is associated with a specific immunological phenotype that is conserved in both the initial wave of WT and the vaccine breakthrough Delta infections.

Prolonged inflammation in patients hospitalized for coronavirus disease 2019 (COVID-19) resolves 2 years after infection.

Long-term complications from coronavirus disease 2019 (COVID-19) are concerning, as survivors can develop subclinical multiorgan dysfunction. It is unknown if such complications are due to prolonged inflammation, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination may reduce sequela. We conducted a prospective longitudinal study on hospitalized patients over 24 months. Clinical symptoms were collected by self-reporting during follow-up, along with blood samples for quantification of inflammatory markers and immune cell frequencies. All patients were given one dose of mRNA vaccine at 12-16 months. Their immune profiles at 12 and 24 months were compared. Approximately 37% and 39% of our patients reported post-COVID-19 symptoms at 12 and 24 months, respectively. The proportion of symptomatic patients with more than one symptom decreased from 69% at 12 months to 56% at 24 months. Longitudinal cytokine profiling revealed a cluster of individuals with persistently high inflammatory cytokine levels 12 months after infection. Patients with prolonged inflammation showed elevated terminally differentiated memory T cells in their blood; 54% had symptoms at 12 months. The majority of inflammatory markers and dysregulated immune cells in vaccinated patients recovered to a healthy baseline at 24 months, even though symptoms persisted. Post-COVID-19 symptoms can linger for 2 years after the initial infection and are associated with prolonged inflammation. Prolonged inflammation in hospitalized patients resolves after 2 years. We define a set of analytes associated with persistent inflammation and presence of symptoms, which could be useful biomarkers for identifying and monitoring high-risk survivors.

Waning of specific antibodies against Delta and Omicron variants five months after a third dose of BNT162b2 SARS-CoV-2 vaccine in elderly individuals.

The emergence of new SARS-CoV-2 variants, such as the more transmissible Delta and Omicron variants, has raised concerns on efficacy of the COVID-19 vaccines. Here, we examined the waning of antibody responses against different variants following primary and booster vaccination. We found that antibody responses against variants were low following primary vaccination. The antibody response against Omicron was almost non-existent. Efficient boosting of antibody response against all variants, including Omicron, was observed following a third dose. The antibody response against the variants tested was significantly higher at one month following booster vaccination, compared with two months following primary vaccination, for all individuals, including the low antibody responders identified at two months following primary vaccination. The antibody response, for all variants tested, was significantly higher at four months post booster than at five months post primary vaccination, and the proportion of low responders remained low (6-11%). However, there was significant waning of antibody response in more than 95% of individuals at four months, compared to one month following booster. We also observed a robust memory B cell response following booster, which remained higher at four months post booster than prior to booster. However, the memory B cell responses were on the decline for 50% of individuals at four months following booster. Similarly, while the T cell response is sustained, at cohort level, at four months post booster, a substantial proportion of individuals (18.8 - 53.8%) exhibited T cell response at four months post booster that has waned to levels below their corresponding levels before booster. The findings show an efficient induction of immune response against SARS-CoV-2 variants following booster vaccination. However, the induced immunity by the third BNT162b2 vaccine dose was transient. The findings suggest that elderly individuals may require a fourth dose to provide protection against SARS-CoV-2.

Efficient recall of SARS-CoV-2 variant-reactive B cells and T responses in the elderly upon heterologous mRNA vaccines as boosters.

Waning antibody levels against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the emergence of variants of concern highlight the need for booster vaccinations. This is particularly important for the elderly population, who are at a higher risk of developing severe coronavirus disease 2019 (COVID-19) disease. While studies have shown increased antibody responses following booster vaccination, understanding the changes in T and B cell compartments induced by a third vaccine dose remains limited. We analyzed the humoral and cellular responses in subjects who received either a homologous messenger RNA(mRNA) booster vaccine (BNT162b2 + BNT162b2 + BNT162b2; ''BBB") or a heterologous mRNA booster vaccine (BNT162b2 + BNT162b2 + mRNA-1273; ''BBM") at Day 0 (prebooster), Day 7, and Day 28 (postbooster). Compared with BBB, elderly individuals (≥60 years old) who received the BBM vaccination regimen display higher levels of neutralizing antibodies against the Wuhan and Delta strains along with a higher boost in immunoglobulin G memory B cells, particularly against the Omicron variant. Circulating T helper type 1(Th1), Th2, Th17, and T follicular helper responses were also increased in elderly individuals given the BBM regimen. While mRNA vaccines increase antibody, T cell, and B cell responses against SARS-CoV-2 1 month after receiving the third dose booster, the efficacy of the booster vaccine strategies may vary depending on age group and regimen combination.

