![](https://webarchive.library.unt.edu/eot2008/20090117115851im_/http://www.cdc.gov/ncidod/eid/images/spacer.gif)
|
![](https://webarchive.library.unt.edu/eot2008/20090117115851im_/http://www.cdc.gov/ncidod/eid/images/spacer.gif) |
Dispatch
Flow Cytometry and T-Cell
Response Monitoring after Smallpox Vaccination
Fabrizio Poccia,* Cristiana Gioia,* Carla Montesano,* Federico Martini,*
Douglas Horejsh,* Concetta Castilletti,* Leopoldo Paolo Pucillo,* Maria
Rosaria Capobianchi,* and Giuseppe Ippolito*
*National Institute for Infectious Diseases "Lazzaro Spallanzani,"
Rome, Italy
Suggested citation
for this article:
Poccia F, Gioia C, Montesano C, Martini F, Horejsh P, Castilletti C,
et al. Flow cytometry and T-cell response monitoring after smallpox
vaccination. Emerg Infect Dis [serial online] 2003 Nov [date cited].
Available from: URL: http://www.cdc.gov/ncidod/EID/vol9no11/03-0349.htm
Orthopoxvirus zoonosis
or smallpox as result of bioterrorism or biological warfare represents
a risk for epidemic spread. By monitoring T-cell responses by flow cytometry,
we observed a recall response after recent vaccination against smallpox.
When the high similarity between the orthopoxviruses is considered,
this rapid assay that uses vaccinia antigens could identify recently
exposures.
Although the last natural case of smallpox was reported in Somalia in
1977, this orthopoxvirus remains a source of concern. No evidence exists
that smallpox will recur as an endemic disease, but the virus may have
been acquired for use in biological warfare or bioterrorist attacks. If
one assumes an average of 15 days is needed for infected persons to become
infectious, delay in intervention will be costly, increasing the total
number of cases (1). Furthermore, the recent outbreak
of the severe acute respiratory syndrome coronavirus and the first documented
outbreak of monkeypoxvirus in the Western Hemisphere underline the ever-present
risk for epidemic extension of zoonosis and raise concerns about the medical
and social effect of reemerging orthopoxvirus infection in humans. During
the epidemic spread of an emerging pathogen, evaluating exposed persons
and containing the infected population should be the first priorities.
A local outbreak of orthopoxvirus infection would require rapid and sensitive
diagnostics, including novel assays based on host responses.
For intracellular pathogens, the antibody titers and neutralization assays
represent routine immunologic tests that provide results after several
weeks of infection. The appearance of a detectable antibody titer takes
place a few days after the induction of a T-cell response (2).
Moreover, antigen-specific T-cell responses could be detected in exposed,
but uninfected persons, as shown in those with HIV infection (3).
Using a rapid flow cytometric test, we previously showed that monitoring
interferon (IFN)-
production by antigen-pulsed T cells provides a quantitative and functional
assessment of HIV- or cytomegalovirus (CMV)-specific CD8(+) and CD4(+)
T cells (4-6). This technique requires that whole proteins
or selected peptide antigens are added to blood cells, allowing the simultaneous
analysis of both major histocompatibility complex class I and II restricted
T-cell responses (7). Because smallpox vaccination was
recently shown to induce a strong vaccinia virus-specific CD8(+) CTL-
and IFN- –producing
T cells detectable by more cumbersome research laboratory methods (cytotoxic,
proliferative, or ELISPOT assays) (8,9), we evaluated
the feasibility of an easy, rapid, and sensitive assay to monitor T-cell
responses after recent vaccination against smallpox; the assay can potentially
be used as a routine diagnostic assay.
The Study
|
Figure |
|
|
![Figure.](images/03-0349_t.gif) |
|
|
Click to view
enlarged image
Figure. Flow cytometric analysis of T-cell
responses to smallpox antigens after recent smallpox vaccination
and in long-term vaccinated or not vaccinated persons...
