CDC - Flow Cytometry and T-Cell Response Monitoring = after Smallpox Vaccination

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Vol.=20 9, No. 11
Nobember = 2003

Comments<= /A>=20 | Emai= l=20 this article


=95	The = Study
=95	Conclusions
=95	References
=95	Figure

Dispatch

Flow Cytometry and = T-Cell=20 Response Monitoring after Smallpox Vaccination

Fabrizio Poccia,* Cristiana Gioia,* Carla Montesano,* = Federico=20 Martini,* Douglas Horejsh,* Concetta Castilletti,* Leopoldo Paolo=20 Pucillo,* Maria Rosaria Capobianchi,* and Giuseppe=20 Ippolito*
*National Institute for Infectious Diseases = "Lazzaro=20 Spallanzani," Rome, Italy

Suggested=20 citation for this article: Poccia F, Gioia = C, Montesano=20 C, Martini F, Horejsh P, Castilletti C, et al. Flow cytometry = and T-cell=20 response monitoring after smallpox vaccination. Emerg Infect Dis = [serial=20 online] 2003 Nov [date cited]. Available from: URL:=20 = http://www.cdc.gov/ncidod/EID/vol9no11/03-0349.htm

Orthopoxvirus=20 zoonosis or smallpox as result of bioterrorism or biological = warfare=20 represents a risk for epidemic spread. By monitoring T-cell = responses by=20 flow cytometry, we observed a recall response after recent = vaccination=20 against smallpox. When the high similarity between the = orthopoxviruses=20 is considered, this rapid assay that uses vaccinia antigens = could=20 identify recently exposures.

Although the last natural case of smallpox was reported in = Somalia in=20 1977, this orthopoxvirus remains a source of concern. No evidence = exists=20 that smallpox will recur as an endemic disease, but the virus may = have=20 been acquired for use in biological warfare or bioterrorist = attacks. If=20 one assumes an average of 15 days is needed for infected persons = to become=20 infectious, delay in intervention will be costly, increasing the = total=20 number of cases (1).=20 Furthermore, the recent outbreak of the severe acute respiratory = syndrome=20 coronavirus and the first documented outbreak of monkeypoxvirus in = the=20 Western Hemisphere underline the ever-present risk for epidemic = extension=20 of zoonosis and raise concerns about the medical and social effect = of=20 reemerging orthopoxvirus infection in humans. During the epidemic = spread=20 of an emerging pathogen, evaluating exposed persons and containing = the=20 infected population should be the first priorities. A local = outbreak of=20 orthopoxvirus infection would require rapid and sensitive = diagnostics,=20 including novel assays based on host responses.

For intracellular pathogens, the antibody titers and = neutralization=20 assays represent routine immunologic tests that provide results = after=20 several weeks of infection. The appearance of a detectable = antibody titer=20 takes place a few days after the induction of a T-cell response = (2).=20 Moreover, antigen-specific T-cell responses could be detected in = exposed,=20 but uninfected persons, as shown in those with HIV infection (3). = Using=20 a rapid flow cytometric test, we previously showed that monitoring = interferon (IFN)- = production=20 by antigen-pulsed T cells provides a quantitative and functional=20 assessment of HIV- or cytomegalovirus (CMV)-specific CD8(+) and = CD4(+) T=20 cells (4-6). = This=20 technique requires that whole proteins or selected peptide = antigens are=20 added to blood cells, allowing the simultaneous analysis of both = major=20 histocompatibility complex class I and II restricted T-cell = responses (7).=20 Because smallpox vaccination was recently shown to induce a strong = vaccinia virus-specific CD8(+) CTL- and IFN-=96producing T=20 cells detectable by more cumbersome research laboratory methods=20 (cytotoxic, proliferative, or ELISPOT assays) (8,9), = we=20 evaluated the feasibility of an easy, rapid, and sensitive assay = to=20 monitor T-cell responses after recent vaccination against = smallpox; the=20 assay can potentially be used as a routine diagnostic assay.

The=20 Study

Figure

Click to view enlarged = image

Figure. Flow = cytometric=20 analysis of T-cell responses to smallpox antigens after = recent=20 smallpox vaccination and in long-term vaccinated or not = vaccinated=20 persons...

