Phase I dose-escalating trial of Escherichia coli purine nucleoside phosphorylase and fludarabine gene therapy for advanced solid tumors.

BACKGROUND: The use of Escherichia coli purine nucleoside phosphorylase (PNP) to activate fludarabine has demonstrated safety and antitumor activity during preclinical analysis and has been approved for clinical investigation. PATIENTS AND METHODS: A first-in-human phase I clinical trial (NCT 01310179; IND 14271) was initiated to evaluate safety and efficacy of an intratumoral injection of adenoviral vector expressing E. coli PNP in combination with intravenous fludarabine for the treatment of solid tumors. The study was designed with escalating doses of fludarabine in the first three cohorts (15, 45, and 75 mg/m(2)) and escalating virus in the fourth (10(11)-10(12) viral particles, VP). RESULTS: All 12 study subjects completed therapy without dose-limiting toxicity. Tumor size change from baseline to final measurement demonstrated a dose-dependent response, with 5 of 6 patients in cohorts 3 and 4 achieving significant tumor regression compared with 0 responsive subjects in cohorts 1 and 2. The overall adverse event rate was not dose-dependent. Most common adverse events included pain at the viral injection site (92%), drainage/itching/burning (50%), fatigue (50%), and fever/chills/influenza-like symptoms (42%). Analysis of serum confirmed the lack of systemic exposure to fluoroadenine. Antibody response to adenovirus was detected in two patients, suggesting that neutralizing immune response is not a barrier to efficacy. CONCLUSIONS: This first-in-human clinical trial found that localized generation of fluoroadenine within tumor tissues using E. coli PNP and fludarabine is safe and effective. The pronounced effect on tumor volume after a single treatment cycle suggests that phase II studies are warranted. CLINICALTRIALSGOV IDENTIFIER: NCT01310179.

Lack of T-cell immunity in humans with preexisting anti-mouse immunoglobulin reactivity.

We have identified five normal individuals without known exposure to mouse immunoglobulin with "preexisting" human anti-mouse antibody (HAMA) against a panel of four mouse monoclonal antibodies and polyclonal mouse IgG. Competitive inhibition by polyclonal mouse IgG of HAMA binding to monoclonal antibody coated beads demonstrates the mouse constant region specificity of these sera. Lack of such inhibition by polyclonal human IgG eliminates polyclonal rheumatoid factors as an explanation. Lymphoblastic transformation studies in these five persons failed to demonstrate a memory T-cell response to the four mouse monoclonals or polyclonal mouse IgG. Positive controls included T-cell responses to streptolysin O in these five individuals as well as responses to monoclonal antibody D612 in three patients following treatment with that antibody. This lack of T-cell immunity to mouse IgG suggests that "preexisting" HAMA is the product of inadvertent cross-reactivity with murine constant region by antibodies directed against other antigens. Therefore, "preexisting" HAMA should pose no risk of anamnestic or allergic response in patients considered for murine monoclonal therapy.

Characterization of a messenger RNA polynucleotide vaccine vector.

We have constructed mRNA transcripts encoding luciferase and human carcinoembryonic antigen (CEA) which are capped, polyadenylated, and stabilized by human beta-globin 5' and 3' untranslated regions. The mRNA construct encoding human CEA directed CEA expression in mouse fibroblasts in vitro following liposome-mediated transfection. The luciferase encoding mRNA transcripts mediated luciferase expression in vivo following i.m. injection. Based on the demonstration of protein expression in vitro and in vivo, the feasibility of using such a vector as a tumor vaccine was examined. In this pilot study, seven mice received 50 micrograms mRNA transcripts encoding CEA twice weekly for 5 weeks by i.m. injection followed by challenge with syngeneic, CEA-expressing tumor cells. This dose and schedule "primed" an immune response to CEA. Five of seven mRNA-immunized mice demonstrated anti-CEA antibody 3 weeks after tumor challenge whereas control mice had no evidence of antibody response. This strategy might be particularly useful to induce an immune response to a proto-oncogene product or growth factor which poses a risk of inducing malignant transformation consequent to prolonged protein expression.

Immune response to a carcinoembryonic antigen polynucleotide vaccine.

