Magnitude Of T-cell Responses Research Articles

Perturbations of the T-cell receptor repertoire in response to SARS-CoV-2 in immunocompetent and immunocompromised individuals

Functional T-cell responses are essential for virus clearance and long-term protection after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, whereas certain clinical factors, such as older age and immunocompromise, are associated with worse outcome. We sought to study the breadth and magnitude of T-cell responses in patients with coronavirus disease 2019 (COVID-19) and in individuals with inborn errors of immunity (IEIs) who had received COVID-19 mRNA vaccine. Using high-throughput sequencing and bioinformatics tools to characterize the T-cell receptor β repertoire signatures in 540 individuals after SARS-CoV-2 infection, 31 IEI recipients of COVID-19 mRNA vaccine, and healthy controls, we quantified HLA class I- and class II-restricted SARS-CoV-2-specific responses and also identified several HLA allele-clonotype motif associations in patients with COVID-19, including a subcohort of anti-type 1 interferon (IFN-1)-positive patients. Our analysis revealed that elderly patients with COVID-19 with critical disease manifested lower SARS-CoV-2 T-cell clonotype diversity as well as T-cell responses with reduced magnitude, whereas the SARS-CoV-2-specific clonotypes targeted a broad range of HLA class I- and class II-restricted epitopes across the viral proteome. The presence of anti-IFN-I antibodies was associated with certain HLA alleles. Finally, COVID-19 mRNA immunization induced an increase in the breadth of SARS-CoV-2-specific clonotypes in patients with IEIs, including those who had failed to seroconvert. Elderly individuals have impaired capacity to develop broad and sustained T-cell responses after SARS-CoV-2 infection. Genetic factors may play a role in the production of anti-IFN-1 antibodies. COVID-19 mRNA vaccines are effective in inducing T-cell responses in patients with IEIs.

MO856: Immunogenicity and Adverse Events After Vaccination With GAM-COVID-VAC (Sputnik V) in Patients on Maintenance Haemodialysis Compared With Healthy Controls: A Preliminary Report

Abstract BACKGROUND AND AIMS Recent studies evaluated safety and efficacy of vaccines against coronavirus disease (COVID-19), but none of them are currently approved in Russia. Here, we report results on immunogenicity of the recombinant adenovirus (rAd) 26 and rAd5 vector-based COVID-19 vaccine Gam-COVID-Vac (Sputnik V, developed by Gamaleya National Research Centre, Russia) in patients, receiving maintenance haemodialysis (HD). We aimed to compare the strength of humoral and cellular immunity after 2 doses of Gam-COVID-Vac in patients receiving HD and individuals with normal kidney function. METHOD The prospective cohort study (NCT: 04 805 632) included 23 patients treated with maintenance HD and 28 volunteers with normal kidney function. All participates were adult, had been fully vaccinated with Gam-COVID-Vac vaccine and had no prior history of suspected or confirmed COVID-19. Subjects were excluded if they had a history of confirmed SARS-CoV-2 infection, had underlying autoimmune disease, malignancies or concomitant immunosuppressive therapy. In all participants the levels of specific IgG were quantified at 4 weeks after second vaccine dose administration using a semi-quantitative SARS-CoV-2 S1 IgG enzyme-linked immunosorbent assay. In all subjects, specific CD4+ and CD8+ T-lymphocyte responses (count of spots to spike structural peptide of SARS-CoV-2 virus in IGRA test) were evaluated at the same timepoint. All the participants were asked to report adverse events (AEs) following first and second vaccine administration, included general malaise, fever, myalgia, headache, allergic reactions and injection site reactions. RESULTS Overall, the incidence of vaccine-associated AEs was less in HD patients than in healthy controls: 32% versus 75% after both first and second vaccine administrations, RR = 0.46 (95% confidence interval 0.24–0.79), P = 009 (Table 1). The most commonly reported AE in both groups was pain in the injection site. No severe or serious AEs occurred in both patients and healthy controls. The seroconversion rate in 4 weeks after second vaccine shot reached 100% (28 of 28) in healthy subjects and 87% (20 of 23) in patients receiving HD. IgG levels did not differ between groups: 4.7 [Q1-Q3: 3.37; 6.25] in HD patients versus 5.5 [Q1-Q3: 3.3; 7.2] in controls, P = 0.219 (Figure 1A). The T-test result was positive in 70% (16 of 23) of HD patients and in 79% (22 of 28) of controls. The magnitude of T-cell response was comparable between groups: 30 spots [Q1-Q3: 11; 48] in HD patients versus 30 spots [Q1-Q3: 14; 51] in controls, P = 0.745 (Figure 1B). CONCLUSION Patients receiving HD develop efficient humoral and cellular immune responses after complete vaccination against COVID-19 with Gam-COVID-Vac vaccine, which is comparable with those in healthy adults.