Conserved longitudinal alterations of anti-S-protein IgG subclasses in disease progression in initial ancestral Wuhan and vaccine breakthrough Delta infections

Introduction COVID-19 has a wide disease spectrum ranging from asymptomatic to severe. While humoral immune responses are critical in preventing infection, the immune mechanisms leading to severe disease, and the identification of biomarkers of disease progression and/or resolution of the infection remains to be determined. Methods Plasma samples were obtained from infections during the initial wave of ancestral wildtype SARS-CoV-2 and from vaccine breakthrough infections during the wave of Delta variant, up to six months post infection. The spike-specific antibody profiles were compared across different severity groups and timepoints. Results We found an association between spike-specific IgM, IgA and IgG and disease severity in unvaccinated infected individuals. In addition to strong IgG1 and IgG3 response, patients with severe disease develop a robust IgG2 and IgG4 response. A comparison of the ratio of IgG1 and IgG3 to IgG2 and IgG4 showed that disease progression is associated with a smaller ratio in both the initial wave of WT and the vaccine breakthrough Delta infections. Time-course analysis revealed that smaller (IgG1 and IgG3)/(IgG2 and IgG4) ratio is associated with disease progression, while the reverse associates with clinical recovery. Discussion While each IgG subclass is associated with disease severity, the balance within the four IgG subclasses may affect disease outcome. Acute disease progression or infection resolution is associated with a specific immunological phenotype that is conserved in both the initial wave of WT and the vaccine breakthrough Delta infections.

Waning of specific antibodies against Delta and Omicron variants five months after a third dose of BNT162b2 SARS-CoV-2 vaccine in elderly individuals

The emergence of new SARS-CoV-2 variants, such as the more transmissible Delta and Omicron variants, has raised concerns on efficacy of the COVID-19 vaccines. Here, we examined the waning of antibody responses against different variants following primary and booster vaccination. We found that antibody responses against variants were low following primary vaccination. The antibody response against Omicron was almost non-existent. Efficient boosting of antibody response against all variants, including Omicron, was observed following a third dose. The antibody response against the variants tested was significantly higher at one month following booster vaccination, compared with two months following primary vaccination, for all individuals, including the low antibody responders identified at two months following primary vaccination. The antibody response, for all variants tested, was significantly higher at four months post booster than at five months post primary vaccination, and the proportion of low responders remained low (6-11%). However, there was significant waning of antibody response in more than 95% of individuals at four months, compared to one month following booster. We also observed a robust memory B cell response following booster, which remained higher at four months post booster than prior to booster. However, the memory B cell responses were on the decline for 50% of individuals at four months following booster. Similarly, while the T cell response is sustained, at cohort level, at four months post booster, a substantial proportion of individuals (18.8 – 53.8%) exhibited T cell response at four months post booster that has waned to levels below their corresponding levels before booster. The findings show an efficient induction of immune response against SARS-CoV-2 variants following booster vaccination. However, the induced immunity by the third BNT162b2 vaccine dose was transient. The findings suggest that elderly individuals may require a fourth dose to provide protection against SARS-CoV-2.

Lower vaccine-acquired immunity in the elderly population following two-dose BNT162b2 vaccination is alleviated by a third vaccine dose.

Understanding the impact of age on vaccinations is essential for the design and delivery of vaccines against SARS-CoV-2. Here, we present findings from a comprehensive analysis of multiple compartments of the memory immune response in 312 individuals vaccinated with the BNT162b2 SARS-CoV-2 mRNA vaccine. Two vaccine doses induce high antibody and T cell responses in most individuals. However, antibody recognition of the Spike protein of the Delta and Omicron variants is less efficient than that of the ancestral Wuhan strain. Age-stratified analyses identify a group of low antibody responders where individuals ≥60 years are overrepresented. Waning of the antibody and cellular responses is observed in 30% of the vaccinees after 6 months. However, age does not influence the waning of these responses. Taken together, while individuals ≥60 years old take longer to acquire vaccine-induced immunity, they develop more sustained acquired immunity at 6 months post-vaccination. A third dose strongly boosts the low antibody responses in the older individuals against the ancestral Wuhan strain, Delta and Omicron variants.

