Immune reconstitution and cidofovir administration rescue human adenovirus hepatitis after allogeneic hematopoietic cell transplantation.

Human adenovirus infection (HAdV) may be fatal in patients undergoing allogeneic hematopoietic cell transplantation (HCT). Cidofovir is effective in only a part of the post-HCT HAdV infection. Therefore, posttransplant immune reconstitution is important for HAdV clearance. We describe the detailed immune reconstitution and response of adenovirus-specific T cells in a patient with inborn errors of immunity who had disseminated HAdV infection with hepatitis post-HCT and was treated with cidofovir. Though the patient received cidofovir for only 19 days starting from Day 72 after HCT because of renal dysfunction, we observed T-cell reconstitution, a decrease in HAdV copy number, and amelioration of the symptoms of HAdV infection after Day 90. We initially observed expanded NK and CD8+CD45RO+ memory subsets and later gradual increase of naïve T cells eveloped after cessation of cidofovir treatment. An increase in adenovirus-specific IFN-γ secretion from 2 to 4 months after HCT was confirmed by ELISpot assay. The progression of immune reconstitution and cidofovir treatment are considered to have contributed to survival in this patient. Optimization of transplantation methods, prompt appropriate antiviral medication, and virus-specific T-cell therapy would be necessary as the better strategy for systemic HAdV infection.

High glutamic acid decarboxylase antibody titers may be associated with a decline in ß-cell function over time and future insulin deficiency in latent autoimmune diabetes in adults.

AIMS: Latent autoimmune diabetes in adults (LADA) is characterized by positive islet-associated autoantibodies including glutamic acid decarboxylase antibody (GADA), and gradual decline in insulin secretion, progressing to insulin dependency. This cross-sectional study aimed to determine whether GADA by enzyme-linked immunosorbent assay (GADA-ELISA) titer of ≥180 U/mL could be associated with decline in ß-cell function in participants with LADA. METHODS: Sixty-three participants with LADA were recruited and an association between insulin secretion capacity and disease duration was investigated. Insulin peptide-specific inflammatory immunoreactivity was investigated to determine the disease's activity. RESULTS: There was a significant inverse correlation between disease duration and C-peptide index in participants with GADA-ELISA titer of ≥180 U/mL (Spearman's r (rs) = -0.516, p < 0.01). The positivity rate of insulin peptide-specific inflammatory immunoreactivity was significantly higher in those with ≥180 U/mL than in those with <180 U/mL (p < 0.05). In participants with human leukocyte antigen (HLA)-DRB1*04:05, a significant inverse correlation was observed between disease duration and C-peptide index in those with ≥180 U/mL (rs = -0.751, p < 0.01). CONCLUSIONS: GADA-ELISA titer of ≥180 U/mL, especially with HLA-DRB1*04:05, might reflect higher disease activity and may be associated with decline in ß-cell function over time and future insulin dependency in LADA.

Strategies to determine positive anti-SARS-CoV-2 memory T lymphocyte response during the evolution of an epidemic.

During SARS-CoV-2 pandemic, the assessment of immune protection of people at risk of severe infection was an important goal. The appearance of VOCs (Variant of Concern) highlighted the limits of evaluating immune protection through the humoral response. While the humoral response partly loses its neutralizing activity, the anti-SARS-CoV-2 memory T cell response strongly cross protects against VOCs becoming an indispensable tool to assess immune protection. We compared two techniques available in laboratory to evaluate anti-SARS-CoV-2 memory T cell response in a cohort of infected or vaccinated patients with different levels of risk to develop a severe disease: the ELISpot assay and the T-Cell Lymphocyte Proliferation Assay respectively exploring IFNγ production and cell proliferation. We showed that the ELISpot assay detected more anti-Spike memory T cell response than the Lymphocyte Proliferation Assay. We next observed that the use of two different suppliers as antigenic source in the ELISpot assay did not affect the detection of anti-Spike memory T cell response. Finally, we explored a new approach for defining the positivity threshold, using unsupervised mixed Gaussian modeling, challenging the traditional ROC curve used by the supplier. That will be helpful in endemic situation where it could be difficult to recruit "negative" patients.