MO836: Patients Receiving Haemodialysis Maintain Sars-Cov-2 Immunity Comparable With Healthy Individuals: A Prospective Cohort Study

Abstract BACKGROUND AND AIMS Patients with end-stage kidney disease receiving maintenance haemodialysis (HD) are at increased risk for mortality after infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) compared with the general population. However, it is currently unknown whether the long-term SARS-CoV-2 humoral and cellular immune responses in patients receiving HD are comparable to individuals with normal kidney function. We aimed to investigate the 6-month kinetics of SARS-CoV-2 IgG antibody and specific T-cell levels in patients receiving HD compared with those in healthy individuals. METHOD The prospective cohort study included 24 patients treated with maintenance HD and 27 healthy controls with confirmed history of coronavirus disease (COVID-19). Nobody of them received vaccination before and during the study period. In all participants, the levels of specific IgG were quantified at three timepoints: 10, 18 and 26 weeks from disease onset. In a subgroup of patients, specific CD4+ and CD8+ T-lymphocyte responses were evaluated using IGRA test. RESULTS The incidence of COVID-19-related symptoms did not differ between the groups, except for the loss of smell, which occurred much more frequently among healthy subjects (70% versus 30.4% in HD group, P = 0.01). The seropositivity rate declined in healthy subjects over time and was 85% and 70.4% at weeks 18 and 26, respectively. All HD patients remained seropositive over the study period. Seropositivity rate at week 26 was greater among patients receiving HD: RR = 1.4 [95% confidence interval (95% CI): 1.17–1.94] (reciprocal of RR = 0.7, 95% CI: 0.52–0.86), P = 0.0064. In both groups, IgG levels decreased from week 10 to week 26, but antibodies vanished more rapidly in controls than in HD group (ANOVA P = 0.0012)—Figure 1. The magnitude of T-cell response was significantly lower in controls than in HD patients at weeks 10 (P = 0.019) and 26 (P = 0.0098) after COVID-19 diagnosis, but not at week 18—Figure 2. CONCLUSION Compared with healthy adults, patients receiving HD maintain significant long-term humoral and cellular immune responses following natural COVID-19. SARS-CoV-2 IgG antibodies did not decline more rapidly in patients receiving HD than in healthy controls over 6-months period.

Pre-existing T cell immunity determines the frequency and magnitude of cellular immune response to two doses of mRNA vaccine against SARS-CoV-2

Little is known about the factors associated with lack of T-cell response to mRNA vaccines against SARS-CoV-2. In a prospective cohort of 61 health care workers (HCWs), 21% and 16% after the first dose of mRNA BNT162b vaccine, and 12% and 7% after the second dose, showed lack of CD4+ and CD8+ T-cell response, respectively. Pre-existing T-cell immunity, due to past infection (46%) or cross-reactive cellular response (26%), was significantly associated with T-cell response in frequency (CD4+ T-cell, 100% vs 82% after two doses; p = 0.049) and in the magnitude of T-cell response during follow up. Furthermore, baseline CD4+ T-cell correlated positively with the titer of specific IgG-antibodies after first and second vaccine dose. Our data demonstrate that cross-reactive T-cells correlate with a better cellular response as well as an enhanced humoral response, and we confirm the close correlation of humoral and cellular response after mRNA vaccination.