T-cell reactivity was analyzed after recent (<2 years ago) = smallpox=20 vaccinations, in long-term vaccinated (>20 years ago) and not=20 vaccinated persons. Briefly, peripheral blood mononuclear cells = (PBMC)=20 were isolated by standard density centrifugation (Ficoll-Hypaque,=20 Pharmacia, Uppsala, Sweden). Stimulation was also performed on = whole blood=20 samples; however, the assay had reduced sensitivity. We cannot = exclude the=20 possibility that whole bloodassay sensitivity could be improved by = changing protocol conditions (data not shown). PBMC were cultured = in=20 complete Roswell Park Memorial Institute 1640 medium, 10% v/v=20 heat-inactivated fetal calf serum, 2 mM L-glutamine, and 10 U/mL=20 penicillin/streptomycin at a concentration of 10⁶ = cells/mL.=20 Stimulation was performed with 40 =B5L/mL (total protein content = of=20 approximately 1 =B5g/mL) of vaccinia viral antigen resuspended = according to=20 the manufacturer's instructions (Maine Biotechnology Services, = Portland,=20 ME), or 2 =B5g of CMV antigen (Biowhittaker, Walkersville, MD), = always in=20 the presence of co-stimulation with both anti-CD28 and CD49d = monoclonal=20 antibodies (1 =B5g/mL, Becton, Dickinson and Company, Franklin = Lakes, NJ).=20 We also tested the T-cell response with live vaccinia-infected = fibroblast=20 or Vero cells. The response against uninfected antigenic = preparations was=20 always above background, reducing the sensitivity of the assay = (data not=20 shown); therefore, the commercially available antigens were used = in=20 subsequent experiments. Cultures were incubated at 37=B0C for 1 h, = followed=20 by an additional overnight incubation with 10 =B5g/mL of the = secretion=20 inhibitor Brefeldin-A (Sigma-Aldrich Corporation, St. Louis, MO). = Cells=20 were washed twice in phosphate-buffered saline, 1% bovine serum = albumin,=20 and 0.1% sodium azide, and stained for 15 min at 4=B0C with = monoclonal=20 antibodies specific for cell surface CD antigens (Becton, = Dickinson and=20 Company). Samples were then fixed in 1% paraformaldehyde for 10 = min at=20 4=B0C, incubated with Phyco-Erithrin-conjugated mouse-anti-human = IFN- (=20 (Becton, Dickinson and Company), washed twice in = phosphate-buffered=20 saline, 1% bovine serum albumin, and 0.1% saponin, and resuspended = in=20 FACSFlow before being acquired on FACScalibur (Becton, Dickinson = and=20 Company), as previously described (4,6).=20 Controls for nonspecific staining were monitored with = isotype-matched=20 monoclonal antibodies (Becton, Dickinson and Company); cells = incubated=20 with only anti-CD28 and -CD49d were included in each experiment = and=20 nonspecific staining was always subtracted from specific results. = In the=20 cytometric panels shown in the Figure= ,=20 the IFN- = production=20 by CD3(-) cells is 1 log lower in intensity compared to the=20 antigen-specific CD3(+) T-cell response, representing an = unspecific=20 response that may involve natural killer cells. To monitor=20 antigen-specific T-cell responses, we collected data only from = CD3(+) T=20 cells producing higher amounts of IFN-. = Negative=20 control antigenic stimulation was always below the detection limit = of the=20 assay (0.02%).

Cytometric panels in the Figure= =20 show the IFN- = synthesis by=20 CD3(+) T cells after in vitro stimulation with vaccinia virus or = CMV=20 antigens. As shown in panels D, E, and F, all donors were strongly = reactive to the CMV antigens (0.87%, 0.20%, and 1.53% of CD3(+) T = cells=20 respectively; the numbers of CMV-specific CD3(+) T cells per blood = milliliter were 13,132, 2,964, and 11,385, respectively). As = previously=20 described (6), = most=20 of the CMV-specific response was related to CD4(+) T cells (96%, = 75%, and=20 59% of CMV-specific T cells, respectively). Both unvaccinated and=20 long-term vaccinated healthy donors had undetectable responses to = smallpox=20 vaccinia antigens (Figure= ,=20 panels A and B). In contrast, a recall response was detectable = after a=20 recent immunization (Figure= ,=20 panel C). In this case, the percentage of T cells specific for = smallpox=20 vaccine antigens was 0.23% among CD3(+) T cell , and the number of = vaccinia antigen-specific cells was 1,725 per blood mL = corresponding to a=20 frequency of 1/667. Most vaccinia-specific T cells detected by = this assay=20 were CD4(+) (vaccinia-specific CD4(+) T cells were 80% of=20 vaccinia-specific T cells). Nevertheless, the sensitivity of this = assay to=20 detect CD8(+) T cells could be improved by using human leukocyte = antigen=20 (HLA) class I=96specific peptides as previously described (4).
=
Conclusions