|
|
T-cell reactivity was analyzed after recent (<2 years ago) smallpox
vaccinations, in long-term vaccinated (>20 years ago) and not vaccinated
persons. Briefly, peripheral blood mononuclear cells (PBMC) were isolated
by standard density centrifugation (Ficoll-Hypaque, Pharmacia, Uppsala,
Sweden). Stimulation was also performed on whole blood samples; however,
the assay had reduced sensitivity. We cannot exclude the possibility that
whole blood assay sensitivity could be improved by changing protocol conditions
(data not shown). PBMC were cultured in complete Roswell Park Memorial
Institute 1640 medium, 10% v/v heat-inactivated fetal calf serum, 2 mM
L-glutamine, and 10 U/mL penicillin/streptomycin at a concentration of
106 cells/mL. Stimulation was performed with 40 µL/mL
(total protein content of approximately 1 µg/mL) of vaccinia viral
antigen resuspended according to the manufacturer's instructions (Maine
Biotechnology Services, Portland, ME), or 2 µg of CMV antigen (Biowhittaker,
Walkersville, MD), always in the presence of co-stimulation with both
anti-CD28 and CD49d monoclonal antibodies (1 µg/mL, Becton, Dickinson
and Company, Franklin Lakes, NJ). We also tested the T-cell response with
live vaccinia-infected fibroblast or Vero cells. The response against
uninfected antigenic preparations was always above background, reducing
the sensitivity of the assay (data not shown); therefore, the commercially
available antigens were used in subsequent experiments. Cultures were
incubated at 37°C for 1 h, followed by an additional overnight incubation
with 10 µg/mL of the secretion inhibitor Brefeldin-A (Sigma-Aldrich
Corporation, St. Louis, MO). Cells were washed twice in phosphate-buffered
saline, 1% bovine serum albumin, and 0.1% sodium azide, and stained for
15 min at 4°C with monoclonal antibodies specific for cell surface
CD antigens (Becton, Dickinson and Company). Samples were then fixed in
1% paraformaldehyde for 10 min at 4°C, incubated with Phyco-Erithrin-conjugated
mouse-anti-human IFN-
(Becton, Dickinson and Company), washed twice in phosphate-buffered saline,
1% bovine serum albumin, and 0.1% saponin, and resuspended in FACSFlow
before being acquired on FACScalibur (Becton, Dickinson and Company),
as previously described (4,6). Controls for nonspecific
staining were monitored with isotype-matched monoclonal antibodies (Becton,
Dickinson and Company); cells incubated with only anti-CD28 and -CD49d
were included in each experiment and nonspecific staining was always subtracted
from specific results. In the cytometric panels shown in the Figure,
the IFN- production
by CD3(-) cells is 1 log lower in intensity compared to the antigen-specific
CD3(+) T-cell response, representing an unspecific response that may involve
natural killer cells. To monitor antigen-specific T-cell responses, we
collected data only from CD3(+) T cells producing higher amounts of IFN- .
Negative control antigenic stimulation was always below the detection
limit of the assay (0.02%).
Cytometric panels in the Figure show the IFN-
synthesis by CD3(+) T cells after in vitro stimulation with vaccinia virus
or CMV 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
respectively; the numbers of CMV-specific CD3(+) T cells per blood milliliter
were 13,132, 2,964, and 11,385, respectively). As previously described
(6), most of the CMV-specific response was related to
CD4(+) T cells (96%, 75%, and 59% of CMV-specific T cells, respectively).
Both unvaccinated and long-term vaccinated healthy donors had undetectable
responses to smallpox vaccinia antigens (Figure,
panels A and B). In contrast, a recall response was detectable after a
recent immunization (Figure, panel C). In this case,
the percentage of T cells specific for smallpox 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 frequency of 1/667. Most vaccinia-specific
T cells detected by this assay were CD4(+) (vaccinia-specific CD4(+) T
cells were 80% of vaccinia-specific T cells). Nevertheless, the sensitivity
of this assay to detect CD8(+) T cells could be improved by using human
leukocyte antigen (HLA) class I–specific 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 be maintained
for decades (10,11). In long-term vaccinated
persons, virus-specific CD4(+) and CD8(+) T-lymphocytes are detectable
only after extensive in vitro culture and restimulation to generate antigen-specific
lines or clones. This limitation is due to the long, but limited, lifespan
of memory T cells and to their low frequency, usually below 1/50,000 (12).
Our in vitro rapid assay based on a short-time primary T-cell response
was unable to show the residual memory T-cell response present in long-term
vaccinated persons since the assay sensitivity is 1 log lower but could
detect the higher frequencies of IFN- –producing
antigen-specific cells appearing a few weeks after smallpox vaccine inoculation
(8). Accordingly, Terajima et al. (13)
demonstrated that T-cell responses to vaccinia and variola conserved epitopes
peak 14 days after primary immunization with vaccinia virus. In this study,
the frequency of antigen-specific T cells was measured as IFN-
production by ELISPOT and HLA/peptide tetramer–staining methods.
Because strong correlations between the data derived from ELISPOT, tetramer
assays, and intracellular cytokine staining for IFN-
were previously observed (14), vaccinia-specific T cells
could be detected by flow cytometry only a few days after immunization
with vaccinia virus. In addition, Pincus and Flick demonstrated the initial
development of delayed hypersensitivity, an index of cell-mediated immunity,
as early as 2 days after smallpox vaccination (15).
During viral infection, high levels of virus-specific T cells are found
in 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 HIV-specific
CD8(+) T cells releasing IFN-
were quantitatively increased a few weeks after viral rebound consequent
to the interruption of antiviral therapy (5). These observations
indicate that the frequency of virus-specific T cells is clinically relevant,
which suggests that this method may be useful in detecting immune response
by monitoring the frequency of virus-specific T cells. In recently vaccinated
persons, memory cells are expanded by antigen reexposure, and their increase
in frequency could be quantitatively detected by the rapid flow cytometric
T-cell assay, confirming the efficacy of vaccination. Moreover, because
of the high similarity between orthopoxviruses, this rapid assay using
vaccinia antigens could be used to identify recently exposed persons.