Immunogenicity of COVID-19 Vaccinations in Hematological Patients: 6-Month Follow-Up and Evaluation of a 3rd Vaccination.

Simple SummaryHere we confirm lower humoral and cellular responses in hematological patients compared with controls and highlight the response in risk-groups such as CAR-T-cell recipients. The main risk factor for a poor humoral response was anti-CD20 therapy. CD19+ B-cell and CD4+ T-cell levels were shown to be additional predictive markers for seroconversion. Further, we demonstrate a decline in antibodies over six months in responders and controls, and question if second vaccination non-responders benefit from of a third vaccination.Here we analyzed SARS-CoV-2-specific antibodies and T-cell responses after two coronavirus disease 2019 vaccinations over a six-month period in patients with hematological malignancies and assessed the effect of a third vaccination in a subgroup. Sixty-six patients and 66 healthy controls were included. After two vaccinations seroconversion was seen in 52% and a T-cell-specific response in 59% of patients compared with 100% in controls (p = 0.001). Risk factors for a poor serological response were age (<65a), history of anti-CD20 therapy within the year preceding vaccination, CD19+ B-cells < 110/µL, and CD4+ T-cells > 310/µL. The magnitude of T-cell response was higher in patients <65a and with CD19+ B-cells < 110/µL. Patients and healthy controls demonstrated a significant decrease in SARS-CoV-2 S antibody levels over the period of six months (p < 0.001). A third vaccination demonstrated a strong serological response in patients who had responded to the previous doses (p < 0.001). The third vaccination yielded seroconversion in three out of 19 patients in those without serological response. We conclude that both humoral and cellular responses after SARS-CoV-2 immunization are impaired in patients with hematological malignancies. A third vaccination enhanced B-cell response in patients who previously responded to the second vaccination but may be of limited benefit in patients without prior seroconversion.

SARS-CoV-2-specific antibody and T-cell responses 1 year after infection in people recovered from COVID-19: a longitudinal cohort study

