Evaluation Of Cellular Immune Responses Research Articles

Immunological Insights: A Multicenter Longitudinal Study on Humoral Response to COVID-19 Vaccines in Greece

Vaccination has emerged as the most effective tool in the battle against COVID-19. To optimize vaccination protocols, a deeper understanding of the immune response to vaccination, including influential factors and its duration, is essential. This study aimed to assess the humoral response in vaccinated individuals with or without prior SARS-CoV-2 infection. A prospective observational study was conducted across 14 private healthcare structures in Greece. Anti-spike IgG titers were measured at different timepoints following the initial vaccination and booster doses of the BNT162b2, mRNA-1273, ChAdOx1 nCoV-19, and Ad26.COV2.S vaccines. A total of 505 participants were included in the first phase, evaluating the humoral response after the initial vaccination, and 311 participants were involved in the second phase, assessing the effects of booster vaccination. All vaccines elicited high anti-S IgG titers initially, followed by a subsequent decline that was addressed by the booster vaccination. The humoral response was sustained up to one year after the booster vaccination. mRNA vaccines induced higher anti-S IgG titers compared to vector vaccines, with mRNA-1273 eliciting higher titers than BNT162b2. Vaccination resulted in higher antibody titers than natural infection alone; however, convalescent patients who received vaccination had significantly higher anti-S IgG titers compared to those who received the booster vaccine without previous SARS-CoV-2 infection. Lower antibody titers were observed in men and older patients (>51.5 years old), as well as smokers, although the decline rate was lower in these subgroups. These results underscore the importance of booster doses and reveal the potential influence of age, gender, smoking habits, and vaccine type on varying humoral responses. Long-term monitoring of antibody persistence, evaluation of cellular immune responses, and assessment of vaccine efficacy against emerging variants should be considered to enhance our understanding of immunity dynamics and inform vaccine development and deployment strategies.

Comparison of different techniques for evaluation of cellular immunity to SARS-CoV-2 virus

Most techniques for evaluation of T-cell immunity are laborious and unsuitable for routine laboratory diagnostics, thus encouraging researchers to look for accessible and reproducible tests. The purpose of our study is to compare three methods aimed for evaluation of cellular immune response levels to the SARS-CoV-2 viral antigens in patients who have been ill and vaccinated against a new coronavirus infection. We have examined 26 persons who experienced mild or moderate COVID-19 (group 1); 19 people vaccinated twice with Sputnik V, who did not have clinical COVID-19 (group 2); 21 subjects who had COVID-19 and were twice vaccinated with Sputnik V (group 3), and 14 persons who had COVID-19 twice (group 4). Peripheral blood mononuclear cells were isolated by gradient centrifugation. The first tested technique was performed as follows: the mononuclear cells were incubated with the S-protein of the SARS-CoV-2 virus, and stained with fluorescently labeled antibodies. The percentage of CD8highCD107a was counted by means of BD FACS Canto II flow cytometer. When assessed by the ELISpot method with “Human IFN-γ ELISpot” kit, IFNγ production was stimulated by SARS-CoV-2 S-protein, or a mixture of SARS-CoV-2 protein peptides in the “Corona-T-test” kit. There were no significant differences in the levels of CD107a expression on CD8high cells between the groups 1, 2, 3, and 4, as well as in amounts of IFNγ producers against SARS-CoV-2 S-protein when using “Human IFN-γ ELISpot” kit. Production of IFN was significantly lower in group 3 (hybrid immunity), i.e., 317.29±19.04 pg/ml compared to groups 1 and 2 (post-infection and post-vaccination immunity), i.e., 454.95±20.32 and 470.77±26.24 pg /ml, respectively. The relative level of IFNγ -producing cells in group 2 was higher (22.34±3.77) versus 16.83±2.35 in group 1, and 15.46±1.83 in group 3, whereas the relative levels of IFNγ did not differ in these groups. Stimulation with full-length S-protein showed a significant reduction in the number of spots in group 4 (breakthrough immunity), i.e., 30.59±2.29 vs 58.97±4.47 in group 3. Stimulation with a mixture of SARS-CoV-2 peptides in group 4 vs group 3 revealed a significantly increased number of IFNγ -producing cells (86.72±7.20 versus 69.38±5.53) and higher IFNγ production (991.25±65.18 pg/ml versus 760.76±50.70 pg/ml). Appropriate relative values were as follows: 10.30±2.77 versus 8.61±2.66, and 68.10±9.41 versus 48.35±8.15, respectively. The results of three methods for evaluation of cellular immune response correlate positively with each other, but at different significance levels.