In Silico Analyses, Experimental Verification and Application in DNA Vaccines of Ebolavirus GP-Derived pan-MHC-II-Restricted Epitopes.

(1) Background and Purpose: Ebola virus (EBOV) is the causative agent of Ebola virus disease (EVD), which causes extremely high mortality and widespread epidemics. The only glycoprotein (GP) on the surface of EBOV particles is the key to mediating viral invasion into host cells. DNA vaccines for EBOV are in development, but their effectiveness is unclear. The lack of immune characteristics resides in antigenic MHC class II reactivity. (2) Methods: We selected MHC-II molecules from four human leukocyte antigen II (HLA-II) superfamilies with 98% population coverage and eight mouse H2-I alleles. IEDB, NetMHCIIpan, SYFPEITHI, and Rankpep were used to screen MHC-II-restricted epitopes with high affinity for EBOV GP. Further immunogenicity and conservation analyses were performed using VaxiJen and BLASTp, respectively. EpiDock was used to simulate molecular docking. Cluster analysis and binding affinity analysis of EBOV GP epitopes and selected MHC-II molecules were performed using data from NetMHCIIpan. The selective GP epitopes were verified by the enzyme-linked immunospot (ELISpot) assay using splenocytes of BALB/c (H2d), C3H, and C57 mice after DNA vaccine pVAX-GPEBO immunization. Subsequently, BALB/c mice were immunized with Protein-GPEBO, plasmid pVAX-GPEBO, and pVAX-LAMP/GPEBO, which encoded EBOV GP. The dominant epitopes of BALB/c (H-2-I-AdEd genotype) mice were verified by the enzyme-linked immunospot (ELISpot) assay. It is also used to evaluate and explore the advantages of pVAX-LAMP/GPEBO and the reasons behind them. (3) Results: Thirty-one HLA-II-restricted and 68 H2-I-restricted selective epitopes were confirmed to have high affinity, immunogenicity, and conservation. Nineteen selective epitopes have cross-species reactivity with good performance in MHC-II molecular docking. The ELISpot results showed that pVAX-GPEBO could induce a cellular immune response to the synthesized selective peptides. The better immunoprotection of the DNA vaccines pVAX-LAMP/GPEBO coincides with the enhancement of the MHC class II response. (4) Conclusions: Promising MHC-II-restricted candidate epitopes of EBOV GP were identified in humans and mice, which is of great significance for the development and evaluation of Ebola vaccines.

Impact of Baseline and Week 2 and Week 4 Posttransplant CMV Cell-Mediated Immunity on Risk of CMV Infections and Mortality in Recipients of Allogeneic Hematopoietic Cell Transplant.

Background: Cytomegalovirus (CMV) infection is a common opportunistic infection after allogeneic hematopoietic cell transplant (alloHCT). We explored whether a change in CMV cell-mediated immunity during the first month after transplant predicts the risk of development of CMV infection and all-cause mortality. Methods: This follow-up analysis is based on data from the REACT study, a multicenter prospective observational study of recipients of alloHCT who were CMV-seropositive. Production of interferon γ following ex vivo stimulation with CMV antigens IE1 (immediate early 1) and pp65 (phosphoprotein 65) was assessed by CMV ELISPOT assay at baseline and 2 and 4 weeks after transplant. Clinically significant CMV infection (CS-CMVi) was defined as CMV viremia and/or disease necessitating antiviral therapy. We evaluated the impact of CMV CMI changes on the risk of CS-CMVi and post transplant mortality. Results: The analysis included 226 recipients of alloHCT with CMV cell-mediated immunity data at baseline and 2 and/or 4 weeks after transplant. CS-CMVi occurred in 64 patients (28%). On Cox regression analyses, independent predictors of CS-CMVi included a negative Δ change from baseline to week 2 of pp65 spot counts (hazard ratio, 3.65 [95% CI, 1.65-8.04]; P = .001) to week 4 of IE1 spot counts (hazard ratio, 2.79 [95% CI, 1.46-5.35]; P = .002), anti-thymocyte globulin conditioning regimen, type of transplant, female sex, and corticosteroid use. Kaplan-Meir analysis showed a significant association of a negative IE1 change from baseline to week 4 and increased all-cause mortality after transplant (log rank test = 0.041). Conclusions: A decrease in CMV-specific T-cell responses during the first month after transplant may predict CS-CMVi and is associated with all-cause mortality in recipients of alloHCT.