BackgroundThe memory immune response is crucial for preventing reinfection or reducing disease severity. However, the robustness and functionality of the humoral and T-cell response to SARS-CoV-2 remains unknown 12 months after initial infection. The aim of this study is to investigate the durability and functionality of the humoral and T-cell response to the original SARS-CoV-2 strain and variants in recovered patients 12 months after infection.MethodsIn this longitudinal cohort study, we recruited participants who had recovered from COVID-19 and who were discharged from the Wuhan Research Center for Communicable Disease Diagnosis and Treatment at the Chinese Academy of Medical Sciences, Wuhan, China, between Jan 7 and May 29, 2020. Patients received a follow-up visit between Dec 16, 2020, and Jan 27, 2021. We evaluated the presence of IgM, IgA, and IgG antibodies against the SARS-CoV-2 nucleoprotein, Spike protein, and the receptor-binding domain 12 months after initial infection, using ELISA. Neutralising antibodies against the original SARS-CoV-2 strain, and the D614G, beta (B.1.351), and delta (B.1.617.2) variants were analysed using a microneutralisation assay in a subset of plasma samples. We analysed the magnitude and breadth of the SARS-CoV-2-specific memory T-cell responses using the interferon γ (IFNγ) enzyme-linked immune absorbent spot (ELISpot) assay and intracellular cytokine staining (ICS) assay. The antibody response and T-cell response (ie, IFN-γ, interleukin-2 [IL-2], and tumour necrosis factor α [TNFα]) were analysed by age and disease severity. Antibody titres were also analysed according to sequelae symptoms.FindingsWe enrolled 1096 patients, including 289 (26·4%) patients with moderate initial disease, 734 (67·0%) with severe initial disease, and 73 (6·7%) with critical initial disease. Paired plasma samples were collected from 141 patients during the follow-up visits for the microneutralisation assay. PBMCs were collected from 92 of 141 individuals at the 12-month follow-up visit, of which 80 were analysed by ELISpot and 92 by ICS assay to detect the SARS-CoV-2-specific memory T-cell responses. N-IgG (899 [82·0%]), S-IgG (1043 [95·2%]), RBD-IgG (1032 [94·2%]), and neutralising (115 [81·6%] of 141) antibodies were detectable 12 months after initial infection in most individuals. Neutralising antibodies remained stable 6 and 12 months after initial infection in most individuals younger than 60 years. Multifunctional T-cell responses were detected for all SARS-CoV-2 viral proteins tested. There was no difference in the magnitude of T-cell responses or cytokine profiles in individuals with different symptom severity. Moreover, we evaluated both antibody and T-cell responses to the D614G, beta, and delta viral strains. The degree of reduced in-vitro neutralising antibody responses to the D614G and delta variants, but not to the beta variant, was associated with the neutralising antibody titres after SARS-CoV-2 infection. We also found poor neutralising antibody responses to the beta variant; 83 (72·2%) of 115 patients showed no response at all. Moreover, the neutralising antibody titre reduction of the recovered patient plasma against the delta variant was similar to that of the D614G variant and lower than that of the beta variant. By contrast, T-cell responses were cross-reactive to the beta variant in most individuals. Importantly, T-cell responses could be detected in all individuals who had lost the neutralising antibody response to SARS-CoV-2 12 months after the initial infection.InterpretationSARS-CoV-2-specific neutralising antibody and T-cell responses were retained 12 months after initial infection. Neutralising antibodies to the D614G, beta, and delta viral strains were reduced compared with those for the original strain, and were diminished in general. Memory T-cell responses to the original strain were not disrupted by new variants. This study suggests that cross-reactive SARS-CoV-2-specific T-cell responses could be particularly important in the protection against severe disease caused by variants of concern whereas neutralising antibody responses seem to reduce over time.FundingChinese Academy of Medical Sciences, National Natural Science Foundation, and UK Medical Research Council.

Humoral and cellular immunogenicity to a second dose of COVID-19 vaccine BNT162b2 in people receiving methotrexate or targeted immunosuppression: a longitudinal cohort study.

SummaryBackgroundCOVID-19 vaccines have robust immunogenicity in the general population. However, data for individuals with immune-mediated inflammatory diseases who are taking immunosuppressants remains scarce. Our previously published cohort study showed that methotrexate, but not targeted biologics, impaired functional humoral immunity to a single dose of COVID-19 vaccine BNT162b2 (Pfizer-BioNTech), whereas cellular responses were similar. Here, we aimed to assess immune responses following the second dose.MethodsIn this longitudinal cohort study, we recruited individuals with psoriasis who were receiving methotrexate or targeted biological monotherapy (ie, tumour necrosis factor [TNF] inhibitors, interleukin [IL]-17 inhibitors, or IL-23 inhibitors) from a specialist psoriasis centre serving London and South-East England. The healthy control cohort were volunteers without psoriasis, not receiving immunosuppression. Immunogenicity was evaluated immediately before, on day 28 after the first BNT162b2 vaccination and on day 14 after the second dose (administered according to an extended interval regimen). Here, we report immune responses following the second dose. The primary outcomes were humoral immunity to the SARS-CoV-2 spike glycoprotein, defined as titres of total spike-specific IgG and of neutralising antibody to wild-type, alpha (B.1.1.7), and delta (B.1.617.2) SARS-CoV-2 variants, and cellular immunity defined as spike-specific T-cell responses (including numbers of cells producing interferon-γ, IL-2, IL-21).FindingsBetween Jan 14 and April 4, 2021, 121 individuals were recruited, and data were available for 82 participants after the second vaccination. The study population included patients with psoriasis receiving methotrexate (n=14), TNF inhibitors (n=19), IL-17 inhibitors (n=14), IL-23 inhibitors (n=20), and 15 healthy controls, who had received both vaccine doses. The median age of the study population was 44 years (IQR 33–52), with 43 (52%) males and 71 (87%) participants of White ethnicity. All participants had detectable spike-specific antibodies following the second dose, and all groups (methotrexate, targeted biologics, and healthy controls) demonstrated similar neutralising antibody titres against wild-type, alpha, and delta variants. By contrast, a lower proportion of participants on methotrexate (eight [62%] of 13, 95% CI 32–86) and targeted biologics (37 [74%] of 50, 60–85; p=0·38) had detectable T-cell responses following the second vaccine dose, compared with controls (14 [100%] of 14, 77–100; p=0·022). There was no difference in the magnitude of T-cell responses between patients receiving methotrexate (median cytokine-secreting cells per 106 cells 160 [IQR 10–625]), targeted biologics (169 [25–503], p=0·56), and controls (185 [133–328], p=0·41).InterpretationFunctional humoral immunity (ie, neutralising antibody responses) at 14 days following a second dose of BNT162b2 was not impaired by methotrexate or targeted biologics. A proportion of patients on immunosuppression did not have detectable T-cell responses following the second dose. The longevity of vaccine-elicited antibody responses is unknown in this population.FundingNIHR Biomedical Research Centre at Guy's and St Thomas' NHS Foundation Trust and King's College London; The Psoriasis Association.