Evaluation of Cellular Immune Response to Adeno-Associated Virus-Based Gene Therapy.

The number of approved or investigational late phase viral vector gene therapies (GTx) has been rapidly growing. The adeno-associated virus vector (AAV) technology continues to be the most used GTx platform of choice. The presence of pre-existing anti-AAV immunity has been firmly established and is broadly viewed as a potential deterrent for successful AAV transduction with a possibility of negative impact on clinical efficacy and a connection to adverse events. Recommendations for the evaluation of humoral, including neutralizing and total antibody based, anti-AAV immune response have been presented elsewhere. This manuscript aims to cover considerations related to the assessment of anti-AAV cellular immune response, including review of correlations between humoral and cellular responses, potential value of cellular immunogenicity assessment, and commonly used analytical methodologies and parameters critical for monitoring assay performance. This manuscript was authored by a group of scientists involved in GTx development who represent several pharma and contract research organizations. It is our intent to provide recommendations and guidance to the industry sponsors, academic laboratories, and regulatory agencies working on AAV-based GTx viral vector modalities with the goal of achieving a more consistent approach to anti-AAV cellular immune response assessment.

Mucosal immunity of mannose-modified chitosan microspheres loaded with the nontyepable Haemophilus influenzae outer membrane protein P6 in BALB/c mice.

Nontypeable Haemophilus influenzae (NTHi) is a common opportunistic pathogen that colonizes the nasopharynx. NTHi infections result in enormous global morbidity in two clinical settings: otitis media in children and acute exacerbation of chronic obstructive pulmonary disease (COPD) in adults. Thus, there is an urgent need to design and develop effective vaccines to prevent morbidity and reduce antibiotic use. The NTHi outer membrane protein P6, a potential vaccine candidate, is highly conserved and effectively induces protective immunity. Here, to enhance mucosal immune responses, P6-loaded mannose-modified chitosan (MC) microspheres (P6-MCMs) were developed for mucosal delivery. MC (18.75%) was synthesized by the reductive amination reaction method using sodium cyanoborohydride (NaBH3CN), and P6-MCMs with an average size of 590.4±16.2 nm were successfully prepared via the tripolyphosphate (TPP) ionotropic gelation process. After intranasal immunization with P6-MCMs, evaluation of humoral immune responses indicated that P6-MCMs enhance both systemic and mucosal immune responses. Evaluation of cellular immune responses indicated that P6-MCMs enhance cellular immunity and trigger a mixed Th1/Th2-type immune response. Importantly, P6-MCMs also trigger a Th17-type immune response. They are effective in promoting lymphocyte proliferation and differentiation without toxicity in vitro. The results also demonstrate that P6-MCMs can effectively induce MHC class I- and II-restricted cross-presentation, promoting CD4+-mediated Th immune responses and CD8+-mediated cytotoxic T lymphocyte (CTL) immune responses. Evaluation of protective immunity indicated that immunization with P6-MCMs can reduce inflammation in the nasal mucosa and the lung and prevent NTHi infection. In conclusion, MCMs are a promising adjuvant-delivery system for vaccines against NTHi.

The Evaluation of Cellular Immunity to Avian Viral Diseases: Methods, Applications, and Challenges.

Cellular immune responses play critical roles in the control of viral infection. However, the immune protection against avian viral diseases (AVDs), a major challenge to poultry industry, is yet mainly evaluated by measuring humoral immune response though antibody-independent immune protection was increasingly evident in the development of vaccines against some of these diseases. The evaluation of cellular immune response to avian viral infection has long been neglected due to limited reagents and methods. Recently, with the availability of more immunological reagents and validated approaches, the evaluation of cellular immunity has become feasible and necessary for AVD. Herein, we reviewed the methods used for evaluating T cell immunity in chickens following infection or vaccination, which are involved in the definition of different cellular subset, the analysis of T cell activation, proliferation and cytokine secretion, and in vitro culture of antigen-presenting cells (APC) and T cells. The pros and cons of each method were discussed, and potential future directions to enhance the studies of avian cellular immunity were suggested. The methodological improvement and standardization in analyzing cellular immune response in birds after viral infection or vaccination would facilitate the dissection of mechanism of immune protection and the development of novel vaccines and therapeutics against AVD.

