Patent Abstract:
this invention relates to methods and systems for generating a safe and effective oral smallpox vaccine for humans using a genetically defective strain of vaccinia virus to confer immunity following oral delivery of the vaccine . this invention is one that expands on current use of vaccinia virus propagation developed for gene therapy applications , and pharmaceuticals and nutraceuticals packaging and formulation technologies . the vaccine invention can be delivered as a live virus with the ability to express viral proteins but unable to achieve complete , lytic virus replication , or it may be derived from such a virus , contain additional immunogens , or be delivered as viral antigens . furthermore , the invention establishes innovative methods for formulation and packaging and for preclinical testing of the vaccine invention for safety , efficacy and potency with the use of human intestinal and other test cells and diagnostic test systems and kits .

Detailed Description:
all cells , virus and reagents are handled according to cgmp ( current good manufacturing practice ) standards . the manufacturing facility ( anteroom , class 10 , 000 room , class 100 hoods ) staff will use sops that meet fda testing , validation , and qa / qc manufacturing standards . these measures are taken to accelerate the process from discovery to product . the bhk - 21 cl 13 cell line ( atcc # ccl - 10 ) is used as the permissive cells to propagate the stock virus . the rationale for choosing this line is that it is permissive for dvv such as mva and is easily grown in culture . an example showing bhk - 21 cells growing in culture is shown in fig1 . cells are maintained as recommended by the atcc in a modified minimal essential medium with earle &# 39 ; s salts [ emem ], 0 . 1 mm non - essential amino acids , and 1 mm sodium pyruvate ( e . g ., gibco or other vendor ) with 10 % v / v fetal bovine serum ( fbs ; e . g ., hyclone or other vendor ), or another suitable growth medium . they are subcultured using 0 . 25 % trypsin , 0 . 03 % edta ( gibco ) at subcultivation ratios of 1 : 2 to 1 : 10 . cells used for vaccine preparation are derived from incell &# 39 ; s reference master cell bank ( mcb ) and working cell bank ( wcb ; n ≧ 200 vials ) stocks . the banked cells have been checked for sterility by standard microbial growth and mycoplasma pcr assays of the mcb and wcb and characterized by dna fingerprinting to assure identity . the incell propagated strain of mva ( atcc # vr01508 ), designated i - mva , has been routinely propagated by standard methods and titered by the preferred method of immunoplaque assay as detailed below . other quantitative methods that have been used include either end point dilution in bhk - 21 cells to obtain a 50 % tissue culture infectious dose ( tcid50 / ml ) or iu ( infectious units ), as detailed by dresden et al . [ 15 ]. for in vitro and in vivo assays , virus has been purified by ultracentrifugation through a 36 % sucrose cushion using standard virus purification methods . the bhk - 21 cells are grown in culture ( also termed “ in vitro ”) and infected at 0 . 1 ffu per cell to generate large lots of virus harvested at 72 +/− 2 hours post infection ( p . i ). the bhk - 21 cell cultures can be monolayers in various types of bioreactor or scale - up cultures , including culture flasks , stacked systems , culture microcarrier beads , or other appropriate substrates to culture the cells . the resultant virus stocks can be concentrated or purified by ultracentrifugation , ultrafiltration , or other standard methods , then titered on bhk - 21 cells and stored for packaging . part of the stock is aliquoted for use in the bioassays and for qa testing . all lot information is entered into the master database and inventory management system which were developed as part of the invention and its use . for immunoplaque assays , bhk - 21 cells are seeded at a density of 4 . 5 × 10 4 cells per well of a 24 - well plate in growth medium ( emem , 10 % fbs , plus additives , as described above ). after overnight attachment , when the cells are 80 - 90 % confluent , they are infected with i - mva by mixing the virus with emem prepared as per the growth medium but with 2 % rather than 10 % fbs (= emem : 2 infectivity medium ). test i - mva source materials are generally diluted in 10 − 3 to 10 − 7 for cell culture derived , unconcentrated supernatants , and 10 − 6 to 10 − 10 for gradient centrifugation purified or otherwise concentrated ( e . g ., ultrafiltered ) virus . dilutions are made in emem : 2 infectivity medium . cells that receive no virus but are otherwise incubated with emem : 2 infectivity medium and treated the same are used as negative controls . reference virus stocks that are known to produce 100 - 200 plaques per well are included in the assays as positive controls to assure performance of the assay . cultures are gently swirled to assure even virus distribution then incubated for 24 hr at 37 ° c ., in a 5 % co 2 , 95 % air environment . at the end of the incubation period , the medium is removed from the wells , the cells are fixed with 0 . 