Immune-checkpoint proteins VISTA and PD-1 nonredundantly regulate murine T-cell responses

V-domain immunoglobulin suppressor of T-cell activation (VISTA) is a negative immune-checkpoint protein that suppresses T-cell responses. To determine whether VISTA synergizes with another immune-checkpoint, programmed death 1 (PD-1), this study characterizes the immune responses in VISTA-deficient, PD-1-deficient (KO) mice and VISTA/PD-1 double KO mice. Chronic inflammation and spontaneous activation of T cells were observed in both single KO mice, demonstrating their nonredundancy. However, the VISTA/PD-1 double KO mice exhibited significantly higher levels of these phenotypes than the single KO mice. When bred onto the 2D2 T-cell receptor transgenic mice, which are predisposed to development of inflammatory autoimmune disease in the CNS, the level of disease penetrance was significantly enhanced in the double KO mice compared with in the single KO mice. Consistently, the magnitude of T-cell response toward foreign antigens was synergistically higher in the VISTA/PD-1 double KO mice. A combinatorial blockade using monoclonal antibodies specific for VISTA and PD-L1 achieved optimal tumor-clearing therapeutic efficacy. In conclusion, our study demonstrates the nonredundant role of VISTA that is distinct from the PD-1/PD-L1 pathway in controlling T-cell activation. These findings provide the rationale to concurrently target VISTA and PD-1 pathways for treating T-cell-regulated diseases such as cancer.

Doubtful Role of IL28B Polymorphism in Occult Hepatitis B Infection

Aims: To investigate the influence of IL28B polymorphism in occult hepatitis B infection (OBI) and whether IL28B genetic variants are associated with hepatitis B virus (HBV)-specific T-cell responses. Patients and Methods: The rs12979860 IL28B genotype was determined in 34 OBI blood donors, 22 spontaneous HBV resolvers, 36 inactive HBV carriers and 25 seronegative donors. T-cell responses to HBV recombinant proteins were assessed by interferon-γ enzyme-linked immunospot assay. Results: The frequency of the IL28B CC genotype among OBI patients was similar to that of inactive carriers [41 vs. 39%, respectively, p = 0.961; odds ratio (OR) = 1.10; 95% confidence interval (CI) = 0.42-2.86; p = 0.845]. The IL28B CC genotype was found more frequently in spontaneous resolvers, although the differences were not significant (45 vs. 39%, spontaneous resolvers and inactive carriers, respectively; p = 0.828; OR = 1.31; 95% CI = 0.45-3.83; p = 0.622). HBV-specific T-cell responses were detected in OBIs, and significantly stronger T-cell responses towards hepatitis B envelope antigen were observed in those with the IL28B CC genotype. In spontaneous resolvers and inactive carriers, IL28B CC did not correlate with the magnitude of T-cell responses. Conclusions: In OBI donors, IL28B CC correlates with the intensity of HBV-specific T-cell responses. In this study, IL28B CC is not statistically associated with OBI or with HBV clearance, but a larger number of cases is needed before completely ruling out its role in HBV infection.