Evaluation of Cellular Immune Responses in Dogs Immunized with Alum-Precipitated Autoclaved Leishmania major along with BCG and Imiquimod.

Background:We aimed to investigate the potential effects of BCG and imiquimod on improvement of current experimental L. major vaccine against dogs in an endemic area of Zoonotic visceral leishmaniasis (ZVL) in Iran.Methods:During 2012 till 2014, seven mixed-breed shepherd dogs with no anti-Leishmania antibodies and no response to Leishmanin reagent were immunized with 2 doses of alum-precipitated autoclaved L. major (Alum-AML) while BCG and imiquimod (for skin pre-treatment) were used as adjuvants. The productions of a few characteristic cytokines of T-helper immune responses and the development of delayed-type hypersensitivity (DTH) of the immunized animals were then evaluated, up to 300 days. Blood samples were collected at 0, 30, 80 and 300 d post-vaccination and the concentrations of IFN-γ, IL10, IL-12 and TGF-β cytokines secreted from PBMCs at these time-points were quantified by ELISA. DTH was evaluated by Leishmanin skin test (LST).Results:Although a similar LST conversion was observed at all time-points, the cytokine measurement results indicated significantly higher levels of IFN-γ at day 80 and elevated levels of IL-10 at days 80 and 300, post-vaccination. Moreover, a significantly higher IFN-γ/IL-10 ratio was observed at day 30 post-vaccination compared to the other time-points.Conclusion:Although a Th1-like response could be observed at day 30 post-vaccination, the development of cytokine profiles was inclined toward mixed Th1 and Th2 responses at days 80 and 300 post-vaccination. This situation may indicate the requirement of an additional boosting by this Alum-AML formula, in order to induce long-lasting protection against ZVL.

Immunomodulatory effect of Kaempferia parviflora against cyclophosphamide-induced immunosuppression in swiss albino mice

Background: Kaempferia parviflora Wall. Ex. Baker (Black ginger) is an important medicinal plant used as a health-promoting tonic i.e., both a stimulant and a vitalizing agent in Thailand. Research findings are available on its aphrodisiac and anti-inflammatory activities. So far, there is no research work conducted on immunomodulatory effect of K. parviflora. Objectives: The present study was carried out to evaluate the immunomodulatory effect of ethanolic extract of rhizomes of K. parviflora in cyclophosphamide-induced immunosuppression in Swiss albino mice. Materials and Methods: Immunomodulatory status was assessed by physiological, hematological, biochemical, and histopathological observations. The weight of organs such as liver and spleen was recorded at the time of sacrifice. Gas chromatography mass spectrophotometry (GC-MS) analysis was performed for profiling compounds present in the extract. Results: Significant increase in body weight was observed on 12th day in K. parviflora-treated immunosuppressed mice. In hematological parameters, there was significantly higher lymphocyte count for K. parviflora-treated immunosuppressed mice. In hemagglutination test, conducted for the evaluation of humoral immune response, both K. parviflora alone and K. parviflora-treated immunosuppressed mice showed significant increase in titer value compared with cyclophosphamide control. Bone marrow cellularity test performed for evaluation of cellular immune response showed cyclophosphamide control group with significant lower bone marrow cellularity on 12th and 19th day while K. parviflora alone-treated and K. parviflora-treated immunosuppressed mice showed a significant increase in the bone marrow cellularity. The result of histopathology of spleen revealed to prevent the depletion of red pulp and white pulp on 12th day, and this prevention was marked on 19th day. GC-MS profiling showed that the extract contained eight compounds. Majority of the compounds belong to flavonoids class which might have helped in immunomodulation. Conclusion: The results of the present study revealed that the test extract possessed promising immunomodulatory activity.