MERS-CoV‒Specific T-Cell Responses in Camels after Single MVA-MERS-S Vaccination.

We developed an ELISPOT assay for evaluating Middle East respiratory syndrome coronavirus (MERS-CoV)‒specific T-cell responses in dromedary camels. After single modified vaccinia virus Ankara-MERS-S vaccination, seropositive camels showed increased levels of MERS-CoV‒specific T cells and antibodies, indicating suitability of camel vaccinations in disease-endemic areas as a promising approach to control infection.

PP19128R, a Multiepitope Vaccine Designed to Prevent Latent Tuberculosis Infection, Induced Immune Responses In Silico and In Vitro Assays.

Background: Latent tuberculosis infection (LTBI) is the primary source of active tuberculosis (ATB), but a preventive vaccine against LTBI is lacking. Methods: In this study, dominant helper T lymphocyte (HTL), cytotoxic T lymphocyte (CTL), and B-cell epitopes were identified from nine antigens related to LTBI and regions of difference (RDs). These epitopes were used to construct a novel multiepitope vaccine (MEV) based on their antigenicity, immunogenicity, sensitization, and toxicity. The immunological characteristics of the MEV were analyzed with immunoinformatics technology and verified by enzyme-linked immunospot assay and Th1/Th2/Th17 cytokine assay in vitro. Results: A novel MEV, designated PP19128R, containing 19 HTL epitopes, 12 CTL epitopes, 8 B-cell epitopes, toll-like receptor (TLR) agonists, and helper peptides, was successfully constructed. Bioinformatics analysis showed that the antigenicity, immunogenicity, and solubility of PP19128R were 0.8067, 9.29811, and 0.900675, respectively. The global population coverage of PP19128R in HLA class I and II alleles reached 82.24% and 93.71%, respectively. The binding energies of the PP19128R-TLR2 and PP19128R-TLR4 complexes were -1324.77 kcal/mol and -1278 kcal/mol, respectively. In vitro experiments showed that the PP19128R vaccine significantly increased the number of interferon gamma-positive (IFN-γ+) T lymphocytes and the levels of cytokines, such as IFN-γ, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-10. Furthermore, positive correlations were observed between PP19128R-specific cytokines in ATB patients and individuals with LTBI. Conclusions: The PP19128R vaccine is a promising MEV with excellent antigenicity and immunogenicity and no toxicity or sensitization that can induce robust immune responses in silico and in vitro. This study provides a vaccine candidate for the prevention of LTBI in the future.