Antiretroviral Pre-Exposure Prophylaxis Does Not Enhance Immune Responses to HIV in Exposed but Uninfected Persons.

Antiretroviral preexposure prophylaxis (PrEP), using daily oral combination tenofovir disoproxil fumarate plus emtricitabine, is an effective human immunodeficiency virus (HIV) prevention strategy for populations at high risk of HIV acquisition. Although the primary mode of action for the protective effect of PrEP is probably direct antiviral activity, nonhuman primate studies suggest that PrEP may also allow for development of HIV-specific immune responses, hypothesized to result from aborted HIV infections providing a source of immunologic priming. We sought to evaluate whether PrEP affects the development of HIV-specific immune response in humans. Within a PrEP clinical trial among high-risk heterosexual African men and women, we detected HIV-specific CD4(+) and CD8(+) peripheral blood T-cell responses in 10%-20% of 247 subjects evaluated. The response rate and magnitude of T-cell responses did not vary significantly between those assigned PrEP versus placebo, and no significant difference between those assigned PrEP and placebo was observed in measures of innate immune function. We found no evidence to support the hypothesis that PrEP alters either the frequency or magnitude of HIV-specific immune responses in HIV-1-exposed seronegative individuals. These results suggest that PrEP is unlikely to serve as an immunologic prime to aid protection by a putative HIV vaccine.

Fusion-expressed CTB improves both systemic and mucosal T-cell responses elicited by an intranasal DNA priming/intramuscular recombinant vaccinia boosting regimen.

Previous study showed that CTB (Cholera toxin subunit B) can be used as a genetic adjuvant to enhance the systemic immune responses. To further investigate whether it can also be used as a genetic adjuvant to improve mucosal immune responses, we constructed DNA and recombinant Tiantan vaccinia (rTTV) vaccines expressing OVA-CTB fusion antigen. Female C57BL/6 mice were immunized with an intranasal DNA priming/intramuscular rTTV boosting regimen. OVA specific T-cell responses were measured by IFN-γ ELISPOT and specific antibody responses were determined by ELISA. Compared to the nonadjuvant group (pSV-OVA intranasal priming/rTTV-OVA intramuscular boosting), pSV-OVA-CTB intranasal priming/rTTV-OVA-CTB intramuscular boosting group significantly improved the magnitudes of T-cell responses at spleen (1562 ± 567 SFCs/106 splenocytes versus 330 ± 182 SFCs/106 splenocytes, P < 0.01), mesenteric LN (96 ± 83 SFCs/106 lymphocytes versus 1 ± 2 SFCs/106 lymphocytes, P < 0.05), draining LNs of respiratory tract (109 ± 60 SFCs/106 lymphocytes versus 2 ± 2 SFCs/106 lymphocytes, P < 0.01) and female genital tract (89 ± 48 SFCs/106 lymphocytes versus 23 ± 21 SFCs/106 lymphocytes, P < 0.01). These results collectively demonstrated that fusion-expressed CTB could act as a potent adjuvant to improve both systemic and mucosal T-cell responses.