Evaluation of cellular immune response provoked by experimental infection with Cryptosporidium spp. in mice

Experimental infection was induced in immunosupreesed and non-immunosupressed mice using inoculation of two different Cryptosporidium spp. isolates: faecal origin and river water origin; then the infectivity rate and cellular immunological status were examined in these groups of mice. The results indicated that these two isolates induced experimental infection in immunosuppressed and non-immunosuppressed mice with no significant differences in infectivity rate between groups. Immunosuppressed mice infected with feacal or water origin oocyst showed low and significant values of transformed lymphocytes 8.68% ± 0.65%, 9.18% ± 1.09 respectively and phagocytic index 10.1% ± 1.66 and 11.2% ± 0.19 respectively. It concludes that lymphocyte transformation and phagocytosis index are good indicators to evaluate the cellular immune response in infected experimental mice with Cryptosporidium.

THU0033 Monitoring Cellular Immune Responses to Influenza Vaccination in Rheumatoid Arthritis Patients: Comparison of Flow Cytometric Analysis of Cytokine Production, Elisa Assay of IFN-Gamma Secretion, and the Granzyme-B Activity Assay

BackgroundMonitoring of immune responses is essential in a number of clinical research areas that address immunosuppressed individuals, including oncologic and rheumatic patients. During the past 10 years, our group has...

Evaluation of Cellular Immune Responses to Mycobacterial Antigens among Immigrants from South-East Asian Countries in South Korea

Evaluation of Cellular Immune Responses to Mycobacterial Antigens among Immigrants from South-East Asian Countries in South Korea

Evaluation of cellular immune response in Golden Hamsters experimentally infected with Leishmania donovani comparing with cellular immune response against chicken Red Blood Cells.

The Evaluation of the immune response in Golden Hamsters experimentally infected with Leishmania donovani was determined in this study, particularly, the cellular immune response. Follow up has maintained to determine the Delayed Type of Hypersensitivity using skin test both in infected and control lab animals. Chicken red blood cells were used as a parameter to evaluate the immune system; they are dull and have the ability of immunization. Two concentrations of chicken R.B.C were examined to determine which gives the higher titration in Hamsters and those were 1.5 X 109 cell/ml and 3 X 109 cell/ml , the second concentration gave the maximum titration where then used in this work. After sensitization with Chicken R.B.C for both infected and control groups, delayed type of hypersensitivity has been used against Leishmania donovani antigen and 4 days of follow up were adopted and they were (14, 30, 60, 90) day after infection. Results showed that skin test against both antigens ( L.donovani and chicken R.B.C) was significantly higher than normal at the first day of follow up ( day 14) then gradual decreasing were noticed till the last day of follow up (90). This can indicate that the infection with L.donovani activated the immune response at the beginning of infection, then leads to cellular immune suppression against both L.donovani antigen and chicken R.B.C., so that this immunosuppression is not specific.

Evaluation of cellular immune response in Golden Hamsters experimentally infected with Leishmania donovani comparing with cellular immune response against chicken Red Blood Cells.

The Evaluation of the immune response in Golden Hamsters experimentally infected with Leishmania donovani was determined in this study, particularly, the cellular immune response. Follow up has maintained to determine the Delayed Type of Hypersensitivity using skin test both in infected and control lab animals. Chicken red blood cells were used as a parameter to evaluate the immune system; they are dull and have the ability of immunization. Two concentrations of chicken R.B.C were examined to determine which gives the higher titration in Hamsters and those were 1.5 X 109 cell/ml and 3 X 109 cell/ml , the second concentration gave the maximum titration where then used in this work. After sensitization with Chicken R.B.C for both infected and control groups, delayed type of hypersensitivity has been used against Leishmania donovani antigen and 4 days of follow up were adopted and they were (14, 30, 60, 90) day after infection. Results showed that skin test against both antigens ( L.donovani and chicken R.B.C) was significantly higher than normal at the first day of follow up ( day 14) then gradual decreasing were noticed till the last day of follow up (90). This can indicate that the infection with L.donovani activated the immune response at the beginning of infection, then leads to cellular immune suppression against both L.donovani antigen and chicken R.B.C., so that this immunosuppression is not specific.