T Cell Responses to Dystrophin in a Natural History Study of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is caused by the lack of dystrophin, but many patients have rare revertant fibers that express dystrophin. The skeletal muscle pathology of DMD patients includes immune cell infiltration and inflammatory cascades. There are several strategies to restore dystrophin in skeletal muscles of patients, including exon skipping and gene therapy. There is some evidence that dystrophin restoration leads to a reduction in immune cells, but dystrophin epitopes expressed in revertant fibers or following genome editing, cell therapy, or microdystrophin delivery after adeno-associated viral gene therapy may elicit T cell production in patients. This may affect the efficacy of the therapeutic intervention, and potentially lead to serious adverse events. To confirm and extend previous studies, we performed annual enzyme- linked immunospot interferon-gamma assays on peripheral blood mononuclear cells from 77 pediatric boys with DMD recruited into a natural history study, 69 of whom (89.6%) were treated with corticosteroids. T cell responses to dystrophin were quantified using a total of 368 peptides spanning the entire dystrophin protein, organized into nine peptide pools. Peptide mapping pools were used to further localize the immune response in one positive patient. Six (7.8%) patients had a T cell-mediated immune response to dystrophin at at least one time point. All patients who had a positive result had been treated with corticosteroids, either prednisolone or prednisone. Our results show that ∼8% of DMD individuals in our cohort have a pre-existing T cell-mediated immune response to dystrophin, despite steroid treatment. Although these responses are relatively low level, this information should be considered a useful immunological baseline before undertaking clinical trials and future DMD studies. We further highlight the importance for a robust, reproducible standard operating procedure for collecting, storing, and shipping samples from multiple centers to minimize the number of inconclusive data.

Long-Term SARS-CoV-2-Specific Humoral and T Cell Responses after the BNT162b2 or BBIBP-CorV Booster and the Incidence of Breakthrough Infections among Healthcare Workers.

The effectiveness of COVID-19 vaccines developed against the original virus strain deteriorated noticeably in efficacy against the Omicron variant (B.1.1.529). Moreover, the immunity developed after vaccination or due to natural infection rapidly waned. In the present study, covering this period, we summarize the incidence of breakthrough infections among healthcare workers (HCWs) with respect to administration of the three vaccine doses. Additionally, we evaluate the long-term SARS-CoV-2-specific humoral and T cell responses at two different time points: six and twelve months after receipt of the third (booster) dose. The spike-protein-specific antibody levels and the quantity of structural-protein-specific T cells were evaluated at these time points and compared with the values measured earlier, 14 days after the booster vaccination. The study participants were categorized into two cohorts: Members of the first cohort received a two-dose BNT162b2 mRNA-based vaccine regimen, followed by an additional BNT162b2 booster six months later. Individuals in the second cohort received an inactivated-virus-based BBIBP-CorV booster six months after the initial two-dose BNT162b2 vaccination. Overall, 64.3% of participants were infected with SARS-CoV-2 confirmed by PCR or antigen test; however, additional subjects from the first cohort (23%) who did not know about their previous infection but had an anti-nucleocapsid T cell response were also considered virus-experienced. According to our results, no statistically significant difference was found between the two cohorts regarding the SARS-CoV-2-specific T cell response, neutralizing anti-RBD IgG, and anti-S IgA serum antibody levels either six or twelve months after receiving the booster, despite the overall higher median values of the first cohort. The only significant difference was the higher anti-S1/S2 IgG antibody level in the first cohort one year after the BNT162b2 booster (p = 0.039). In summary, the BNT162b2 and BBIBP-CorV boosters maintain durable humoral and T cell-mediated immune memory even one year after application. Although the booster provided limited protection against Omicron breakthrough infections, as 73.6% of these infections occurred after the booster vaccination, which means 53.5% cumulative incidence, it still offered excellent protection against severe disease and hospitalization in both cohorts.

Comparison between enzyme-linked immunospot assay and intracellular cytokine flow cytometry assays for the evaluation of T cell response to SARS-CoV-2 after symptomatic COVID-19.