A Randomized Phase II Study of Immunization With Dendritic Cells Modified With Poxvectors Encoding CEA and MUC1 Compared With the Same Poxvectors Plus GM-CSF for Resected Metastatic Colorectal Cancer

To determine whether 1 of 2 vaccines based on dendritic cells (DCs) and poxvectors encoding CEA (carcinoembryonic antigen) and MUC1 (PANVAC) would lengthen survival in patients with resected metastases of colorectal cancer (CRC). Recurrences after complete resections of metastatic CRC remain frequent. Immune responses to CRC are associated with fewer recurrences, suggesting a role for cancer vaccines as adjuvant therapy. Both DCs and poxvectors are potent stimulators of immune responses against cancer antigens. Patients, disease-free after CRC metastasectomy and perioperative chemotherapy (n = 74), were randomized to injections of autologous DCs modified with PANVAC (DC/PANVAC) or PANVAC with per injection GM-CSF (granulocyte-macrophage colony-stimulating factor). Endpoints were recurrence-free survival overall survival, and rate of CEA-specific immune responses. Clinical outcome was compared with that of an unvaccinated, contemporary group of patients who had undergone CRC metastasectomy, received similar perioperative therapy, and would have otherwise been eligible for the study. Recurrence-free survival at 2 years was similar (47% and 55% for DC/PANVAC and PANVAC/GM-CSF, respectively) (χ P = 0.48). At a median follow-up of 35.7 months, there were 2 of 37 deaths in the DC/PANVAC arm and 5 of 37 deaths in the PANVAC/GM-CSF arm. The rate and magnitude of T-cell responses against CEA was statistically similar between study arms. As a group, vaccinated patients had superior survival compared with the contemporary unvaccinated group. Both DC and poxvector vaccines have similar activity. Survival was longer for vaccinated patients than for a contemporary unvaccinated group, suggesting that a randomized trial of poxvector vaccinations compared with standard follow-up after metastasectomy is warranted. (NCT00103142).

Capsid-specific T-cell Responses to Natural Infections With Adeno-associated Viruses in Humans Differ From Those of Nonhuman Primates

Hepatic adeno-associated virus serotype 2 (AAV2)-mediated gene transfer failed to achieve sustained transgene product expression in human subjects. We formulated the hypothesis that rejection of AAV-transduced hepatocytes is caused by AAV capsid-specific CD8(+) T cells that become reactivated upon gene transfer. Although this hypothesis was compatible with clinical data, which showed a rise in circulating AAV capsid-specific T cells following injection of AAV vectors, it did not explain that AAV vectors achieved long-term transgene expression in rhesus macaques, which are naturally infected with AAV serotypes closely related to those of humans. To address this apparent contradiction, we tested human and rhesus macaque samples for AAV capsid-specific T cells by intracellular cytokine staining combined with staining for T-cell subset and differentiation markers. This highly sensitive method, which could provide a tool to monitor adverse T-cell responses in gene transfer trials, showed that AAV capsid-specific CD8(+) and CD4(+) T cells can be detected in blood of naturally infected humans and rhesus macaques. They are present at higher frequencies in rhesus macaques. Furthermore, T cells from humans and rhesus macaques exhibit striking differences in their differentiation status and in their functions, which may explain the disparate duration of AAV-mediated gene transfer in these two species.

Cross-Reactive T-Cell Responses to the Nonstructural Regions of Dengue Viruses among Dengue Fever and Dengue Hemorrhagic Fever Patients in Malaysia