5 ml 1 : 1 acetone : methanol for 5 min , the fixative is removed and 1 ml cmf - pbs is added to each well . the rinse solution is removed and anti - vaccinia virus primary antibodies ( e . g ., rabbit , sheep , human or other source ) and the biotin or other chromagen - labeled secondary antibodies used at an effective dilution ( e . g ., 1 : 500 to 1 : 1000 ) to easily visualize the immunoplaques . for most studies , 1 : 500 dilutions of each of the primary ( rabbit anti - vaccinia ; accurate chemical or incell - prepared ) and secondary ( hrp anti - rabbit igg ; sigma or other vendor ) antibodies were used . an example showing bhk - 21 cells and the appearance of plaques in the immunoplaque assay is shown in fig2 . for rabbit immunizations , 10 8 ffu in 0 . 5 ml pbs were combined with 0 . 5 ml titermax gold using a double hub emulsification needle ( push antigen into titermax first , aqueous into oil phase ) for mixing . the emulsion was injected into 4 sites ( 0 . 2 ml each ) over both shoulders and both hind quadriceps . for sheep immunizations , 2 × 10 8 ffu in 1 ml pbs were combined with 1 ml titermax gold as above and inject 0 . 4 ml twice into each hind quadriceps . animals were bled periodically to test antibody production . good antibody titers are present within 4 - 6 weeks and remain high for several months . a variety of oral immunization formulae can be used for immunization . oral immunization is done by preparing a formula in which the virus remains viable ( as determined by infectivity of released virus from the orally delivered paste and separate components of the paste formulae listed below ) and is captured in nanoparticles and micelles as part of the protective formulation that includes aqueous and oil - based components , as well as suspending agents and carriers that protect the virus from degradation and allow it to be absorbed from the oral cavity and the intestine . as an example of the formulation used for the the studies shown in the figures , virus is prepared ( at 10 8 per rabbit or 2 × 10 8 per sheep ) by mixing virus in a solution of hetastarch ( hydroxyethyl starch , clinical grade ; 6 % w / v ; baxter ), 40 % ( v / v ) mannitol [ ups grade higher ; sigma or other vendor ], 0 . 15 % ( v / v ) aafa ™ ( nutritional supplement grade fish oil ; incell 5 % ( v / v ) glycerol ( ups grade ; sigma or other vendor ), 0 . 5 % ( w / v ) gelatin ( sigma ) at a volume that will achieve a final concentration of 5 × 10 4 to 2 × 10 8 infectious ffu , depending on the effective or test dose expected ( e . g ., 10 6 to 10 8 for humans , depending on immunization status ). in the animal studies , doses were at 10 8 per rabbit and 2 × 10 8 per sheep . gel - sol virus carrier ( gsvc ) excipient components were prepared as an equal mixture ( 1 : 1 : 1 ; avicel ® ce - 15 ( microcrystalline cellulose and guar gum ), avicel ® 591 ( water - dispersible microcrystalline cellulose containing sodium carboxymethylcellulose ( nacmc ) and ac - di - sol ® ( internally - crosslinked , water insoluble sodium carboxymethylcellulose ( nacmc )) [ source of all components : fmc products ]) which was slowly added ( with vortexing ) to a final concentration of 10 % ( w / v ). taste - testing ( humans and animals ) revealed that the formulae was palatable as a slightly sweet paste - gel type of formulae that caused no aftertaste and which could be subsequently dried ( e . g ., for tableting ) and still maintain infectious virus as measured by infection of dissolved materials on bhk - 21 cells after they had been dried and stored for various time periods , supporting long - term storage as a tablet or paste - gel material that maintains biological activity . a variety of bioassays and biochemical analyses are done to evaluate the vaccine . these include : ( a ) human cell line nonpermissiveness with expression of vaccine antigens ( a safety test ); ( b ) viral antigen expression and production compared to previous lots and reference standards ( i . e ., potency ); and ( c ) activation of humoral and cell - mediated immunity ( e . g ., potency and efficacy ) in infected animals . these are imperative types of assays to evaluate each virus lot and the overall potential variability between lots of virus . 