Large-Scale HLA Tetramer Tracking of T Cells during Dengue Infection Reveals Broad Acute Activation and Differentiation into Two Memory Cell Fates.

T cells play important multifaceted roles during dengue infection, and understanding their responses is important for defining correlates of protective immunity and identifying effective vaccine antigens. Using mass cytometry and a highly multiplexed peptide-HLA (human leukocyte antigen) tetramer staining strategy, we probed T cells from dengue patients-a total of 430 dengue and control candidate epitopes-together with key markers of activation, trafficking, and differentiation. During acute disease, dengue-specific CD8+ T cells expressed a distinct profile of activation and trafficking receptors that distinguished them from non-dengue-specific T cells. During convalescence, dengue-specific T cells differentiated into two major cell fates, CD57+ CD127--resembling terminally differentiated senescent memory cells and CD127+ CD57--resembling proliferation-capable memory cells. Validation in an independent cohort showed that these subsets remained at elevated frequencies up to one year after infection. These analyses aid our understanding of the generation of T cell memory in dengue infection or vaccination.

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.

Triggering of TLR-3, -4, NOD2, and DC-SIGN reduces viral replication and increases T-cell activation capacity of HIV-infected human dendritic cells.

A variety of signals influence the capacity of dendritic cells (DCs) to mount potent antiviral cytotoxic T-cell (CTL) responses. In particular, innate immune sensing by pathogen recognition receptors, such as TLR and C-type lectines, influences DC biology and affects their susceptibility to HIV infection. Yet, whether the combined effects of PPRs triggering and HIV infection influence HIV-specific (HS) CTL responses remain enigmatic. Here, we dissect the impact of innate immune sensing by pathogen recognition receptors on DC maturation, HIV infection, and on the quality of HS CTL activation. Remarkably, ligand-driven triggering of TLR-3, -4, NOD2, and DC-SIGN, despite reducing viral replication, markedly increased the capacity of infected DCs to stimulate HS CTLs. This was exemplified by the diversity and the quantity of cytokines produced by HS CTLs primed by these DCs. Infecting DCs with viruses harboring members of the APOBEC family of antiviral factors enhanced the antigen-presenting skills of infected DCs. Our results highlight the tight interplay between innate and adaptive immunity and may help develop innovative immunotherapies against viral infections.

HIV-Infected Dendritic Cells Present Endogenous MHC Class II-Restricted Antigens to HIV-Specific CD4+ T Cells.

It is widely assumed that CD4(+) T cells recognize antigenic peptides (epitopes) derived solely from incoming, exogenous, viral particles or proteins. However, alternative sources of MHC class II (MHC-II)-restricted Ags have been described, in particular epitopes derived from newly synthesized proteins (so-called endogenous). In this study, we show that HIV-infected dendritic cells (DC) present MHC-II-restricted endogenous viral Ags to HIV-specific (HS) CD4(+) T cells. This endogenous pathway functions independently of the exogenous route for HIV Ag presentation and offers a distinct possibility for the immune system to activate HS CD4(+) T cells. We examined the implication of autophagy, which plays a crucial role in endogenous viral Ag presentation and thymic selection of CD4(+) T cells, in HIV endogenous presentation. We show that infected DC do not use autophagy to process MHC-II-restricted HIV Ags. This is unlikely to correspond to a viral escape from autophagic degradation, as infecting DC with Nef- or Env-deficient HIV strains did not impact HS T cell activation. However, we demonstrate that, in DC, specific targeting of HIV Ags to autophagosomes using a microtubule-associated protein L chain 3 (LC3) fusion protein effectively enhances and broadens HS CD4(+) T cell responses, thus favoring an endogenous MHC-II-restricted presentation. In summary, in DC, multiple endogenous presentation pathways lead to the activation of HS CD4(+) T cell responses. These findings will help in designing novel strategies to activate HS CD4(+) T cells that are required for CTL activation/maintenance and B cell maturation.

Effector T cells boost regulatory T cell expansion by IL-2, TNF, OX40, and plasmacytoid dendritic cells depending on the immune context.

CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells play a major role in peripheral tolerance. Multiple environmental factors and cell types affect their biology. Among them, activated effector CD4(+) T cells can boost Treg cell expansion through TNF or IL-2. In this study, we further characterized this effector T (Teff) cell-dependent Treg cell boost in vivo in mice. This phenomenon was observed when both Treg and Teff cells were activated by their cognate Ag, with the latter being the same or different. Also, when Treg cells highly proliferated on their own, there was no additional Treg cell boost by Teff cells. In a condition of low inflammation, the Teff cell-mediated Treg cell boost involved TNF, OX40L, and plasmacytoid dendritic cells, whereas in a condition of high inflammation, it involved TNF and IL-2. Thus, this feedback mechanism in which Treg cells are highly activated by their Teff cell counterparts depends on the immune context for its effectiveness and mechanism. This Teff cell-dependent Treg cell boost may be crucial to limit inflammatory and autoimmune responses.