INTRODUCTION: Evaluation of different cell-based assays for the study of adaptive immune responses against SARS-CoV-2 is crucial for studying long-term and vaccine-induced immunity. METHODS: Enzyme-linked immunospot assay (ELISpot) and intracellular cytokine staining (ICS) using peptide pools spanning the spike protein and nucleoprotein of SARS-CoV-2 were performed in 25 patients who recovered from paucisymptomatic (n = 19) or severe COVID-19 (n = 6). RESULTS: The proportion of paucisymptomatic patients with detectable SARS-CoV-2 T cells was low, as only 44% exhibit a positive T cell response with the ICS and 67% with the ELISpot. The magnitude of SARS-CoV-2 T cell responses was low, both with ICS (median at 0.12% among total T cells) and ELISpot (median at 61 SFCs/million peripheral blood mononuclear cells [PBMC]) assays. Moreover, T cell responses in paucisymptomatic patients seemed lower than among patients with severe disease. In the paucisymptomatic patients, the two assays were well correlated with 76% of concordant responses and a Cohen's kappa of 55. Furthermore, in four patients SARS-CoV-2 T cells were detected by ELISpot but not with ICS. Short-term culture could improve the detection of specific T cells. CONCLUSIONS: In patients who recovered from paucisymptomatic COVID-19, the proportion of detectable anti-SARS-CoV-2 responses and their magnitude seemed lower than in patients with more severe symptoms. The ELISpot appeared to be more sensitive than the ICS assay. Short-term culture revealed that paucisymptomatic patients had nonetheless few SARS-CoV-2 T cells at a very low rate in peripheral blood. These data indicate that various ex-vivo assays may lead to different conclusions about the presence or absence of SARS-CoV-2 T cell immunity.

ELISpot Assay for Gene Therapy in Large Animal Studies.

Formation of neutralizing antibodies and cellular immune response with repeat adeno-associated virus (AAV) gene therapy dosing are critical concerns in translational, large animal studies. The enzyme-linked immunospot/immunosorbent spot (ELISpot) assay introduced a way to track B- and/or T-cell response to therapy over time at a protein level. We describe the protocol for this assay looking at relative interferon (IFN)-γ secretion in pre- and post-AAV injections in a pig model.

Specific Antibody and the T-Cell Response Elicited by BNT162b2 Boosting After Two ChAdOx1 nCoV-19 in Common Variable Immunodeficiency.

Common variable immunodeficiency (CVID) patients have markedly decreased immune response to vaccinations. In this study we evaluated humoral and T cell-mediated responses against severe acute respiratory syndrome coronavirus-2 (SARS-Cov-2) with additional flow cytometric changes in CVID patients receiving booster vaccination with BNT162b2 after two ChAdOx1 nCoV-19. The BNT162b2 vaccine raised the anti-spike protein S immunoglobulin G over the cut-off value from 70% to 83% in CVID, anti-neutralizing antibody had been raised over a cut-off value from 70% to 80% but levels after boosting were significantly less in both tests than in healthy controls (*p=0.02; **p=0.009 respectively). Anti-SARS-CoV-2 immunoglobulin A became less positive in CVID after boosting, but the difference was not significant. The cumulative interferon-γ positive T cell response by ELISpot was over the cut-off value in 53% of the tested individuals and raised to 83% after boosting. This and flow cytometric control of cumulative CD4+ and CD8+ virus-specific T cell absolute counts in CVID were also statistically not different from healthy individuals after boosting. Additional flow cytometric measures for CD45+ lymphocytes, CD3+, and CD19+ cells have not shown significant differences from controls except for lower CD4+T cell counts at both time points (**p=0.003; **p=0.002), in parallel CD4+ virus-specific T-cell ratio was significantly lower in CVID patients at the first time point (*p: 0.03). After boosting, in more than 33% of both CVID patients and also in their healthy controls we detected a decrease in absolute CD45+, CD3+, CD3+CD4+, and CD3+CD8+, CD19+, and CD16+56+ cell counts. CD16+CD56+ cell counts were significantly lower compared to controls before and after boosting (*p=0.02, *p=0.02). CVID patients receiving immunosuppressive therapy throughout the previous year or autologous stem cell transplantation two years before vaccination had worse responses in anti-spike, anti-neutralizing antibody, CD3+CD4+T, CD19+ B, and natural killer cell counts than the whole CVID group. Vaccinations had few side effects. Based on these data, CVID patients receiving booster vaccination with BNT162b2 after two ChadOx1 can effectively elevate the levels of protection against COVID-19 infection, but the duration of the immune response together with COVID-19 morbidity data needs further investigation among these patients.