1 . safety and potency bioassays : i - mva infection of human cells in vitro incell has the only long - term continuous cell lines derived from human intestine ( hi ). as part of the pre - clinical testing , the hi cells were be grown in m3 : 10 ™ medium ( incell ) as monolayer cultures using standard methods so that they maintained functional cell and organ - specific markers that make them useful in vitro surrogates for orally administered products , including vaccines or drugs . master and working cell banks of these cells were banked in the incell repository prior to initiating these studies . as part of the evaluation of i - mva lots of oral vaccine , the hi test cell line ( s ) lines were seeded into culture vessels in m3 : 10 ™ ( incell ) growth medium , allowed to attach , then infected with test lots of virus essentially as described above for the ffu immunoplaque assays or as detailed elsewhere ( 15 ) for alternate cell infectivity studies . for each set , parallel cultures of uninfected and infected permissive bhk - 21 cell controls , and dilutions of prepared reference virus , were tested to validate the bioactivity of the virus stocks . an example of the study showing comparative infectivity of i - mva for human intestinal and other human cells compared to the permissive bhk - 21 cells are shown in table 1 . the important vaccine safety - related conclusion from the results shown in this table is that the i - mva strain used to prepare the vaccine does not grow in human cells but readily replicates in the permissive bhk - 21 cells . three methods are used to evaluate production of the viral antigens and anti - viral antibodies as a measure of potency of the lots produced : ( a ) elisa assays , ( b ) western blots , and ( c ) immunocytology . elisa plate assays are done to quantitate the amount of anti - virus antibodies produced against the virus or the amount of virus antigen produced by infected cells . such assays have many variables and methodologies . an example test method is as follows . the virus stocks are diluted to 0 . 1 to 4 μg / ml in carbonate buffer and coated onto elisa 96 - well plates at 25 μl / well for 4 hours to overnight at 37 ° c . and then washed 3 times with pbs - tween40 ( pbs - t ). the antigen - coated plates are blocked with 3 % bsa at 200 μl / ml for 1 hr at 37 ° c . ( on a rocker platform ) and washed 3 times with pbs - t . virus reference test antigens and reference antibody dilutions are used at known positive concentrations and ratios as positive controls . test antibodies are bracketed for assay at multiple dilutions , based on expected ranges , in replicates of n = 4 , with test dilution samples added to the plates at 25 μl / well . after incubation for 2 hr at 37 ° c ., the plates are washed with pbs - t . all comparative values are analyzed using incell &# 39 ; s customized plate analysis software in concert with statistical and graphics programs . importantly , antibodies have been consistently demonstrable in all of the immunized animals . in general the orally immunized animals had a somewhat lower titer than the animals that received intramuscular depot immunization with the titermax . however , as shown below , the circulating antibody titer did not necessarily correlate with neutralization differences and cmi may actually be higher in the orally immunized animals . for western blot analyses , cell protein lysates were prepared and resolved by electrophoresis on a sds - 8 % polyacrylamide gel , then transferred onto nitrocellulose for 2 h in a buffer containing 25 mm tris , 192 mm glycine , and 20 % methanol ( ph 8 . 6 ). the blots were blocked overnight at 4 ° c . in a pbs blocking buffer containing 1 % bsa and 0 . 1 % np40 and then incubated for 1 h at room temperature with rabbit or sheep anti - vaccinia antibody diluted 100 - fold in blocking buffer . after being washed with 0 . 1 % np40 in pbs , the blots were incubated for 1 h at room temperature with goat anti - rabbit or anti - sheep igg light ( amersham ) diluted 1000 - fold in blocking buffer , washed again , and exposed to x - ray film for comparative evaluation and image analysis to quantify the samples . as shown in the examples of western blots in fig1 and 11 , the immunized rabbits and sheep were able to elicit antibodies that could recognize viral proteins on a western blot analysis . this further verifies the specificity of the antibodies for the virus , as was demonstrated with sera from all of the test animals . immunocytology assays may have many variables and methodologies . an example test method is as follows . to analyze cells for visualizing the expression of viral or cell antigens , the cells are grown as monolayers . this is done on multi - well plates or on lab - tek ( corning ) slides or attached to coated slides by standard cytocentrifugation protocols . for immunodetection assays , standard protocols have primary antibody diluted in pbs to an optimal working dilution followed by incubation with the cells that have been fixed in omnifix or another antigen appropriate fixative . for immunocytology assays , the cells are fixed with 10 % formalin ( sigma ) for 1 hr at 4 ° c . and blocked with 3 % bovine serum albumin ( bsa ; sigma ) in calcium - and magnesium - free phosphate buffered saline with 0 . 