We have constructed a DNA plasmid encoding the full length complementary DNA for human carcinoembryonic antigen (CEA) driven by the cytomegalovirus early promoter/enhancer (plasmid DNA encoding human CEA) and demonstrated that this plasmid can function as a polynucleotide vaccine. This polynucleotide vaccine induced humoral and/or cellular immune responses specific for human CEA in all 5 immunized mice. Lymphoblastic transformation data with the use of enriched T-cell populations detected the presence of CEA-specific memory T-cells in 3 of 5 mice. Lymphocytes from 2 of 5 mice had interleukin 2/interleukin 4 release in response to CEA. CEA specificity was confirmed by the absence of reactivity to a control antigen and lack of CEA reactivity among mice vaccinated with a control plasmid encoding chloramphenicol acetyltransferase. Four of 5 mice vaccinated with plasmid DNA encoding human CEA demonstrated anti-CEA antibody responses. This immune response compared favorably with a positive control group of mice immunized with vaccinia-CEA by a dose and schedule previously shown to induce immunoprotection and therapy against a human CEA expressing syngeneic murine colon carcinoma model. Studies are ongoing to establish the construct, dose, and schedule to elicit optimal CEA-specific immune response as well as immunoprotection and therapy against human CEA expressing syngeneic murine adenocarcinoma models.

Characterization of poliovirus replicons encoding carcinoembryonic antigen.

Recombinant vaccines hold great promise for the prevention and therapy of infections diseases and cancer. We have explored the use of poliovirus as a recombinant vector to deliver genes into cells for the purpose of vaccination. For our studies, we have chosen to express the gene-encoding carcinoembryonic antigen (CEA) using a novel poliovirus vector. We have constructed a recombinant CEA-poliovirus replicon in which the CEA gene was substituted for the poliovirus capsid gene. Following in vitro transcription, the RNA was transfected into cells to demonstrate CEA expression. We found that a genome in which the region encoding the signal sequence of the CEA protein (amino acids 1-34) was removed was replication competent (i.e., referred to as a replicon). We encapsidated the CEA-poliovirus replicon by transfecting this RNA into cells previously infected with a recombinant vaccinia virus (VV-P1) which expresses the poliovirus capsid protein (P1). Serial passage in the presence of VV-P1 resulted in the generation of stocks of these encapsidated replicons. Infection of cells with the encapsidated replicon containing the CEA-poliovirus genome resulted in expression of the CEA protein. To test immunogenicity, mice susceptible to poliovirus were given three doses of the encapsidated replicons via the i.m. route. By the third administration, a CEA-specific antibody response was detected. Potential future use of the poliovirus replicon system as both a parenteral and oral vaccine vector is discussed.

Human autoantibodies to carcinoembryonic antigen (CEA) induced by a vaccinia-CEA vaccine.

Carcinoembryonic antigen (CEA) is a well-characterized oncofetal glycoprotein whose overexpression by human adenocarcinomas has been a target for cancer immunotherapy. Limited information is available regarding the ability of patients to mount an antibody response to this self-antigen following vaccination. Recombinant vaccinia viruses encoding full-length or internally deleted cDNAs for human CEA were used to vaccinate 32 patients with CEA-expressing adenocarcinomas, predominantly of colorectal origin. CEA-specific autoantibodies were induced by vaccination in 7 of 32 patients. None of the patients had CEA antibodies detected before vaccination. CEA specificity of the antibodies initially identified by ELISA was confirmed by competitive inhibition analysis as well as recognition of recombinant CEA produced in baculovirus-infected insect cell cultures and human cell cultures by Western blot. The CEA autoantibodies were predominantly IgG1, with a minority of patients also demonstrating IgM autoantibodies. CEA antibodies were of low titer and low avidity, based on competitive inhibition assays. These autoantibodies did not affect clinical serum CEA protein quantitation. Furthermore, elevated serum CEA levels commonly encountered in patients with advanced adenocarcinoma did not hinder detection of low avidity polyclonal CEA antibodies. CEA antibodies such as those induced in these pilot trials are projected to have modest antitumor activity. Thus, additional Phase I/II trials of recombinant vaccinia-CEA with alternative prime-boost approaches and/or augmentation strategies are warranted in an effort to enhance the frequency and avidity of CEA-specific autoantibodies and cytolytic T cells before Phase III trials.

Phase I trial of a recombinant vaccinia virus encoding carcinoembryonic antigen in metastatic adenocarcinoma: comparison of intradermal versus subcutaneous administration.