B Cells Loaded with Synthetic Particulate Antigens: A Versatile Platform To Generate Antigen-Specific Helper T Cells for Cell Therapy.

Adoptive cell therapy represents a promising approach for several chronic diseases. This study describes an innovative strategy for biofunctionalization of nanoparticles, allowing the generation of synthetic particulate antigens (SPAg). SPAg activate polyclonal B cells and vectorize noncognate proteins into their endosomes, generating highly efficient stimulators for ex vivo expansion of antigen-specific CD4+ T cells. This method also allows harnessing the ability of B cells to polarize CD4+ T cells into effectors or regulators.

Immunogenicity of mRNA vs. BBV152 vaccine boosters against Omicron subvariants: Final results from Phase B of the PRIBIVAC study, a randomized clinical trial.

BACKGROUND: BBV152 (Covaxin™) is a whole-virion inactivated SARS-CoV-2 vaccine mixed with an immune adjuvant. We aimed to compare immune responses after booster vaccination with heterologous BBV152 versus homologous mRNA vaccine. METHODS: We conducted a randomized, participant-blinded, controlled trial. Fifty mRNA-vaccinated participants were enrolled and randomized to receive an mRNA booster (n = 26) or BBV152 (n = 24). Blood samples were collected pre-vaccination, and at Day 7, 28, 180 and 360 post-booster for analysis of humoral and cellular immune responses. Primary end point was the SARS-CoV-2 anti-spike antibody titer at day 28. RESULTS: Recruitment began in January 2022 and was terminated early due to the BBV152 group meeting pre-specified criteria for futility. At Day 28 post-boost, mean SARS-CoV-2 spike antibody titers were lower with BBV152 (2004 IU/mL; 95 % confidence interval [CI], 1132-3548) vs mRNA (26,669 IU/mL; 95 % CI, 21,330-33,266; p < 0.0001), but comparable levels of spike-specific CD4 and cytotoxic T-cells were observed. Anti-spike antibody titers remained significantly different at Day 180: BBV152 4467 IU/mL (95 % CI, 1959-10,186) vs mRNA 20,749 IU/mL (95 % CI, 12,303-35,075; p = 0.0017). Levels of surrogate virus neutralizing antibodies against ancestral and Omicron subvariants BA.1 and BA.2 were significantly higher among mRNA recipients at Day 180, including after adjusting for intercurrent infection. By Day 360, anti-spike antibody titers and neutralizing antibody levels against Omicron subvariants became similar between vaccine groups. By the end of the study, 16 in each arm (mRNA 64 % and BBV152 69.6 %) had breakthrough infections and time to COVID-19 infection between vaccine groups were similar (p = 0.63). CONCLUSIONS: Wild-type SARS-CoV-2 anti-spike antibody titer and surrogate virus neutralizing test levels against wild-type SARS-CoV-2 and Omicron subvariants BA.1/BA.2/BA.5 were significantly higher at Day 28 and 180 in individuals who received booster vaccination with an mRNA vaccine compared with BBV152. CLINICAL TRIAL REGISTRATION NUMBER: NCT05142319.

First SARS-CoV-2 Omicron infection as an effective immune booster among mRNA vaccinated individuals: final results from the first phase of the PRIBIVAC randomised clinical trial.

BACKGROUND: Understanding how SARS-CoV-2 breakthrough infections impacts the breadth of immune responses against existing and pre-emergent SARS-CoV-2 strains is needed to develop an evidence-based long-term immunisation strategy. METHODS: We performed a randomised, controlled trial to assess the immunogenicity of homologous (BNT162b2) versus heterologous (mRNA-1273) booster vaccination in 100 BNT162b2-vaccinated infection-naïve individuals enrolled from October 2021. Post hoc analysis was performed to assess the impact of SARS-CoV-2 infection on humoral and cellular immune responses against wild-type SARS-CoV-2 and/or Omicron subvariants. FINDINGS: 93 participants completed the study at day 360. 71% (66/93) of participants reported first SARS-CoV-2 Omicron infection by the end of the study with similar proportions of infections between homologous and heterologous booster groups (72.3% [34/47] vs 69.6% [32/46]; p = 0.82). Mean wildtype SARS-CoV-2 anti-S-RBD antibody level was significantly higher in heterologous booster group compared with homologous group at day 180 (14,588 IU/mL; 95% CI, 10,186-20,893 vs 7447 IU/mL; 4646-11,912; p = 0.025). Participants who experienced breakthrough infections during the Omicron BA.1/2 wave had significantly higher anti-S-RBD antibody levels against wildtype SARS-CoV-2 and antibody neutralisation against BA.1 and pre-emergent BA.5 compared with infection-naïve participants. Regardless of hybrid immunity status, wildtype SARS-CoV-2 anti-S-RBD antibody level declined significantly after six months post-booster or post-SARS-CoV-2 infection. INTERPRETATION: Booster vaccination with mRNA-1273 was associated with significantly higher antibody levels compared with BNT162b2. Antibody responses are narrower and decline faster among uninfected, vaccinated individuals. Boosters may be more effective if administered shortly before infection outbreaks and at least six months after last infection or booster. FUNDING: Singapore NMRC, USFDA, MRC.