01 % tween - 20 ( cmf - pbs - t ). specific anti - vaccinia polyclonal rabbit antibody ( accurate chemical ) or newly derived antibodies are added to the fixed and rinsed cells . cells are stained according to the general procedures detailed in the vectastain ® elite abc kit by the manufacturer ( vector laboratories ). briefly , each sample is incubated with test antibody at the appropriate working dilution of the antibody followed by a biotinylated goat anti - rabbit or anti - mouse secondary antibody ( sigma : 1 : 2000 ). the samples are quenched with 0 . 3 % hydrogen peroxide and developed with a combination of an avidin - linked peroxidase conjugate and the 3 , 3 ′- diaminobenzidine ( dab ) chromagen . the slides are counterstained with hematoxylin ( biomeda corp . ), mounted with aqueous mounting medium ( biomeda corp ), and visualized with a nikon microscope . photographs are taken using a digitized format and photo capture software . the stained cells look similar to the individual stained cells shown in fig2 of the immunoplaque assay . when such assays are done using antibodies from i . m . or orally immunized animals , the immunostained cells look similar . for immunoplaque reduction or neutralization assays , the methods are the same for immunoplaque assays through the set - up step , but the virus inoculum is pre - incubated for at least 1 hr with serum or purified igg prior to adding the virus - antibody inoculum . otherwise , the remaining steps of the protocol are they same . when the virus is pre - incubated with the test serum containing antibody the incubation step is at 37 c and the serum is usually heat - inactivated at 56 c for 30 min prior to incubation with the virus . fig1 shows that orally immunized animals could neutralize infectious i - mva as measured by inhibition of plaque formation compared to the controls ( 100 %). similar results were obtained with i . m . immunized animals . it was concluded that all orally immunized animals produced neutralizing antibody . in the example , rabbits showed a stronger effect but sera were collected 37 days pi vs . only 19 days pi for the sheep . this work complements studies done with mice in which orally immunized mice are challenged with an infectious vaccinia strain , such as wr , and the immunized animals are protected from the associate morbidity and mortality of the challenge virus . the peripheral blood mononuclear cells ( pbmcs ) are obtained from peripheral blood and separated using standard methods . either fresh pbmcs or pre - qualified ( known responder ) pbmcs from cryopreservation are used for testing . cell separation methods and cell - mediated immunity assays may have many variables and methodologies as described below . one example method is to take pbmcs from immunized donors and determine whether or not they can respond to stimulation with the immunizing antigen , in this case , i - mva . to that end , pbmcs from a sheep immunized intramuscularly ( i . e ., “ sheep 1 ”) and a sheep immunized orally ( i . e ., “ sheep 2 ”) were added to rpmi culture medium containing 10 % ( v / v ) autologous plasma . quadruplicate cultures of 10 5 cells per well of a 96 - well plate with or without mva antigen , or control wells without cells , were compared to assess a cellular response to mva antigen as measured by stimulation of dna synthesis using a brdu elisa - based assay as detailed below . results of an example study are shown in fig1 , where it is clear that both sheep had demonstrable cell stimulation . the conclusion form these studies is that oral immunization can effectively induce cell - mediated immunity against i - mva and , thus , presumably against a related invading poxvirus , such as smallpox . another example method is as follows . the collected blood cells or cultured cells are layered over warm histopaque and centrifuged at 400 × g to separate dead from viable cells . the number and viability of the cell population is assayed by standard 0 . 25 % trypan blue dye exclusion using incell &# 39 ; s sop . on day zero , 5 × 10 6 viable cells are seeded into 6 - well plates containing a final volume of 5 ml mr : 20 ™ ( incell ) or other suitable culture medium . after an overnight culture adaptation period , a subset of pbmcs are infected with i - mva at an moi of 1 tcid / cell for 2 h and the remaining autologus pbmcs are readied for co - culture as described below . after washing twice , 8 × 10 5 i - mva - infected pbmcs are irradiated or treated with mitomycin c ( 25 mcg / ml ) so they can no longer divide . for cmi activation studies , the infected cells are then added to 5 × 10 6 autologous pbmcs , which had also been cultured overnight . after subsequent culture for 4 - 7 days , dna synthesis is determined by adding radiolabeled 3 h - thymidine or 5 - bromo - 2 - deoxyuridine ( brdu ) for the final 4 to 18 hr of culture period to measure incorporation into dna . the 3 h - thymidine is