Correction: Quantitative PCR Evaluation of Cellular Immune Responses in Kenyan Children Vaccinated with a Candidate Malaria Vaccine

Correction: Quantitative PCR Evaluation of Cellular Immune Responses in Kenyan Children Vaccinated with a Candidate Malaria Vaccine

Evaluation of cellular immune responses in mice elicited by Chinese AIDS candidate vaccines

Objective To compare cellular immune responses in mice elicited by Chinese different AIDS candidate vaccines.Methods According to their different immunization procedures,BALB/c mice were immunized with 6 AIDS candidate vaccines,separately.Spleen cells were isolated for the detection of cellular immune response to HIV-specific peptides using enzyme-linked immunosorbent spot(ELISPOT)assay and intracellular cytokine staining(ICS)method.Results AIDS vaccines were evaluated by using potential T-cell epitopes(PTE)Gag,Env and Pol peptides pool and ELISPOT.The positive conversion rates for cellular immune response of 1#-6# vaccines fluctuated from 70% to 100%.The vaccine-induced cellular immune responses to specific peptides pool are different not only in magnitude but also in breadth.The Th1type cytokines,IFN-γand IL-2,were detected with ELISPOT in 1# and 2# vaccines.The productions of IFN-γand IL-2 induced by both of the two vaccines showed a moderate correlation(r1 =0.62,P1 ＜0.01 ;r2=0.79,P2 ＜ 0.01).The positive conversion rate of IFN-γ secreting cells of 1 # vaccine was 66.7%(10/15)mice detected with both ELISPOT and ICS.And the results tested by ELISPOT and ICS showed moderate correlation(r = 0.55,P ＜ 0.05).Conclusion The magnitude and breadth of cellular immune responses induced by different AIDS candidate vaccines are different.Being induced by different AIDS candidate vaccines,the IFN-γand other Th1 type cytokines detected by ELISPOT or ICS could be used to evaluate the cellular immune responses in mice. Key words: AIDS; Vaccine; Cellular immune responses

Evaluation of cellular immune responses in cancer vaccine recipients: lessons from NY-ESO-1

The rigorous evaluation of cancer vaccination requires evidence of benefit to patients with cancer or those at risk of relapse from the disease. Clinical trials are expensive and require considerable human and clinical resources in order to demonstrate this benefit. In the era of defined cancer antigens, it is possible to evaluate immunogenic targets, and assess the quality and magnitude of immune responses against these antigens following vaccination. Analyzing these surrogate end points complements clinical assessment and provides a depth of understanding to better inform trial evaluation and design. We have used the immunogenic cancer testis antigen NY-ESO-1 as a model antigen. This article summarizes our experience in monitoring immunity against NY-ESO-1.