Finally, an important aspect in developing a diagnostic assay is to use
a rapid and easily automated system that works on virtually all persons
who carry the disease. In this context, the intracellular T-cell cytokine
staining by flow cytometry presents several advantages in comparison to
other techniques, such as tetramer staining and ELISpot (4).
In fact, flow cytometry allows for testing multiple proteins or peptides
at a single time and provides at the same time a quantitative and phenotypic
assessment of CD8(+) and CD4(+) responding T cells. Moreover, optimization
of antigen preparation with peptide pools designed to be virus-specific,
highly conserved, and independent of HLA haplotypes may allow for the
development of a second generation of more sensitive flow cytometric T-cell
assays, extending the possibility to perform routine analysis on cryopreserved
samples (4). The technique could be easily automated
through the use of analytical instruments already available in most clinical
laboratories that use flow cytometry. In comparison with other analytical
systems for assessing antigen-specific responses, this method is economically
advantageous. The recent availability of mobile flow-cytometer units may
allow use of this assay under field investigation conditions.
This study was supported
by grants from the "Ministero della Salute."
Dr. Poccia is senior
scientist at the National Institute for Infectious Diseases "Lazzaro
Spallanzani" of Rome. His research activity is related to emerging
and reemerging infections, focusing on innate immunity and host-pathogen
interactions. His main interests are translational research to develop
novel diagnostic assays based on physiological and immune host responses,
tools for clinical monitoring of immune reconstitution, and broad-spectrum
immunostimulants.
References
- Meltzer MI, Damon I, LeDuc JW, Millar JD. Modeling
potential responses to smallpox as a bioterrorist weapon. Emerg
Infect Dis 2001;7:959–69.
- Doherty PC, Topham DJ, Tripp RA, Cardin RD, Brooks JW, Stevenson PG.
Effector
CD4+ and CD8+ T-cell mechanisms in the control of respiratory virus
infections. Immunol Rev 1997;159:105–17.
- Pinto LA, Sullivan J, Berzofsky JA, Clerici M, Kessler HA, Landay
AL, et al. ENV-specific
cytotoxic T lymphocyte responses in HIV seronegative health care workers
occupationally exposed to HIV-contaminated body fluids. J Clin Invest
1995;96:867–76.
- Amicosante M, Gioia C, Montesano C, Casetti R, Topino S, D'Offizi
G, et al. Computer-based
design of an HLA-haplotype and HIV-clade independent cytotoxic T-lymphocyte
assay for monitoring HIV-specific immunity. Mol Med 2002;8:798–807.
- D'Offizi G, Montesano C, Agrati C, Gioia C, Amicosante M, Topino S,
et al. Expansion
of pre-terminally differentiated CD8 T cells in chronic HIV-positive
patients presenting a rapid viral rebound during structured treatment
interruption. AIDS 2002;16:2431–8.
- D'Offizi G, Ciapparoni V, Gioia C, Goletti D, Agrati C, Pucillo LP,
et al. The
loss of CMV-specific CD27(-) T-cell effectors in a patient with recurrences
of CMV retinitis is independent of HIV-1 viremia. Infection 2002;30:323–5.
- Betts MR, Casazza JP, Koup RA. Monitoring
HIV-specific CD8+ T cell responses by intracellular cytokine production.
Immunol Lett 2001;79:117–25.
- Ennis FA, Cruz J, Demkowicz WE Jr, Rothman AL, McClain DJ. Primary
induction of human CD8+ cytotoxic T lymphocytes and interferon-gamma-producing
T cells after smallpox vaccination. J Infect Dis 2002;185:1657–9.
- Frey SE, Newman FK, Cruz J, Shelton WB, Tennant JM, Polach T, et al.
Dose-related
effects of smallpox vaccine. N Engl J Med 2002;346:1275–80.
- Demkowicz WE Jr, Ennis FA. Vaccinia
virus-specific CD8+ cytotoxic T lymphocytes in humans. J Virol 1993;67:1538–44.
- Littaua RA, Takeda A, Cruz J, Ennis FA. Vaccinia
virus-specific human CD4+ cytotoxic T-lymphocyte clones. J Virol
1992;66:2274–80.
- Demkowicz WE Jr, Littaua RA, Wang J, Ennis FA. Human
cytotoxic T-cell memory: long-lived responses to vaccinia virus.
J Virol 1996;70:2627–31.
- Terajima M, Cruz J, Raines G, Kilpatrick ED, Kennedy JS, Rothman AL,
et al. Quantitation
of CD8+ T cell responses to newly identified HLA-A*0201-restricted T
cell epitopes conserved among vaccinia and variola (smallpox) viruses.
J Exp Med 2003;197:927–32.
- Goulder PJ, Tang Y, Brander C, Betts MR, Altfeld M, Annamalai K, et
al. Functionally
inert HIV-specific cytotoxic T lymphocytes do not play a major role
in chronically infected adults and children. J Exp Med 2000;192:1819–32.
- Pincus WB, Flick JA. The role of hypersensitivity in the pathogenesis
of vaccinia virus in humans. J Pediatr 1963;62:57–62.
|