Variant-specific IgA protects against Omicron infection.

BACKGROUND: The emergence of rapidly evolving SARS-CoV-2 variants, coupled with waning vaccine-induced immunity, has contributed to the rise of vaccine breakthrough infections. It is crucial to understand how vaccine-induced protection is mediated. METHODS: We examined two prospective cohorts of mRNA-vaccinated-and-boosted individuals during the Omicron wave of infection in Singapore. RESULTS: We found that, individuals, who remain uninfected over the follow-up period, had a higher variant-specific IgA, but not IgG, antibody response at 1-month post booster vaccination, compared with individuals who became infected. CONCLUSIONS: We conclude that IgA may have a potential contributory role in protection against Omicron infection.

Higher Delta variant-specific neutralizing antibodies prevented infection in close contacts vaccinated with ancestral mRNA vaccines during the SARS-CoV-2 Delta wave.

Identification of the risk factors and the high-risk groups which are most vulnerable is critical in COVID-19 disease management at a population level. Evaluating the efficacy of vaccination against infections is necessary to determine booster vaccination strategies for better protection in high-risk groups. In this study, we recruited 158 mRNA-vaccinated individuals during the Delta wave of SARS-CoV-2 infections in Singapore and examined the antibody profiles of infected individuals. We found that, despite high exposure due to communal living conditions in proximity, 4% of individuals (6/158) had PCR-confirmed infections and 96% (152/158) remained uninfected. Time-course analysis of the antibody profile at the start and the end of quarantine period showed Delta-specific boosting of anti-spike antibody response in 57% of the uninfected individuals (86/152). In the remaining 43% of the uninfected individuals (66/152) with no Delta-specific antibody boost, we found a higher Delta-specific antibody response at the start of quarantine period, which correlated with higher Delta pseudovirus neutralizing capacity. Our findings indicate that a higher basal variant-specific antibody response in the mRNA-vaccinated individuals contributes to better protection against infections by the new emerging SARS-CoV-2 variants.

A versatile genomic transgenesis platform with enhanced λ integrase for human Expi293F cells.

Reliable cell-based platforms to test and/or produce biologics in a sustainable manner are important for the biotech industry. Utilizing enhanced λ integrase, a sequence-specific DNA recombinase, we developed a novel transgenesis platform involving a fully characterized single genomic locus as an artificial landing pad for transgene insertion in human Expi293F cells. Importantly, transgene instability and variation in expression were not observed in the absence of selection pressure, thus enabling reliable long-term biotherapeutics testing or production. The artificial landing pad for λ integrase can be targeted with multi-transgene constructs and offers future modularity involving additional genome manipulation tools to generate sequential or nearly seamless insertions. We demonstrated broad utility with expression constructs for anti PD-1 monoclonal antibodies and showed that the orientation of heavy and light chain transcription units profoundly affected antibody expression levels. In addition, we demonstrated encapsulation of our PD-1 platform cells into bio-compatible mini-bioreactors and the continued secretion of antibodies, thus providing a basis for future cell-based applications for more effective and affordable therapies.

Third dose of BNT162b2 improves immune response in liver transplant recipients to ancestral strain but not Omicron BA.1 and XBB.

Vaccine immunogenicity in transplant recipients can be impacted by the immunosuppressive (IS) regimens they receive. While BNT162b2 vaccination has been shown to induce an immune response in liver transplant recipients (LTRs), it remains unclear how different IS regimens may affect vaccine immunogenicity after a third BNT162b2 dose in LTRs, which is especially important given the emergence of the Omicron sublineages of SARS-CoV-2. A total of 95 LTRs receiving single and multiple IS regimens were recruited and offered three doses of BNT162b2 during the study period. Blood samples were collected on days 0, 90, and 180 after the first BNT162b2 dose. At each time point, levels of anti-spike antibodies, their neutralizing activity, and specific memory B and T cell responses were assessed. LTRs receiving single IS regimens showed an absence of poor immunogenicity, while LTRs receiving multiple IS regimens showed lower levels of spike-specific antibodies and immunological memory compared to vaccinated healthy controls after two doses of BNT162b2. With a third dose of BNT162b2, spike-specific humoral, memory B, and T cell responses in LTR significantly improved against the ancestral strain of SARS-CoV-2 and were comparable to those seen in healthy controls who received only two doses of BNT162b2. However, LTRs receiving multiple IS regimens still showed poor antibody responses against Omicron sublineages BA.1 and XBB. A third dose of BNT162b2 may be beneficial in boosting antibody, memory B, and T cell responses in LTRs receiving multiple IS regimens, especially against the ancestral Wuhan strain of SARS-CoV-2. However, due to the continued vulnerability of LTRs to presently circulating Omicron variants, antiviral treatments such as medications need to be considered to prevent severe COVID-19 in these individuals.