Heterologous booster vaccination with CoronaVac following prime vaccination with mRNA vaccine.

Objective: Despite the high vaccine efficacy of mRNA COVID-19 vaccines, there are individuals who developed excessive reactogenic and/or allergic responses after the first mRNA dose and were considered ineligible for further mRNA doses. CoronaVac, an inactivated SARS-CoV-2 vaccine, is recommended in Singapore as an alternative. Methods: Individuals, ineligible for further mRNA vaccines (BNT162b2 or mRNA-1273) because of excessive reactive responses to prime mRNA vaccination, were recruited and offered two doses of CoronaVac as booster vaccination 38-224 days post their mRNA vaccine dose. Individuals who did not develop any excessive reactive responses after the prime mRNA vaccination were also recruited and given another mRNA vaccine as booster vaccination. Blood samples were collected at days 0, 21 and 90 post first CoronaVac dose and mRNA dose, respectively, for analysis. Results: We showed that two CoronaVac booster doses induced specific immunity in these mRNA vaccine-primed individuals. Although the spike-specific antibody response was lower, their memory B cell response against the receptor-binding domain (RBD) of the spike protein was similar, compared with individuals who received two BNT162b2 injections. The spike-specific memory T cell response also increased following CoronaVac booster doses. However, specific immunity against the Omicron variant was low, similar to individuals with two BNT162b2 doses. Conclusion: Our findings showed that while mRNA vaccine-primed individuals can opt for two subsequent doses of CoronaVac, an additional dose may be necessary to achieve protection, especially against newly emerging immune escape variants such as Omicron.

Decreased memory B cell frequencies in COVID-19 delta variant vaccine breakthrough infection.

The SARS-CoV-2 Delta (B.1.617.2) variant is capable of infecting vaccinated persons. An open question remains as to whether deficiencies in specific vaccine-elicited immune responses result in susceptibility to vaccine breakthrough infection. We investigated 55 vaccine breakthrough infection cases (mostly Delta) in Singapore, comparing them against 86 vaccinated close contacts who did not contract infection. Vaccine breakthrough cases showed lower memory B cell frequencies against SARS-CoV-2 receptor-binding domain (RBD). Compared to plasma antibodies, antibodies secreted by memory B cells retained a higher fraction of neutralizing properties against the Delta variant. Inflammatory cytokines including IL-1ß and TNF were lower in vaccine breakthrough infections than primary infection of similar disease severity, underscoring the usefulness of vaccination in preventing inflammation. This report highlights the importance of memory B cells against vaccine breakthrough and suggests that lower memory B cell levels may be a correlate of risk for Delta vaccine breakthrough infection.

Robust Virus-Specific Adaptive Immunity in COVID-19 Patients with SARS-CoV-2 Δ382 Variant Infection.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) that have become dominant as the pandemic progresses bear the ORF8 mutation together with multiple spike mutations. A 382-nucleotide deletion (Δ382) in the ORF7b and ORF8 regions has been associated with milder disease phenotype and less systemic inflammation in COVID-19 patients. However, its impact on host immunity against SARS-CoV-2 remains undefined. Here, RNA-sequencing was performed to elucidate whole blood transcriptomic profiles and identify contrasting immune signatures between patients infected with either wildtype or Δ382 SARS-CoV-2 variant. Interestingly, the immune landscape of Δ382 SARS-CoV-2 infected patients featured an increased adaptive immune response, evidenced by enrichment of genes related to T cell functionality, a more robust SARS-CoV-2-specific T cell immunity, as well as a more rapid antibody response. At the molecular level, eukaryotic initiation factor 2 signaling was found to be upregulated in patients bearing Δ382, and its associated genes were correlated with systemic levels of T cell-associated and pro-inflammatory cytokines. This study provides more in-depth insight into the host-pathogen interactions of ORF8 with great promise as a therapeutic target to combat SARS-CoV-2 infection.