Longitudinal Assessment of SARS-CoV-2-Specific T Cell Cytokine-Producing Responses for 1 Year Reveals Persistence of Multicytokine Proliferative Responses, with Greater Immunity Associated with Disease Severity.

Cell-mediated immunity is critical for long-term protection against most viral infections, including coronaviruses. We studied 23 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected survivors over a 1-year post-symptom onset (PSO) interval by ex vivo cytokine enzyme-linked immunosorbent spot assay (ELISpot) assay. All subjects demonstrated SARS-CoV-2-specific gamma interferon (IFN-γ), interleukin 2 (IL-2), and granzyme B (GzmB) T cell responses at presentation, with greater frequencies in severe disease. Cytokines, mainly produced by CD4+ T cells, targeted all structural proteins (nucleocapsid, membrane, and spike) except envelope, with GzmB and IL-2 greater than IFN-γ. Mathematical modeling predicted that (i) cytokine responses peaked at 6 days for IFN-γ, 36 days for IL-2, and 7 days for GzmB, (ii) severe illness was associated with reduced IFN-γ and GzmB but increased IL-2 production rates, and (iii) males displayed greater production of IFN-γ, whereas females produced more GzmB. Ex vivo responses declined over time, with persistence of IL-2 in 86% and of IFN-γ and GzmB in 70% of subjects at a median of 336 days PSO. The average half-life of SARS-CoV-2-specific cytokine-producing cells was modeled to be 139 days (~4.6 months). Potent T cell proliferative responses persisted throughout observation, were CD4 dominant, and were capable of producing all 3 cytokines. Several immunodominant CD4 and CD8 epitopes identified in this study were shared by seasonal coronaviruses or SARS-CoV-1 in the nucleocapsid and membrane regions. Both SARS-CoV-2-specific CD4+ and CD8+ T cell clones were able to kill target cells, though CD8 tended to be more potent. IMPORTANCE Our findings highlight the relative importance of SARS-CoV-2-specific GzmB-producing T cell responses in SARS-CoV-2 control and shared CD4 and CD8 immunodominant epitopes in seasonal coronaviruses or SARS-CoV-1, and they indicate robust persistence of T cell memory at least 1 year after infection. Our findings should inform future strategies to induce T cell vaccines against SARS-CoV-2 and other coronaviruses.

An adjusted ELISpot-based immunoassay for evaluation of SARS-CoV-2-specific T-cell responses.

Like antibody evaluation, using an effective antigen-specific T-cell immunity assessment method in coronavirus disease 2019 (COVID-19) patients, survivors and vaccinees is crucial for understanding the immune persistence, prognosis assessment, and vaccine development for COVID-19. This study evaluated an empirically adjusted enzyme-linked immunospot assay for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T-cell immunity in 175 peripheral blood samples from COVID-19 convalescents and healthy individuals. Results of viral nucleic acid were used as the gold standard of infection confirmation. The SARS-CoV-2M peptide pool had higher sensitivity of 85% and specificity of 71% for the single peptide pool. For combined peptide pools, the parallel evaluation (at least one of the peptide pools is positive) of total peptide pools (S1&S2&M&N) had higher sensitivity (up to 93%), and the serial evaluation (all peptide pools are positive) of total peptide pools had higher specificity (up to 100%). The result of the serial evaluation was better than that of the parallel evaluation as a whole. The detection efficiency of M and N peptide pool serial evaluation appeared the highest, with a sensitivity of 80% and specificity of 93%. This T-cell immunity detection assay introduced in this report can achieve high operability and applicability. Therefore, it can be an effective SARS-CoV-2-specific cellular immune function evaluation method.

Vaccine-induced humoral response against SARS-CoV-2 dramatically declined but cellular immunity possibly remained at 6 months post BNT162b2 vaccination.