B-cell ELISpot assay to analyze human memory B cell and plasmablast responses specific to SARS-CoV-2 receptor-binding domain.

B-cell ELISpot is an extremely sensitive assay based on the secretion of antibodies by B cells that requires the differentiation of B cells into antibody-secreting cells. Here, we describe the procedure to analyze both plasmablast (PB) and memory B cell (MBC) responses specific to SARS-CoV-2 receptor-binding domain (RBD) in the context of acute SARS-CoV-2 infection and vaccination. We detail steps for MBC stimulation, MBC and PB plating, detection, and counting of total IgG and RBD-specific spots. For complete details on the use and execution of this protocol, please refer to Tay et al. (2022).1.

A nonstructural protein 1 capture enzyme-linked immunosorbent assay specific for dengue viruses.

Dengue non-structural protein (NS1) is an important diagnostic marker during the acute phase of infection. Because NS1 is partially conserved across the flaviviruses, a highly specific DENV NS-1 diagnostic test is needed to differentiate dengue infection from Zika virus (ZIKV) infection. In this study, we characterized three newly isolated antibodies against NS1 (A2, D6 and D8) from a dengue-infected patient and a previously published human anti-NS1 antibody (Den3). All four antibodies recognized multimeric forms of NS1 from different serotypes. A2 bound to NS1 from DENV-1, -2, and -3, D6 bound to NS1 from DENV-1, -2, and -4, and D8 and Den3 interacted with NS1 from all four dengue serotypes. Using a competition ELISA, we found that A2 and D6 bound to overlapping epitopes on NS1 whereas D8 recognized an epitope distinct from A2 and D6. In addition, we developed a capture ELISA that specifically detected NS1 from dengue viruses, but not ZIKV, using Den3 as the capture antibody and D8 as the detecting antibody. This assay detected NS1 from all the tested dengue virus strains and dengue-infected patients. In conclusion, we established a dengue-specific capture ELISA using human antibodies against NS1. This assay has the potential to be developed as a point-of-care diagnostic tool.

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.

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.

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.

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.

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.

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.

CD27hiCD38hi plasmablasts are activated B cells of mixed origin with distinct function.

Clinically important broadly reactive B cells evolve during multiple infections, with B cells re-activated after secondary infection differing from B cells activated after a primary infection. Here we studied CD27highCD38high plasmablasts from patients with a primary or secondary dengue virus infection. Three transcriptionally and functionally distinct clusters were identified. The largest cluster 0/1 was plasma cell-related, with cells coding for serotype cross-reactive antibodies of the IgG1 isotype, consistent with memory B cell activation during an extrafollicular response. Cells in clusters 2 and 3 expressed low levels of antibody genes and high levels of genes associated with oxidative phosphorylation, EIF2 pathway, and mitochondrial dysfunction. Clusters 2 and 3 showed a transcriptional footprint of T cell help, in line with activation from naive B cells or memory B cells. Our results contribute to the understanding of the parallel B cell activation events that occur in humans after natural primary and secondary infection.

Immune cell phenotypes associated with disease severity and long-term neutralizing antibody titers after natural dengue virus infection.

Prior immunological exposure to dengue virus can be both protective and disease-enhancing during subsequent infections with different dengue virus serotypes. We provide here a systematic, longitudinal analysis of B cell, T cell, and antibody responses in the same patients. Antibody responses as well as T and B cell activation differentiate primary from secondary responses. Hospitalization is associated with lower frequencies of activated, terminally differentiated T cells and higher percentages of effector memory CD4 T cells. Patients with more severe disease tend to have higher percentages of plasmablasts. This does not translate into long-term antibody titers, since neutralizing titers after 6 months correlate with percentages of specific memory B cells, but not with acute plasmablast activation. Overall, our unbiased analysis reveals associations between cellular profiles and disease severity, opening opportunities to study immunopathology in dengue disease and the potential predictive value of these parameters.

The NF-κB RelA Transcription Factor Is Critical for Regulatory T Cell Activation and Stability.

Regulatory T cells (Tregs) play a major role in immune homeostasis and in the prevention of autoimmune diseases. It has been shown that c-Rel is critical in Treg thymic differentiation, but little is known on the role of NF-κB on mature Treg biology. We thus generated mice with a specific knockout of RelA, a key member of NF-κB, in Tregs. These mice developed a severe autoimmune syndrome with multi-organ immune infiltration and high activation of lymphoid and myeloid cells. Phenotypic and transcriptomic analyses showed that RelA is critical in the acquisition of the effector Treg state independently of surrounding inflammatory environment. Unexpectedly, RelA-deficient Tregs also displayed reduced stability and cells that had lost Foxp3 produced inflammatory cytokines. Overall, we show that RelA is critical for Treg biology as it promotes both the generation of their effector phenotype and the maintenance of their identity.