measured by liquid scintillation counting of trichloroacetic acid [ tca ] ( 10 %)- precipitated cellular dna added to scintillation fluid and counted . the brdu is measured using an elisa assay and highly specific anti - brdu antibody linked to a chromagen ( e . g ., biotin ). control cultures ( cells and media only ) are grown at the same time but do not receive i - mva or i - mva infected cells . the control values are either subtracted from the test assays with infected cells to give specific incorporation numbers or they are used to determine the relative labeling index by the equation ( dna [ t ]- dna [ c ])/ dna [ c ]). to amplify the response or to initiate a de novo response , cultures can be re - stimulated weekly using freshly prepared i - mva infected and autologous pbmcs at a responder to stimulator ratio of 2 : 1 and supplemented with 25 iu / ml il - 2 . after four cycles of re - stimulation , bulk cultures can be further tested for immune activation and many test parameters . controls include purified virus and uninfected cells . comparative test parameters of immune activation include cytokine production , cytotoxicity against target cells , and cell activation ( including target cell death and mixed lymphocyte reaction [ mlr ]) using the methods described in more detail below . for these studies , anova is used to statistically compare the groups . inflammatory mediators or cytokines ( e . g ., tnf - α ; ifn - gamma ) generated by pbmcs after in vitro stimulation with i - mva , i - mva - infected cells , or no ( control ) stimulus . cells are assayed by immunoassays of culture supernatants from multi - well plates or by elispot assays in which cells are attached to plates containing an antibody against the cytokine of interest ( e . g ., ifn - gamma ). supernatants from stimulated pbmcs ( infected cells or purified virus ) are compared to control cultures ( i . e ., media only and uninfected cells with no stimulus ) elisa , western or dot - blot assays can be used to compare cytokine production by cells in the test groups . values of stimulated cells are adjusted for background and baseline values of the control groups so that induced or increased cytokine production is measured . cytotoxic t lymphocytes ( ctls ) are also tested for their production of tnf - α or ifn - gamma following co - culture with selected lines of i - mva - infected compared to uninfected cells . cytokine assays may have many variables and methodologies . an example test method is as follows . stimulator cells are infected for 4 hr with i - mva at an moi of 1 ffu / cell , extensively washed , and plated in 6 - well plates at 8 × 10 5 cells / well . after an overnight incubation 12 - 15 hr at 37 ° c ., effector t cells ( 5 × 10 6 cells / well ) are added . effector cells co - cultured with uninfected cells are used as negative reference controls . all assays are done at least in triplicate . for cytokine ( e . g ., tnf - α or ifn - gamma ) assays , supernatants are harvested after 40 h , and the cytokine content ( ng / ml ) is determined by multi - well plate elisa or elispot assays ( e . g ., r & amp ; d systems ; cell systems ). mlr assays may have many variables and methodologies . an example test method is as follows . “ responder ” pbmcs ( 1 × 10 5 cells / well ) are seeded into 96 - well culture plates . i - mva - infected autologous pbmcs ( 1 - 3 hr pi ), and mock - infected control cells are treated with mitomycin c ( 25 ug / ml ) then added ( 1 × 10 5 cells / well ) to the responder cells ( 1 : 1 ratio ). cell proliferation is measured after 96 hrs with a brdu elisa assay as described above . cras may have many variables and methodologies . an example test method is as follows . the lytic activity of either in vitro - stimulated “ effector ” { e } cytotoxic t lymphocytes ( ctls ) are tested against i - mva - infected or uninfected target { t } cells in a 4 - hr standard 51 cr release assay . target cells are infected for 2 h with i - mva at an moi of 1 ffu / cell , washed once , then labeled with 100 μci na 51 cro 4 for 1 h at 37 ° c . after 4 washes with pbs , labeled target cells are plated in u - bottomed 96 - well plates at 1 × 10 4 cells / well and incubated at 37 ° c . at 15 - 18 hr after infection , effector cells are incubated with the target cells at various e : t ratios ( 0 : 1 , 25 : 1 , 50 : 1 , 100 : 1 ). after 4 hr , the plates are centrifuged to pellet the cells , 100 μl of supernatant per well is collected , counted , and recorded as mean +/− sd counts per minute ( cpm ) of replicate samples ( n = 4 ). the specific 51 cr release is determined by subtracting the background counts of cells in the 0 : 1 { e : t } group where there are no effector cells added . results of the test groups are compared by anova statistical analyses to determine differences between groups at a p value & lt ; 0 . 