To evaluate vaccine-induced humoral and cell-mediated immunity at 6 months after completion of two doses of BNT162b2 vaccination, immunoglobulin G against SARS-CoV-2 spike protein (SP IgG), 50% neutralizing antibody (NT50), and spot-forming cell (SFC) counts were evaluated by interferon-γ releasing ELISpot assay of 98 healthy subjects (median age, 43 years). The geometric mean titers of SP IgG and NT50 decreased from 95.2 (95% confidence interval (CI) 79.8-113.4) to 5.7 (95% CI 4.9-6.7) and from 680.4 (588.0-787.2) to 130.4 (95% CI 104.2-163.1), respectively, at 3 weeks and 6 months after the vaccination. SP IgG titer was negatively correlated with age and alcohol consumption. Spot-forming cell counts at 6 months did not correlate with age, gender, and other parameters of the patients. SP IgG, NT50, and SFC titers were elevated in the breakthrough infected subjects. Although the levels of vaccine-induced antibodies dramatically declined at 6 months after vaccination, a certain degree of cellular immunity was observed irrespective of the age.

Antibody and T Cell Responses against SARS-CoV-2 Elicited by the Third Dose of BBIBP-CorV (Sinopharm) and BNT162b2 (Pfizer-BioNTech) Vaccines Using a Homologous or Heterologous Booster Vaccination Strategy.

In the present study, antibody and T cell-mediated immune responses elicited by BBIBP-CorV and BNT162b2 vaccines were compared 6 months after the two-dose immunization of healthy individuals. Additionally, antibody and T cell responses after the third dose of BBIBP-CorV or BNT162b2 were compared using a homologous or heterologous vaccination strategy. The third dose was consistently administered 6 months after the second dose. Six months following the two-dose vaccination, the cumulative IFNγ-positive T cell response was almost identical in participants immunized with either two doses of BNT162b2 or BBIBP-CorV vaccines; however, significant differences were revealed regarding humoral immunity: the two-dose BNT162b2 vaccine maintained a significantly higher antireceptor-binding domain (RBD) IgG, anti-spike (S1/S2) IgG, and IgA antibody levels. The BNT162b2 + BNT162b2 + BBIBP-CorV vaccine series elicited significantly lower anti-RBD IgG and anti-S1/S2 IgG levels than three doses of BNT162b2, while the anti-S IgA level was equally negligible in both groups. Importantly, the cumulative IFNγ-positive T cell response was highly similar in both groups. Surprisingly, the BBIBP-CorV + BBIBP-CorV + BNT162b2 vaccination series provided a much higher cumulative IFNγ-positive T cell response than that elicited by three doses of BNT162b2; moreover, the levels of anti-RBD IgG and anti-S IgA were almost identical. Only the mean anti-S1/S2 IgG levels were higher after receiving three mRNA vaccines. Based on these data, we can conclude that administering a third dose of BNT162b2 after two doses of BBIBP-CorV is an effective strategy to significantly enhance both humoral and T cell-mediated immune response, and its effectiveness is comparable to that of three BNT162b2 vaccines.

Radiotherapy and High-Dose Interleukin-2: Clinical and Immunological Results of a Proof of Principle Study in Metastatic Melanoma and Renal Cell Carcinoma.