Methods to measure vaccine immunity

The biggest triumph in vaccination is the eradication of smallpox. Ironically, no methods to measure vaccine immunity were set in place. Protection and immunity was achieved without detailed analysis nor knowledge of cellular or humoral immunity induction. Today, owing to strict health and safety issues of vaccine manufacturing, vaccines must meet higher standards of safety and biochemical characterization than they did in the past. This has led to the production of highly purified vaccines, and the identification and isolation of the antigens responsible for protection. A vaccine cannot move forward without the demonstration of effective immunity induced by the regime. Moreover, contrary to the vaccination strategy that eradicated smallpox and other acute viral infections, several modern vaccines aim at the eradication of established chronic diseases such as chronic viral infections or cancer. As the pathogens or cancer antigens are generally intracellular, modern vaccines can rely less effectively on the neutralizing properties of antibody responses that cannot cross the cell membrane, and depend predominantly on T-cell-based recognition of affected cells for their elimination. Thus, new technologies have to be developed to measure vaccine immunity. Are we going round and round in circles? Are we making it harder for ourselves? Is vaccine development getting too hard? Why do we have to demonstrate immunity, if we can show protection? No regulatory agency will take a vaccine forward that is not highly purified and can demonstrate immunity induction. Several methods of measuring humoral and cellular immunity have been required to be developed to meet this criteria. Some methods include ELISA, cytotoxic T-lymphocyte (CTL) assay, CTL precursor frequency assay, T-cell proliferation assays, carboxyfluorescein diacetate succinimidyl ester assays, intracellular and extracellular cytokine production by cells in culture using either ELISA or multiplex and flow cytometry, polyfunctional T-cell assays, ELISpot and MHC class I/II tetramers. Thus, there is an enormous amount of information and reagents available for guiding vaccine and immunotherapeutics development. This special issue on ‘Methods to measure vaccine immunity’ focuses on a number of recent and promising approaches used to measure immunity induced following vaccination. Topics included in this special focus issue are as follows: Gene-expression profiling in vaccine therapy and immunotherapy for cancer [1]. The use of microarrays is described in order to understand the mechanism by which tumors are rejected; Antibody-profiling technologies for studying humoral responses to infectious agents [2]. Protein microarrays are used to evaluate antibody responses to thousands of antigens at one time. The luciferase immunoprecipitation system overcomes some drawbacks of the conventional protein microarray assay. These new technologies offer a new tool for understanding humoral immunity induction to proteins; Vaccine-induced antibody responses in patients with carcinoma [3]. A nice overview of current methods utilized for measuring antibody responses and for assessing their antitumor efficacy in the context of clinical trials; Methods to measure T-cell responses [4]. A mini review on recent methods used to measure T-cell immunity, such as the use of CTL, CTL precursor frequency assays, carboxyfluorescein diacetate succinimidyl ester, flow cytometric approaches, spectrophotometric approaches and use of ovalbumin-specific CD8+ T cells from OTI and OTII T-cell receptor tansgenic mice; New flow cytometric assays for monitoring cell-mediated cytotoxicity [5]. A comparison of IFN-γ ELISpot, granzyme B ELISpot and conventional 51Cr-release assays is given. A detailed analysis of the use of flow cytometric methods to measure T-cell immunity induction is demonstrated; Evaluation of cellular immune responses in cancer vaccine recipients [6]. Measurement of cellular immunity is addressed using NY-ESO-1 as an example and techniques such as delayed-type hypersensitivity, measurement of T cells from peripheral blood, use of overlapping peptides to measure CD4 and CD8 T cells, and the role of Treg cells in vaccine immunity induction are described; Development and application of phosphoflow as a tool for immunomonitoring [7]. This article describes the new flow cytometry technology, phosphoflow for measuring multiple intracellular signaling molecules in the immune system at a single-cell level for lymphocyte immune monitoring; Surface plasmon resonance for vaccine design and efficacy studies [8]. An outline of how surface plasmon resonance biosensors are emerging to understand how serum is used to monitor antibody immunity; The challenges of assessing infant vaccine responses in resource-poor settings [9]. A very nice and comprehensive article on methods to measure vaccine immune responses where only poor settings are available. There is a vast amount of information already available to promote a greater understanding into the role of specific immune induction to a vaccination regime. The use of newer immune monitoring tools as described in this special focus issue will serve to improve immune monitoring and determine the effectiveness of vaccine strategies for the treatment or immunotherapeutic approaches to diseases.

Quantitative PCR Evaluation of Cellular Immune Responses in Kenyan Children Vaccinated with a Candidate Malaria Vaccine

BackgroundThe T-cell mediated immune response plays a central role in the control of malaria after natural infection or vaccination. There is increasing evidence that T-cell responses are heterogeneous and that both the quality of the immune response and the balance between pro-inflammatory and regulatory T-cells determines the outcome of an infection. As Malaria parasites have been shown to induce immunosuppressive responses to the parasite and non-related antigens this study examined T-cell mediated pro-inflammatory and regulatory immune responses induced by malaria vaccination in children in an endemic area to determine if these responses were associated with vaccine immunogenicity.MethodsUsing real–time RT- PCR we profiled the expression of a panel of key markers of immunogenecity at different time points after vaccination with two viral vector vaccines expressing the malaria TRAP antigen (FP9-TRAP and MVA-TRAP) or following rabies vaccination as a control.Principal FindingsThe vaccine induced modest levels of IFN-γ mRNA one week after vaccination. There was also an increase in FoxP3 mRNA expression in both TRAP stimulated and media stimulated cells in the FFM ME-TRAP vaccine group; however, this may have been driven by natural exposure to parasite rather than by vaccination.ConclusionQuantitative PCR is a useful method for evaluating vaccine induced cell mediated immune responses in frozen PBMC from children in a malaria endemic country. Future studies should seek to use vaccine vectors that increase the magnitude and quality of the IFN-γ immune response in naturally exposed populations and should monitor the induction of a regulatory T cell response.