The principal objectives of this trial were twofold: (a) to examine the safety and relative efficacy of intradermal needle injection versus s.c. jet administration of a carcinoembryonic antigen (CEA)-encoding recombinant vaccinia virus (rV-CEA) over a limited dose range and (b) to evaluate CEA-specific immune responses or antitumor effects induced by rV-CEA vaccination. Patients were randomly assigned to one of two groups, depending upon the technique of vaccine administration. All 20 patients received two doses of 10(7) or 10(8) pfu of rV-CEA at a 4-week interval. Toxicity was limited to modest local inflammation at the inoculation site as well as low-grade fever and increased fatigue, each affecting fewer than 20% of the patients. No evidence of CEA-specific lymphoproliferation, interleukin 2 release, delayed-type hypersensitivity, or antibody response was observed. Thus, the efficacy comparison between the two administration techniques was based upon the induction of immune responses to the vaccinia virus vector. Both techniques induced vaccinia-specific lymphoproliferation, interleukin 2 release, and antibody responses of comparable magnitude and frequency as well as protected 80% of patients against pustule formation following vaccinia scarification. Thus, there is no compelling reason to recommend one administration technique over the other based upon toxicity or efficacy. We have selected s.c. jet injection for subsequent trials of rV-CEA based on the ability to accommodate larger injection volumes, enhanced standardization between clinicians, and avoidance of needles that could transmit the vaccine or blood-borne pathogens to health care workers. We recommend use of 10(8) pfu doses for subsequent trials of recombinant vaccinia virus vaccines based upon the favorable toxicity profile and more consistent local pustule formation indicative of an adequate inoculation of live virus. No objective clinical responses to the rV-CEA vaccine were observed among this population of patients with widely metastatic adenocarcinoma.

Polynucleotide immunization of nonhuman primates against carcinoembryonic antigen.

In preparation for a Phase I trial of DNA immunization against carcinoembryonic antigen (CEA) in patients with colorectal carcinoma, we have produced a single plasmid DNA encoding CEA and hepatitis B surface antigen (HBsAg) under transcriptional regulatory control of two separate cytomegalovirus promoters within separate eukaryotic expression cassettes, designated pCEA/HBsAg. Hepatitis B surface antigen was included to provide an internal positive control for the efficacy of this immunization strategy without regard to the issue of breaking tolerance to a self-antigen. In the present work, we sought to examine the immunogenicity of this plasmid in a nonhuman primate model with close phylogenetic relationship to humans. Groups of pig-tailed macaques were immunized with pCEA/ HBsAg by i.m. injection or particle bombardment of the skin according to a dose and schedule thought to be optimal for the respective technique of DNA immunization. Both administration techniques produced humoral and lympho-proliferative responses of comparable magnitude. However, delayed type hypersensitivity to CEA and CEA-specific interleukin-2 release were observed only in the i.m. group, suggesting a qualitative difference in the character of the immune response elicited by the two techniques of DNA immunization. The antibody responses to CEA and HBsAg were surprisingly persistent in that all immunized animals maintained moderate antibody titers against both antigens for more than 15 months after the last boost. No toxicity was observed during 2 years of follow-up, including no measurable levels of anti-DNA antibody. This antitumor immunization strategy is presently being examined in patients with metastatic colorectal carcinoma using pCEA/HBsAg administered by i.m. injection.

Optimization of methods to achieve mRNA-mediated transfection of tumor cells in vitro and in vivo employing cationic liposome vectors.

Direct in vivo transfection of tumor nodules in situ via liposome-DNA complexes has been employed as a strategy to accomplish antitumor immunization. To circumvent the potential safety hazards associated with systemic localization of delivered DNA, the utility of mRNA transcript-mediated gene delivery was explored. Capped, polyadenylated mRNA transcripts encoding the firefly luciferase and Escherichia coli lacZ reporter genes were derived by in vitro transcription. Transfection of the human breast cancer cell line MDA-MB-435 in vitro was accomplished employing cationic liposome-mRNA complexes. Evaluation of a panel of cationic liposome preparations demonstrated significant differences in the capacity of the various preparations to accomplish mRNA-mediated transfection. Quantitative evaluation of in vitro transfection demonstrated that target cells could be transfected at a high level of efficiency. The mRNA liposome-complexes were evaluated for in vivo transfection of tumor nodules in human xenografts in athymic nude mice. It could be demonstrated the liposome-mRNA complexes were comparable in efficacy to liposome-DNA complexes in accomplishing in situ tumor transfection. Thus, mRNA may be considered as an alternative to plasmid DNA as a gene transfer vector for genetic immunopotentiation applications.