Resistance of SARS-CoV-2 variants to neutralization by convalescent plasma from early COVID-19 outbreak in Singapore.

The rapid spreading of SARS-CoV-2 variants B.1.1.7 originated from the United Kingdom and B.1.351 from South Africa has contributed to the second wave of COVID-19 cases in the respective countries and also around the world. In this study, we employed advanced biochemical and virological methodologies to evaluate the impact of Spike mutations of these strains on the degree of protection afforded by humoral immune responses following natural infection of the ancestral SARS-CoV-2 strain during the early stages of the outbreak. We found that antibody-mediated neutralization activity was partially reduced for B.1.1.7 variant and significantly attenuated for the B.1.351 strain. We also found that mutations outside the receptor-binding domain (RBD) can strongly influence antibody binding and neutralization, cautioning the use of solely RBD mutations in evaluating vaccine efficacy. These findings highlight an urgent need to develop new SARS-CoV-2 vaccines that are not based exclusively on the ancestral SARS-CoV-2 Spike gene sequence.

Differential Cytokine Responses in Hospitalized COVID-19 Patients Limit Efficacy of Remdesivir.

A significant proportion of COVID-19 patients will progress to critical illness requiring invasive mechanical ventilation. This accentuates the need for a therapy that can reduce the severity of COVID-19. Clinical trials have shown the effectiveness of remdesivir in shortening recovery time and decreasing progression to respiratory failure and mechanical ventilation. However, some studies have highlighted its lack of efficacy in patients on high-flow oxygen and mechanical ventilation. This study uncovers some underlying immune response differences between responders and non-responders to remdesivir treatment. Immunological analyses revealed an upregulation of tissue repair factors BDNF, PDGF-BB and PIGF-1, as well as an increase in ratio of Th2-associated cytokine IL-4 to Th1-associated cytokine IFN-γ. Serological profiling of IgG subclasses corroborated this observation, with significantly higher magnitude of increase in Th2-associated IgG2 and IgG4 responses. These findings help to identify the mechanisms of immune regulation accompanying successful remdesivir treatment in severe COVID-19 patients.

A flow cytometry-based assay for serological detection of anti-spike antibodies in COVID-19 patients.

One of the key public health strategies in coronavirus 2019 disease (COVID-19) management is the early detection of infected individuals to limit the transmission. As a result, serological assays have been developed to complement PCR-based assays. Here, we report the development of a flow cytometry-based assay to detect antibodies against full-length SARS-CoV-2 spike protein (S protein) in patients with COVID-19. The assay is time-efficient and sensitive, being able to capture the wider repertoire of antibodies against the S protein. For complete details on the use and execution of this protocol, please refer to Goh et al. (2021).

Asymptomatic COVID-19: disease tolerance with efficient anti-viral immunity against SARS-CoV-2.

The immune responses and mechanisms limiting symptom progression in asymptomatic cases of SARS-CoV-2 infection remain unclear. We comprehensively characterized transcriptomic profiles, cytokine responses, neutralization capacity of antibodies, and cellular immune phenotypes of asymptomatic patients with acute SARS-CoV-2 infection to identify potential protective mechanisms. Compared to symptomatic patients, asymptomatic patients had higher counts of mature neutrophils and lower proportion of CD169+ expressing monocytes in the peripheral blood. Systemic levels of pro-inflammatory cytokines were also lower in asymptomatic patients, accompanied by milder pro-inflammatory gene signatures. Mechanistically, a more robust systemic Th2 cell signature with a higher level of virus-specific Th17 cells and a weaker yet sufficient neutralizing antibody profile against SARS-CoV-2 was observed in asymptomatic patients. In addition, asymptomatic COVID-19 patients had higher systemic levels of growth factors that are associated with cellular repair. Together, the data suggest that asymptomatic patients mount less pro-inflammatory and more protective immune responses against SARS-CoV-2 indicative of disease tolerance. Insights from this study highlight key immune pathways that could serve as therapeutic targets to prevent disease progression in COVID-19.

Human neutralising antibodies elicited by SARS-CoV-2 non-D614G variants offer cross-protection against the SARS-CoV-2 D614G variant.