Comparison of Intracellular Cytokine Flow Cytometry and an Enzyme Immunoassay for Evaluation of Cellular Immune Response to Active Tuberculosis

A prospective cross-sectional blinded study of 28 patients (21 male and 7 female patients; mean age, 44 years) with suspected active tuberculosis (TB) attending a TB and chest clinic is described. Blood was taken for immune cell enumeration, a whole-blood enzyme-linked immunosorbent assay (ELISA) for the detection of gamma interferon (IFN-gamma) by the QuantiFERON-TB Gold (QFT-G) assay, and intracellular cytokine flow cytometry (ICC) analysis; and sputum was simultaneously taken for bacteriological culture for Mycobacterium tuberculosis. Twelve healthy subjects were included as controls. The performance characteristics of the QFT-G and ICC assays for the detection of active TB were compared. Among the patients with active TB, we found (i) normal to slightly elevated peripheral CD4(+) and CD8(+) T-cell counts but a significant reduction in the number of NK cells; (ii) CD4(+) T cells were the major cell type producing IFN-gamma, a type 1 cytokine; (iii) small percentages of CD8(+) T cells were also primed for IFN-gamma production; (iv) the production of interleukin-4 (IL-4), a type 2 cytokine, was not prominent; and (v) the sensitivity and the specificity of the QFT-G assay were 88.2% and 18%, respectively, and those of the ICC assay were 94.1% and 36.4%, respectively. The specificities of the blood tests were likely underestimated due to cross-reaction to a non-M. tuberculosis mycobacterial infection and the lack of a confirmatory test that could be used to diagnose latent M. tuberculosis infection. Flow cytometry accurately locates the pool of immunological effector cells responsible for cytokine production during active TB. The ICC assay is an additional useful tool for the diagnosis of active TB.

Evaluation of cellular immune responses induced in mice by alphavirus replicon particle vaccines for SARS-CoV (B190)

Abstract An RNA replicon vector system derived from an attenuated strain of VEE virus was used to produce virus-like replicon particles (VRP) expressing SARS-CoV genes. We previously showed in mouse studies that complete protection was conferred by SARS S VRP and partial protection with SARS M VRP. In this study, two assays for cellular immunity were used to characterize the T cell phenotypes and cytokine profiles induced after SARS VRP vaccination. Following SARS M, N, S or E VRP immunization of BALB/c mice, splenocytes were harvested for antigen specific ICS and ELISPOTs. The SARS S VRP induced both CD4 and CD8 responses with the latter being the dominant component as measured by ICS for IFN-γ or TNF-α. Polyfunctional CD8 cells secreting both IFN-γ and TNF-α were also detected. Following SARS N VRP immunization, T cells secreting IFN-γ or TNF-α were readily detected with cells secreting both predominately identified as CD4 cells. Th1 cytokine secretion was not detected in cells from SARS M VRP inoculated mice. SARS E VRP elicited a robust CD8 response as shown by cytokine expression in cells stimulated with a 9mer peptide. IL-4 was not detectable after any SARS VRP vaccination. The IFN-γ ELISPOT results paralleled those from ICS, indicating that the SARS VRP vaccines induced Th1 biased responses, although the cytokines elicited differed depending on the SARS gene expressed. This study was supported by NIH grant #UC1-AI62582

P2010 Evaluation of cellular immune response against purified antigen 85 in patients with tuberculosis

Background: Tuberculosis (TB) remains an important health problem throughout the world. Despite its significance in public health, mechanisms of protective immunity against Mycobacyerium Tuberculosis in humans have not yet been understood. Objective: To evaluate cell mediated immune response against purified Ag 85, PPD and Phytohemagglutinin (PHA) in patients with tuberculosis and healthy tuberculin positive and negative individuals. Methods: Thirty patients with tuberculosis and 60 healthy tuberculin skin test positive and negative volunteers were participated in this study. Cell mediated immunity was assessed by measuring [³H]-thymidine uptake and detection of IFN- γ in the culture supernatant using commercial ELISA test. Results: In the present study, we showed that IFN- γ production and cell proliferation response to Ag 85 were significantly higher in tuberculin positive than tuberculin negative individuals (P<0.01). Among tuberculous patients, IFN-γ production and cell proliferative responses to Ag 85 was significantly lower in contrast to healthy tuberculin positive individuals (P<0.01). In addition, IFN- γ response in patients with cavitary tuberculosis was lower than patients without cavitation (P<0.05). Conclusion: Based on the higher cell mediated immune responses to Ag 85 in healthy tuberculin positive volunteers compared to patients (especially with advanced disease), purified Ag 85 can be used as a sensitive marker for analysis of immune responses in tuberculosis.