A carcinoembryonic antigen polynucleotide vaccine for human clinical use.

We have constructed a plasmid DNA encoding the full-length complementary DNA for human carcinoembryonic antigen (CEA) under transcriptional regulatory control of the cytomegalovirus early promoter/enhancer (pCEA) and demonstrated that this plasmid can function as a polynucleotide vaccine to elicit a CEA-specific immune response. This immune response protects against tumor challenge with syngeneic CEA-transduced colon carcinoma cells in mice. In the present work, the pCEA construct and purification method were modified to eliminate nonessential viral sequences, the ampicillin selectable marker, mutagens, and endotoxin to produce a reagent suitable for human clinical trials. The human use plasmid (pGT37) directs CEA expression at levels comparable with the original pCEA plasmid and can be propagated to yield large quantities of plasmid DNA based on kanamycin selection. A simple extraction technique greatly reduces contamination by endotoxin. Six weekly intramuscular injections of pGT37 elicited CEA-specific lymphoblastic transformation and antibody response in five of five mice and fully protected 10 of 10 mice against tumor challenge with syngeneic CEA-expressing colon cancer cells 42 days from the first plasmid injection. Thus, pGT37 encoding a tumor-associated antigen (CEA) has been shown to elicit cellular and humoral immune responses and mediate antitumor effects in vivo. This plasmid is suitable for human use and can be easily propagated in the laboratory.

SEREX analysis for tumor antigen identification in a mouse model of adenocarcinoma.

Evaluation of immunotherapy strategies in mouse models of carcinoma is hampered by the limited number of known murine tumor antigens (Ags). Although tumor Ags can be identified based on cytotoxic T-cell activation, this approach is not readily accomplished for many tumor types. We applied an alternative strategy based on a humoral immune response, SEREX, to the identification of tumor Ags in the murine colon adenocarcinoma cell line MC38. Immunization of syngeneic C57BL/6 mice with MC38 cells by three different methods induced a protective immune response with concomitant production of anti-MC38 antibodies. Immunoscreening of an MC38-derived expression library resulted in the identification of the endogenous ecotropic leukemia virus envelope (env) protein and the murine ATRX protein as candidate tumor Ags. Northern blot analysis demonstrated high levels of expression of the env transcript in MC38 cells and in several other murine tumor cell lines, whereas expression in normal colonic epithelium was absent. ATRX was found to be variably expressed in tumor cell lines and in normal tissue. Further analysis of the expressed env sequence indicated that it represents a nonmutated tumor Ag. Polynucleotide immunization with DNA encoding the env polypeptide resulted in strong and specific antibody responses to this self Ag in all immunized mice. Thus, SEREX offers a rapid means of identifying tumor Ags in murine cancer models.

Polynucleotide-mediated immunization therapy of cancer.

The novel observations that intramuscular injection of plasmid DNA preparations could result in myocyte gene expression and induce immune responses to encoded immunogens has generated intense interest in the form of gene therapy. This phenomena can occur with both DNA and RNA reagents, and can be used in immune protection (vaccine) or therapy strategies. Immunization with DNA plasmids has generated protective immunity to a wide variety of pathogens and tumor cells in murine animal models. Immune response has occurred in a broad range of animal species following intramuscular injection of plasmid DNA encoding various immunogens as well as following other routes of administration (intravenous, intradermal, etc). The mechanisms responsible for induction of the immune response are as yet unclear, but responses include antibody production, T-cell proliferation, lymphokine release, generation of cytolytic T cells, and delayed hypersensitivity reactions. Plasmid DNA production and purification methods are relatively easy to standardize, and dual expressing plasmids allow incorporation of immune enhancement molecules or second immunogens. Plasmid DNA encoding nontransforming tumor-associated antigens are in development with a National Institutes of Health-approved protocol for carcinoembryonic antigen in colorectal cancer patients. Transforming tumor-associated antigens (eg, HER2/neu) may be approached with RNA or replicative RNA constructs for immunization. The efficacy of this immune approach will soon be examined in clinical trials in patients with cancer and the acquired immunodeficiency syndrome.

Human immune response to monoclonal antibodies.