OBJECTIVES: The emergence of a SARS-CoV-2 variant with a point mutation in the spike (S) protein, D614G, has taken precedence over the original Wuhan isolate by May 2020. With an increased infection and transmission rate, it is imperative to determine whether antibodies induced against the D614 isolate may cross-neutralise against the G614 variant. METHODS: Antibody profiling against the SARS-CoV-2 S protein of the D614 variant by flow cytometry and assessment of neutralising antibody titres using pseudotyped lentiviruses expressing the SARS-CoV-2 S protein of either the D614 or G614 variant tagged with a luciferase reporter were performed on plasma samples from COVID-19 patients with known D614G status (n = 44 infected with D614, n = 6 infected with G614, n = 7 containing all other clades: O, S, L, V, G, GH or GR). RESULTS: Profiling of the anti-SARS-CoV-2 humoral immunity reveals similar neutralisation profiles against both S protein variants, albeit waning neutralising antibody capacity at the later phase of infection. Of clinical importance, patients infected with either the D614 or G614 clade elicited a similar degree of neutralisation against both pseudoviruses, suggesting that the D614G mutation does not impact the neutralisation capacity of the elicited antibodies. CONCLUSIONS: Cross-reactivity occurs at the functional level of the humoral response on both the S protein variants, which suggests that existing serological assays will be able to detect both D614 and G614 clades of SARS-CoV-2. More importantly, there should be negligible impact towards the efficacy of antibody-based therapies and vaccines that are currently being developed.

Sensitive detection of total anti-Spike antibodies and isotype switching in asymptomatic and symptomatic individuals with COVID-19.

Early detection of infection is crucial to limit the spread of coronavirus disease 2019 (COVID-19). Here we develop a flow cytometry-based assay to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein antibodies in individuals with COVID-19. The assay detects specific immunoglobulin M (IgM), IgA, and IgG in individuals with COVID-19 and also acquisition of all IgG subclasses, with IgG1 being the most dominant. The antibody response is significantly higher at a later stage of infection. Furthermore, asymptomatic individuals with COVID-19 also develop specific IgM, IgA, and IgG, with IgG1 being the most dominant subclass. Although the antibody levels are lower in asymptomatic infection, the assay is highly sensitive and detects 97% of asymptomatic infections. These findings demonstrate that the assay can be used for serological analysis of symptomatic and asymptomatic infections, which may otherwise remain undetected.

Effects of a major deletion in the SARS-CoV-2 genome on the severity of infection and the inflammatory response: an observational cohort study.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with a 382-nucleotide deletion (∆382) in the open reading frame 8 (ORF8) region of the genome have been detected in Singapore and other countries. We investigated the effect of this deletion on the clinical features of infection. METHODS: We retrospectively identified patients who had been screened for the ∆382 variant and recruited to the PROTECT study-a prospective observational cohort study conducted at seven public hospitals in Singapore. We collected clinical, laboratory, and radiological data from patients' electronic medical records and serial blood and respiratory samples taken during hospitalisation and after discharge. Individuals infected with the ∆382 variant were compared with those infected with wild-type SARS-CoV-2. Exact logistic regression was used to examine the association between the infection groups and the development of hypoxia requiring supplemental oxygen (an indicator of severe COVID-19, the primary endpoint). Follow-up for the study's primary endpoint is completed. FINDINGS: Between Jan 22 and March 21, 2020, 278 patients with PCR-confirmed SARS-CoV-2 infection were screened for the ∆382 deletion and 131 were enrolled onto the study, of whom 92 (70%) were infected with the wild-type virus, ten (8%) had a mix of wild-type and ∆382-variant viruses, and 29 (22%) had only the ∆382 variant. Development of hypoxia requiring supplemental oxygen was less frequent in the ∆382 variant group (0 [0%] of 29 patients) than in the wild-type only group (26 [28%] of 92; absolute difference 28% [95% CI 14-28]). After adjusting for age and presence of comorbidities, infection with the ∆382 variant only was associated with lower odds of developing hypoxia requiring supplemental oxygen (adjusted odds ratio 0·07 [95% CI 0·00-0·48]) compared with infection with wild-type virus only. INTERPRETATION: The ∆382 variant of SARS-CoV-2 seems to be associated with a milder infection. The observed clinical effects of deletions in ORF8 could have implications for the development of treatments and vaccines. FUNDING: National Medical Research Council Singapore.