High-dose interleukin-2 (HD IL-2) has curative potential in metastatic melanoma (MM) and renal cell carcinoma (RCC). Radiotherapy (RT) kills cancer cells and induces immunomodulatory effects. Prospective trials exploring clinical and immunological properties of combined RT/HD IL-2 are still needed. We designed a phase II, single-arm clinical trial for patients with MM and RCC. The treatment schedule consisted of 3 daily doses of 6-12 Gy of RT to 1-5 non-index metastatic fields, before IL-2 at the first and third treatment cycle. HD IL-2 was administered by continuous infusion for 72 hours and repeated every 3 weeks for up to 4 cycles, thereafter every 4 weeks for a maximum of 2 cycles. The primary endpoint was the immunological efficacy of the combined RT/HD IL-2 treatment (assessed by IFN-γ ELISPOT). Nineteen out of 22 patients were evaluable for immunological and clinical response. Partial response occurred in 3 (15.7%) patients and stable disease was observed in 7 (36.8%). The disease control rate was 52.6% after a median follow up of 39.2 months. According to Common Terminology Criteria for Adverse Events 4.0 (CTCAE 4.0), the majority of toxicities were grade 1-2. Immunological responses were frequent and detected in 16 (84.2%) patients. Increased levels of IL-8 and IL-10 in melanoma, circulating effector memory CD4+ and intratumoral CD8+ T cells in both tumor types were detected after therapy. Overall the treatment was well tolerated and immunologically active. Immunomonitoring and correlative data on tumor and peripheral blood cell subsets suggest that this combination treatment could be a promising strategy for patients progressing after standard treatments.

Comparison of antibody and T cell responses elicited by BBIBP-CorV (Sinopharm) and BNT162b2 (Pfizer-BioNTech) vaccines against SARS-CoV-2 in healthy adult humans.

In the present study, humoral and T cell-mediated immune responses elicited by BBIBP-CorV (inactivated virus) and BNT162b2 (mRNA-based) vaccines against SARS-CoV-2 virus were compared. Convalescent volunteers were also investigated to evaluate adaptive immunity induced by live virus. Although both vaccines induced antibody- and T cell-mediated immune responses, our analysis revealed significant quantitative and qualitative differences between the two types of challenges. The BBIBP-CorV vaccine elicited antireceptor-binding domain (RBD) IgG, as well as anti-spike protein (S) IgG and IgA antibodies in healthy individuals, the levels of which were much lower than after BNT162b2 vaccination but still higher than in the convalescent patients. The cumulative IFNγ-positive T cell response, however, was only twofold higher in participants injected with BNT162b2 compared to those who were primed and boosted with BBIBP-CorV vaccine. Moreover, the inactivated virus vaccine induced T cell response that targets not only the S but also the nucleocapsid (N) and membrane (M) proteins, whereas the mRNA vaccine was able to elicit a much narrower response that targets the S protein epitopes only. Thus, the pattern of BBIBP-CorV-induced T cell response in virus-naive participants was similar to the cell-mediated anti-SARS-CoV-2 response observed in convalescent patients. Based on these data, we can conclude that the BBIBP-CorV inactivated virus vaccine is immunologically effective. However, the duration of BBIBP-CorV-induced integrated, antibody, and T cell-mediated, immune responses needs further investigation.

Monitoring cell-mediated immune responses in AAV gene therapy clinical trials using a validated IFN-γ ELISpot method.

Adeno-associated virus (AAV)-based gene therapies have recently shown promise as a novel treatment for hereditary diseases. Due to the viral origin of the vector capsid, however, cellular immune response may be elicited that could eliminate transduced target cells. To monitor cellular immune responses in clinical trials, we optimized and bioanalytically validated a sensitive, robust, and reliable interferon-γ (IFN-γ) enzyme-linked immunospot (ELISpot) assay. For method performance validation, human peripheral blood mononuclear cells (PBMCs) were stimulated with peptides derived from AAV5 capsid proteins and the encoded transgene product, human blood clotting factor VIII (FVIII), in addition to positive controls, such as peptides from the 65-kDa phosphoprotein of cytomegalovirus. We statistically assessed the limit of detection and confirmatory cutpoint, evaluated precision and linearity, and confirmed specificity using HIV peptides. Robustness parameter ranges and sample stability periods were established. The validated IFN-γ ELISpot assay was then implemented in an AAV5-FVIII gene therapy clinical trial. Cellular immune responses against the AAV5 capsid were observed in most participants as soon as 2 weeks following dose administration; only limited responses against the transgene product were detected. These data underscore the value of using validated methods for monitoring cellular immunity in AAV gene therapy trials.