Route of Immunization and the Therapeutic Impact of Recombinant Anticancer Vaccines (1997)

Active immunization with recombi-nant poxviruses, e.g., recombinant vac-cinia virus (rVV), containing a varietyof experimental tumor-associated anti-gens can result in prolonged survival ofanimals bearing established tumors (1-3). Poxviruses are currently being testedin patients with cancer [Rosenberg SA,et al.: unpublished data; (4)]. The firstrecombinant to be employed was vac-cinia virus, which demonstrated itssafety and efficacy in the worldwidecampaign to eliminate smallpox (5). An-other category of poxviruses, avipox vi-rus, is currently being explored as a vec-tor for the immunotherapy for cancerand infectious disease. These virusescan cause slow-spreading pox diseasesin birds but do not productively replicatein mammalian cells and thus are beinginvestigated as a safer alternative to rVVfor immunization (6).Since the early days of the war onsmallpox, the traditional route of immu-nization has been scarification, and re-cent clinical trials with recombinantvaccinia viruses have followed suit. Asit is currently done, scarification in-volves the use of a bifurcated needle tomake multiple small and superficial in-cisions in the skin onto which a drop ofinoculum is placed and allowed to ad-sorb into the site. As advocated by theWorld Health Organization, the safetyand efficacy of scarification as a route ofadministration of vaccinia virus hasbeen demonstrated in millions of people.Vaccination against cancer and infec-tious diseases with the use of recombi-nant viruses can also be accomplishedvia scarification (1,7). Our own early at-tempts to generate primary immune re-sponses against vaccinia viral antigensby the use of this method of immuniza-tion, however, were unsuccessful. Toexplore whether the route of immuniza-tion affected the efficacy of tumor treat-ment, we used the experimental murinecolon adenocarcinoma (CT26.WT)transduced with the gene for the modelTAA, b-galactosidase (b-gal), desig-nated CT26.CL25. Previously, Wang etal. (3) have shown that recombinantfowlpox virus (rFPV) expressing b-galwhen injected intravenously can specifi-cally reduce the number of pulmonarynodules in mice bearing 3-day estab-lished tumor metastases. Likewise, rVVcan also mediate partial therapeutic re-sponses in some cases (2). Active treat-ment of pulmonary metastases with theuse of rVV or rFPV was enhanced bythe systemic administration of interleu-kin2(2).BALB/c mice were given intrave-nous injections of CT26.CL25 (b-gal+)tumor cells. After 3 days, tumor-bearingmice were administered rVV expressingb-gal or a control vaccinia virus ex-pressing nucleoprotein from the influ-enza virus by use of either intravenous,intramuscular, subcutaneous, or intra-dermal tail scarification routes of injec-tion. Nine days later, lungs were har-vested and grossly visible metastasespresent on the surface of the lungs wereenumerated in a coded, blinded fashionas previously described (2,3,8). Fig. 1represents the pooled averages of threeexperiments done with the same proto-col. As shown in Fig. 1, injection of thecontrol rVV, V69, regardless of theroute of immunization, had little to noeffect on the number of tumor noduleswhen compared with the no-treatmentgroup. By contrast, all of the mice thatreceived the rVV expressing b-gal,VJS6, had a statistically significantly re-duced number of pulmonary metastasescompared with untreated mice (overallmean ± standard error [SE], 244 ± 9.5metastases or mean by experiment, >250,233, and >250, respectively). The micethat were immunized with VJS6 intrave-nously (overall mean ± SE, 20 ± 13.2metastases or mean by experiment, 11,0.2, and 49, respectively) showed a sta-tistically significantly enhanced reduc-tion in the average number of metastasescompared with either subcutaneously(overall mean ± SE, 123 ± 43 metastasesor mean by experiment, 154, 25, and191, respectively) or intradermal tailscarification (overall mean ± SE, 143 ±47 metastases or mean by experiment,150, 84, and 191) routes of injection.The mice that received VJS6 intramus-cularly had an overall mean ± SE of 46± 37.6 metastases (mean by experiment,56, 11, and 72, respectively) that wasnot statistically different from VJS6 im-munization by the subcutaneous or theintradermal tail scarification route whenan adjustment was made for the numberof tests accomplished. Both the intrave-nous and the intramuscular routes con-sistently demonstrated statistically sig-nificant reductions in the numbers ofmetastases compared with the no-treatment group in all three experiments.Together, these experiments indicatedthat it may be more efficacious to im-munize by the use of either the intrave-nous or the intramuscular route of injec-tion.In these studies, rVV alone mediatedan antitumor effect. This observation isin apparent conflict with an earlier re-port from our laboratory (2). Previously,Bronte et al. (2) used a crude preparationconsisting of a cellular lysate of virallyinfected BSC-1 monkey kidney cells.However, the method of viral processinghas been significantly improved sincethis paper was published. Now, crudelysates are purified over a sucrose cush-ion, thus reducing the presence of cel-lular membranes and cytosolic andnuclear components (9). Furthermore,purification of the virus reduces the in-duction of irrelevant immune responsesagainst these additional elements withinthe crude lysate. This procedure mayalso eliminate potentially immunosup-pressive molecules present in the cellu-lar preparation.