Monoclonal antibodies either of mouse, mouse/human chimeric, or human origin have been used safely in human trials for a decade. Considerable effort has been committed in investigations of manipulating endogenous immunological activity against tumors and targeting various cytotoxic agents to cancers. These studies have identified several problems that need to be resolved before any such reagents can be used routinely in patients. One of these problems has been the immunogenicity of these monoclonal antibodies. This review article discusses what is known regarding human immune response to monoclonal antibodies and their clinical consequences.

Development of a polynucleotide vaccine from melanoma antigen recognized by T cells-1 and recombinant protein from melanoma antigen recognized by T cells-1 for melanoma vaccine clinical trials.

MART-1, a melanoma antigen recognized by T cells-1, is a melanocyte lineage-differentiation antigen expressed only in melanocytes and melanoma cells. This protein is recognized by many T-lymphocyte lines that are human leukocyte antigen (HLA)-A2 restricted and melanoma reactive. These observations have culminated in an array of clinical trials of MART-1 immunization using recombinant viruses or MART-1 immunodominant peptides. Polynucleotide immunization is a promising alternative to recombinant viral vaccines that allows delivery of the full-length cDNA encoding all potential peptide epitopes in a vector that is uncompromised by anti-viral immunity. In preparation for a phase I clinical trial of MART-1 polynucleotide immunization in patients with resected melanoma who were at significant risk for recurrence, the authors constructed a plasmid DNA encoding the MART-1 cDNA under transcriptional regulatory control of the cytomegalovirus immediate early promoter-enhancer and partially deleted intron A. This plasmid directs high-level MART-1 expression in transduced myoblasts and maturing myocytes diffusely throughout the cytoplasm. Immunization of mice with this construct by intramuscular injection elicited MART-1-specific immune responses in all animals. Previous trials of MART-1 immunization have been unable to examine the humoral immune response to MART-1 because of a lack of sufficient, highly purified protein. We have produced and purified Escherichia coli recombinant MART-1 protein using a glutathione-S-transferase fusion protein expression system. Protein staining of a sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a band of MART-1 protein at approximately 20 kD; and Western immunoblotting with an anti-MART-1 monoclonal antibody confirmed a doublet at approximately 20 kD. These findings are consistent with previous reports using different expression systems for recombinant MART-1. This protein preparation functioned well in enzyme-linked immunosorbent assays (ELISAs) to detect anti-MART-1 antibody responses in a mouse model; and a panel of healthy donor human sera showed minimal binding to ELISA plates coated with the protein, supporting its utility in monitoring human anti-MART-1 antibody responses. The glutathione-S-transferase fusion method yielded approximately 200 micrograms MART-1 per 2-L bacterial culture, enough to coat 100 ELISA plates.

Humoral and cellular immune responses of dogs immunized with a nucleic acid vaccine encoding human carcinoembryonic antigen.

A nucleic acid vaccine encoding human carcinoembryonic antigen (CEA) was administered to 10 juvenile dogs, 10-15 weeks of age, by four parenteral routes. The routes tested were intramuscular injection using a needle and syringe, intramuscular injection using the Biojector needleless injection device, intradermal injection or intravenous injection. All groups received 150 micrograms of plasmid DNA on weeks 0, 4, 7 and 13. All dogs were bled weekly for 17 weeks and tested for antibody against human CEA. Dogs given plasmid intramuscularly either by needle and syringe or Biojector showed significant antibody responses by week 9 which peaked by week 15. Dogs receiving plasmid intravenously showed slight, unsustained increases in antibody titers while dogs receiving plasmid intradermally had significant titers, but at levels approximately one log less than those induced by intramuscular injection. The five dogs immunized by intramuscular delivery of plasmid DNA were examined for cellular immune responses to human CEA by lymphoblast transformation (LBT) assay. All five showed significant CEA-specific lymphoproliferation when compared with unvaccinated dogs. Physical examination, clinical chemistry, hematology and histopathology examinations revealed no abnormalities associated with nucleic acid immunization.

Intracellular single-chain antibody directed against erbB2 down-regulates cell surface erbB2 and exhibits a selective anti-proliferative effect in erbB2 overexpressing cancer cell lines.