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.

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.

Comprehensive engineering of a therapeutic neutralizing antibody targeting SARS-CoV-2 spike protein to neutralize escape variants.

The emergence of escape variants of SARS-CoV-2 carrying mutations in the spike protein poses a challenge for therapeutic antibodies. Here, we show that through the comprehensive engineering of the variable region of the neutralizing monoclonal antibody 5A6, the engineered antibody, 5A6CCS1, is able to neutralize SARS-CoV-2 variants that escaped neutralization by the original 5A6 antibody. In addition to the improved affinity against variants, 5A6CCS1 was also optimized to achieve high solubility and low viscosity, enabling a high concentration formulation for subcutaneous injection. In cynomolgus monkeys, 5A6CCS1 showed a long plasma half-life and good subcutaneous bioavailability through engineering of the variable and constant region. These data demonstrate that 5A6CCS1 is a promising antibody for development against SARS-CoV-2 and highlight the importance of antibody engineering as a potential method to counteract escape variants.

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.

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.

Structural insight into SARS-CoV-2 neutralizing antibodies and modulation of syncytia.

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is initiated by binding of the viral Spike protein to host receptor angiotensin-converting enzyme 2 (ACE2), followed by fusion of viral and host membranes. Although antibodies that block this interaction are in emergency use as early coronavirus disease 2019 (COVID-19) therapies, the precise determinants of neutralization potency remain unknown. We discovered a series of antibodies that potently block ACE2 binding but exhibit divergent neutralization efficacy against the live virus. Strikingly, these neutralizing antibodies can inhibit or enhance Spike-mediated membrane fusion and formation of syncytia, which are associated with chronic tissue damage in individuals with COVID-19. As revealed by cryoelectron microscopy, multiple structures of Spike-antibody complexes have distinct binding modes that not only block ACE2 binding but also alter the Spike protein conformational cycle triggered by ACE2 binding. We show that stabilization of different Spike conformations leads to modulation of Spike-mediated membrane fusion with profound implications for COVID-19 pathology and immunity.

Bivalent binding of a fully human IgG to the SARS-CoV-2 spike proteins reveals mechanisms of potent neutralization.

In vitro antibody selection against pathogens from naïve combinatorial libraries can yield various classes of antigen-specific binders that are distinct from those evolved from natural infection1-4. Also, rapid neutralizing antibody discovery can be made possible by a strategy that selects for those interfering with pathogen and host interaction5. Here we report the discovery of antibodies that neutralize SARS-CoV-2, the virus responsible for the COVID-19 pandemic, from a highly diverse naïve human Fab library. Lead antibody 5A6 blocks the receptor binding domain (RBD) of the viral spike from binding to the host receptor angiotensin converting enzyme 2 (ACE2), neutralizes SARS-CoV-2 infection of Vero E6 cells, and reduces viral replication in reconstituted human nasal and bronchial epithelium models. 5A6 has a high occupancy on the viral surface and exerts its neutralization activity via a bivalent binding mode to the tip of two neighbouring RBDs at the ACE2 interaction interface, one in the "up" and the other in the "down" position, explaining its superior neutralization capacity. Furthermore, 5A6 is insensitive to several spike mutations identified in clinical isolates, including the D614G mutant that has become dominant worldwide. Our results suggest that 5A6 could be an effective prophylactic and therapeutic treatment of COVID-19.