05 . the manufacturing steps for production of the vaccine will include the use of existing disposable cell propagation devices , connectors , and other closed system technologies that are adaptable from laboratory cell culture to scale - up manufacturing of large batches . fig1 is an example of the overall manufacturing approach from virus propagation to packaging . in this example , the mva virus is propagated on bhk - 21 cells that are cultured to high culture density on microcarrier beads in plastic cell culture bags , followed by concentration and purification of the virus , combining the virus with a proprietary oral delivery formulation as the “ vaccine mixture ”, processing the vaccine mixture to a tablet , paste , gel , liquid or other oral delivery form , then packaging it in a foil package , blister pack , or other standard form as a single unit dose . all procedures and materials for virus propagation and handling at all steps of the manufacturing are selected with the notion that they can be scaled up from laboratory lots to 200 or more liters , then discarded after use , to remove the validation and other aspects related to cleaning and sterilization of vessels and other manufacturing components . fig1 shows examples of the types of closed and fda approved , disposable products that will be obtained from qualified vendors and used for manufacturing steps . they include a variety of plasticware disposables that can be scaled larger manufacturing needs . fig1 shows an example of how fda approved cgmp components , connectors , and closed , integrated systems might be combined as a manufacturing step . fig1 shows an example of an oravax ™ sample package prepared as a unit dose package for oral delivery . the unit dose would include i - mva in a formulation that has been tableted or is prepared as a paste or gel that can be squeezed from the foil or other packaging . the package can be made as foil or blister packages to which tablets are added or it can be made as a form , fill , seal method whereby the package is formed , filled with the vaccine product ( gel , paste , liquid , then sealed in a package that can be opened for single use consumption . features of the package are that it does not allow light or moisture and , preferably , the product can be stored at room temperature or refrigerated , but does not require freezer temperatures to maintain stability . manufacturing approvals and outcomes include evaluation of product : safety , potency , efficacy , stability , shelf life and other measures that include innovative and unique elements for this application . safety of the virus lots to be used in the oral delivery formulation is tested by using appropriate human target cells , i . e ., normal human cells from multiple donors and several regions of the alimentary tract . potency is measured by determining that the vaccine virus lot is infectious for permissive cells with a quantified titer as determined by immunoplaque or other assay . efficacy is measured by the production of protective immunity , such as virus neutralization with associated lack of infectivity for the host target cells or animals , following oral delivery of the vaccine with the immunizing virus strain included in a formulation that protects the virus and augments immune responsiveness , and has stability and a shelf life of at least a year , with preferable storage at room temperature . said formulation may include gelatin , cellulose , or a variety of other excipients as ingredients , or the formulation may be a gel or a food carrier such as a pudding or similar formulation that would include the virus as a component . as another embodiment flavorings , emulsifiers , or other additives may be included in the formulation of the product , the delivery vehicle or other components of the packaged material . the i - mva immunogen can be packaged as a solution , as single doses , as a paste or gel , or in a food or nutritional substance in a plastic container , pillow - pack , tear - pack , straw tube packaging or other suitable packaging for the liquid , gel or food carrier formulation . 1 . kortepeter , m . and g . parker , potential biological weapons threats . emerg infect dis , 1999 . 5 : p . 523 - 7 . 2 . henderson , d ., looming threat of bioterrorism . science , 1999 . 283 : p . 1279 - 82 . 3 . fenner , f ., et al ., smallpox and its eradication . 1988 , geneva : world health organization . 4 . cohen , j ., smallpox vaccinations : how much protection remains ? science , 2001 . 294 : p . 985 . 5 . littaua , r ., et al ., vaccinia virus - specific human cd 4 + cytotoxic t - lymphocyte clones . j virol , 1992 . 66 ( 4 ): p . 2274 - 2280 . 6 . demkowicz jr , w ., et al ., human cytotoxic t - cell memory : long - lived responses to vaccinia virus . j virol , 1996 . 70 ( 4 ): p . 2627 - 2631 . 7 . demkowicz jr , w . and f . ennis , vaccinia virus - 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