Vigorous and long-lasting protective immune responses have been = associated with smallpox vaccination, and specific immunity is = believed to=20 be maintained for decades (10,11). = In=20 long-term vaccinated persons, virus-specific CD4(+) and CD8(+)=20 T-lymphocytes are detectable only after extensive in vitro culture = and=20 restimulation to generate antigen-specific lines or clones. This=20 limitation is due to the long, but limited, lifespan of memory T = cells and=20 to their low frequency, usually below 1/50,000 (12). = Our=20 in vitro rapid assay based on a short-time primary T-cell response = was=20 unable to show the residual memory T-cell response present in = long-term=20 vaccinated persons since the assay sensitivity is 1 log lower but = could=20 detect the higher frequencies of IFN-=96producing=20 antigen-specific cells appearing a few weeks after smallpox = vaccine=20 inoculation (8).=20 Accordingly, Terajima et al. (13)=20 demonstrated that T-cell responses to vaccinia and variola = conserved=20 epitopes peak 14 days after primary immunization with vaccinia = virus. In=20 this study, the frequency of antigen-specific T cells was measured = as=20 IFN- production by ELISPOT and HLA/peptide = tetramer=96staining methods.=20 Because strong correlations between the data derived from ELISPOT, = tetramer assays, and intracellular cytokine staining for IFN-=20 were previously observed (14),=20 vaccinia-specific T cells could be detected by flow cytometry only = a few=20 days after immunization with vaccinia virus. In addition, Pincus = and Flick=20 demonstrated the initial development of delayed hypersensitivity, = an index=20 of cell-mediated immunity, as early as 2 days after smallpox = vaccination=20 (15).=20 During viral infection, high levels of virus-specific T cells are = found in=20 acute infection, falling below detectable limits as the viral load = decreases and reappearing in chronic infections during episodes of = transient viremia. Accordingly, we observed that the frequencies = of=20 HIV-specific CD8(+) T cells releasing IFN- = were=20 quantitatively increased a few weeks after viral rebound = consequent to the=20 interruption of antiviral therapy (5). = These=20 observations indicate that the frequency of virus-specific T cells = is=20 clinically relevant, which suggests that this method may be useful = in=20 detecting immune response by monitoring the frequency of = virus-specific T=20 cells. In recently vaccinated persons, memory cells are expanded = by=20 antigen reexposure, and their increase in frequency could be=20 quantitatively detected by the rapid flow cytometric T-cell assay, = confirming the efficacy of vaccination. Moreover, because of the = high=20 similarity between orthopoxviruses, this rapid assay using = vaccinia=20 antigens could be used to identify recently exposed persons.

Finally, an important aspect in developing a diagnostic assay = is to use=20 a rapid and easily automated system that works on virtually all = persons=20 who carry the disease. In this context, the intracellular T-cell = cytokine=20 staining by flow cytometry presents several advantages in = comparison to=20 other techniques, such as tetramer staining and ELISpot (4). In = fact, flow cytometry allows for testing multiple proteins or = peptides at a=20 single time and provides at the same time a quantitative and = phenotypic=20 assessment of CD8(+) and CD4(+) responding T cells. Moreover, = optimization=20 of antigen preparation with peptide pools designed to be = virus-specific,=20 highly conserved, and independent of HLA haplotypes may allow for = the=20 development of a second generation of more sensitive flow = cytometric=20 T-cell assays, extending the possibility to perform routine = analysis on=20 cryopreserved samples (4). = The=20 technique could be easily automated through the use of analytical=20 instruments already available in most clinical laboratories that = use flow=20 cytometry. In comparison with other analytical systems for = assessing=20 antigen-specific responses, this method is economically = advantageous. The=20 recent availability of mobile flow-cytometer units may allow use = of this=20 assay under field investigation conditions.

This = study was=20 supported by grants from the "Ministero della = Salute."

Dr. = Poccia was an=20 European Molecular Biology Organization fellow at the Viral = Oncology=20 Unit of Pasteur Institute in Paris and faculty member in the = Department=20 of Medical Microbiology & Immunology, University of = Wisconsin,=20 Madison, Wisconsin His present position is senior scientist at = the=20 National Institute for Infectious Diseases "Lazzaro Spallanzani" = of=20 Rome. His research activity is related to emerging and = reemerging=20 infections, focusing on innate immunity and host-pathogen = interactions.=20 His main interests are translational research to develop novel=20 diagnostic assays based on physiological and immune host = responses,=20 tools for clinical monitoring of immune reconstitution, and=20 broad-spectrum immunostimulants.

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Terajima M, Cruz J, Raines G, Kilpatrick ED, Kennedy JS, = Rothman AL,=20 et al. Quantitation of CD8+ T cell responses to newly = identified=20 HLA-A*0201-restricted T cell epitopes conserved among vaccinia = and=20 variola (smallpox) viruses. J Exp Med 2003;197:927=9632.=20
Goulder PJ, Tang Y, Brander C, Betts MR, Altfeld M, = Annamalai K, et=20 al. Functionally inert HIV-specific cytotoxic T = lymphocytes do=20 not play a major role in chronically infected adults and = children. J=20 Exp Med 2000;192:1819=9632.=20
Pincus WB, Flick JA. The role of hypersensitivity in the=20 pathogenesis of vaccinia virus in humans. J Pediatr = 1963;62:57=9662.=20

Comments to the = Authors

Please use=20 the form below to submit correspondence to the authors = or=20 contact them at the following address:

Fabrizio=20 Poccia, National Institute for Infectious Diseases "L. = Spallanzani," I.R.C.C.S., Padiglione Del Vecchio, Via=20 Portuense 292, I-00149 Rome, Italy; fax: = ++39-06-55170-904;=20 email: poccia@inmi.it

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