Dengue virus infections are a major cause of morbidity and mortality in tropical and subtropical areas in the world. Attempts to develop effective vaccines have been hampered by the lack of understanding of the pathogenesis of the disease and the absence of suitable experimental models for dengue viral infection. The magnitude of T-cell responses has been reported to correlate with dengue disease severity. Sixty Malaysian adults with dengue viral infections were investigated for their dengue virus-specific T-cell responses to 32 peptides antigens from the structural and nonstructural regions from a dengue virus isolate. Seventeen different peptides from the C, E, NS2B, NS3, NS4A, NS4B, and NS5 regions were found to evoke significant responses in a gamma interferon enzyme-linked immunospot (ELISPOT) assay of samples from 13 selected patients with dengue fever (DF) and dengue hemorrhagic fever (DHF). NS3 and predominantly NS3(422-431) were found to be important T-cell targets. The highest peaks of T-cell responses observed were in responses to NS3(422-431) and NS5(563-571) in DHF patients. We also found almost a sevenfold increase in T-cell response in three DHF patients compared to three DF patient responses to peptide NS3(422-431). A large number of patients' T cells also responded to the NS2B(97-106) region. The ELISPOT analyses also revealed high frequencies of T cells that recognize both serotype-specific and cross-reactive dengue virus antigens in patients with DHF.

Early T-Cell Responses to Dengue Virus Epitopes in Vietnamese Adults with Secondary Dengue Virus Infections

T-cell responses to dengue viruses may be important in both protective immunity and pathogenesis. This study of 48 Vietnamese adults with secondary dengue virus infections defined the breadth and magnitude of peripheral T-cell responses to 260 overlapping peptide antigens derived from a dengue virus serotype 2 (DV2) isolate. Forty-seven different peptides evoked significant gamma interferon enzyme-linked immunospot (ELISPOT) assay responses in 39 patients; of these, 34 peptides contained potentially novel T-cell epitopes. NS3 and particularly NS3200-324 were important T-cell targets. The breadth and magnitude of ELISPOT responses to DV2 peptides were independent of the infecting dengue virus serotype, suggesting that cross-reactive T cells dominate the acute response during secondary infection. Acute ELISPOT responses were weakly correlated with the extent of hemoconcentration in individual patients but not with the nadir of thrombocytopenia or overall clinical disease grade. NS3556-564 and Env414-422 were identified as novel HLA-A*24 and B*07-restricted CD8+ T-cell epitopes, respectively. Acute T-cell responses to natural variants of Env414-422 and NS3556-564 were largely cross-reactive and peaked during disease convalescence. The results highlight the importance of NS3 and cross-reactive T cells during acute secondary infection but suggest that the overall breadth and magnitude of the T-cell response is not significantly related to clinical disease grade.

Immunologic and Clinical Outcomes of Vaccination With a Multiepitope Melanoma Peptide Vaccine Plus Low-Dose Interleukin-2 Administered Either Concurrently or on a Delayed Schedule

A phase II trial was performed to test whether systemic low-dose interleukin-2 (IL-2) augments T-cell immune responses to a multipeptide melanoma vaccine. Forty patients with resected stage IIB-IV melanoma were randomly assigned to vaccination with four gp100- and tyrosinase-derived peptides restricted by human leukocyte antigen (HLA) -A1, HLA-A2, and HLA-A3, and a tetanus helper peptide plus IL-2 administered daily either beginning day 7 (group 1), or beginning day 28 (group 2). T-cell responses were assessed by an interferon gamma ELIspot assay in peripheral blood lymphocytes (PBL) and in a lymph node draining a vaccination site (sentinel immunized node [SIN]). Patients were followed for disease-free and overall survival. T-cell responses to the melanoma peptides were observed in 37% of PBL and 38% of SINs in group 1, and in 53% of PBL and 83% of SINs in group 2. The magnitude of T-cell response was higher in group 2. The tyrosinase peptides DAEKSDICTDEY and YMDGTMSQV were more immunogenic than the gp100 peptides YLEPGPVTA and ALLAVGATK. T-cell responses were detected in the SINs more frequently, and with higher magnitude, than responses in the PBL. Disease-free survival estimates at 2 years were 39% (95% CI, 18% to 61%) for group 1, and 50% (95% CI, 28% to 72%) for group 2 (P = .32). The results of this study support the safety and immunogenicity of a vaccine composed of four peptides derived from gp100 and tyrosinase. The low-dose IL-2 regimen used for group 1 paradoxically diminishes the magnitude and frequency of cytotoxic T lymphocyte responses to these peptides.