Overexpression of the tyrosine kinase receptor erbB2 is important in the pathogenesis of a variety of neoplasms. Based on this concept, targeted anti-cancer strategies have been designed to selectively eradicate erbB2 overexpressing tumor cells. These strategies have employed either monoclonal antibodies or antibody toxin molecules with specificity for the cell surface erbB2 protein. As an alternative strategy, anti-erbB2 single-chain immunoglobulin (sFv) genes were constructed to direct expression of intracellular anti-erbB2 antibodies. Expression of an endoplasmic reticulum (ER) form of the anti-erbB2 sFv resulted in a profound down-regulation of cell surface erbB2 in an erbB2 overexpressing ovarian carcinoma cell line. In addition, expression of the intracellular antibody resulted in marked inhibition of tumor cell proliferation. Whereas stable transfectants expressing the anti-erbB2 sFv could be dervied from non-erbB2 overexpressing cancer cell lines, expression of the intracellular antibody was incompatible with long-term survival of the erbB2 overpressing tumor cells. The ability to selectively 'knock-out' erbB2 demonstrates that cell surface localization of erbB2 is essential to its ability to induce aberrant cellular proliferation in tumor cells.(ABSTRACT TRUNCATED AT 250 WORDS)

An animal model to predict the immunogenicity of murine V regions in humans.

Clinical trials with genetically engineered chimeric mouse/human monoclonal antibodies have demonstrated that mouse variable regions differ dramatically in their degree of immunogenicity. These observations led us to search for an animal model that could predict mouse variable region (V region) immunogenicity prior to human clinical trials. We selected monoclonal antibodies 17-1A and B72.3 for study because human trials have demonstrated the very low immunogenicity of the mouse 17-1A V region and the high immunogenicity of the mouse B72.3 V region. Random-bred New Zealand white rabbits were injected intravenously with mouse 17-1A, B72.3, or both using a dose and schedule comparable to human trials. After initial injection only two of ten rabbits developed an antibody response to mouse 17-1A, while all five animals receiving a second injection developed antibody that was entirely mouse constant region-specific. On the other hand, nine of ten rabbits demonstrated an antibody response to initial infusion of mouse B72.3 that was greater than 90% specific for the complementarity-determining region components of the V regions. Competitive inhibition with isolated heavy and light chains demonstrated specificity for heavy or light chains (approximately 60%) or a requirement for both (40%). Thus, as in humans, the V region of 17-1A elicited little or no immune response in rabbits, while the V region of B72.3 was highly immunogenic.(ABSTRACT TRUNCATED AT 250 WORDS)

Incorporation of beta-globin untranslated regions into a Sindbis virus vector for augmentation of heterologous mRNA expression.

Polynucleotide immunization has been employed as a means of inducing immune responses through the introduction of antigen-encoding DNA. While immunization against specific tumor antigens may be achieved through this strategy, various candidate tumor antigens may not be approached via DNA-based vaccines as they represent transforming oncogenes. As an alternative approach, we have explored the utility of mRNA vectors for polynucleotide immunization. The transient expression achieved by mRNA may provide an efficient and safe system for stimulating immune responses to tumor-specific antigens. Our previous work demonstrated that a self-replicating RNA enhances the magnitude and duration of transgene expression for this application. Here we further modify the vector for optimal use in gene therapy through the incorporation of untranslated regions flanking the encoded transgene. The beta-globin 5' and 3' untranslated regions (UTRs) were inserted directly flanking the luciferase gene in both nonreplicative and replicative RNA constructs. In both cases, elevated and prolonged levels of luciferase expression were detected from the beta-globin UTR-flanked luciferase as compared to luciferase without these sequences. These modifications improve the ability of replicative RNA vectors to produce high, yet transient transgene expression for cancer immunotherapy strategies.

Immunization with a plasmid expressing pneumococcal surface protein A (PspA) can elicit protection against fatal infection with Streptococcus pneumoniae.

Pneumococcal surface protein A (PspA) is a protectioneliciting protein of Streptococcus pneumoniae. We observed that immunization of BALB/c mice with a plasmid expressing PspA significantly protected the mice from lethal challenge with S. pneumoniae when compared to control mice that received injections of the plasmid vector alone. The plasmid construct expressing PspA has been designated pKSD2601. Mice immunized intramuscularly with pKSD2601 had a mean log of colony-forming units of 2.67 +/- 0.25 pneumococci circulating in their blood at 24 h after challenge as compared with control mice that had a mean log of colony-forming units of 4.95 +/- 0.59. Those mice with lower numbers of pneumococci subsequently survived the challenge. Given the quantitative nature and ultimate end point (ie live versus dead) our mouse model should be useful in working out optimum expression of bacterial genes for DNA immunization.