A helper-dependent capsid-modified adenovirus vector expressing adeno-associated virus rep78 mediates site-specific integration of a 27-kilobase transgene cassette

Random integration of viral gene therapy vectors and subsequent activation or disruption of cellular genes poses safety risks. Major efforts in the field are aimed toward targeting vector integration to specific sites in the host genome. The adeno-associated virus (AAV) Rep78 protein is able to target AAV integration to a specific site on human chromosome 19, called AAVS1. We studied whether this ability could be harnessed to achieve site-specific integration of a 27-kb transgene cassette into a model cell line for human hematopoietic cells (Mo7e). To deliver rep78 and the transgene to Mo7e cells, we used helper-dependent adenovirus (Ad) vectors containing Ad serotype 35 fiber knob domains (HD-Ad). An HD-Ad vector containing the rep78 gene under the control of the globin locus control region (LCR) (Ad.LCR-rep78) conferred Rep78 expression on Mo7e cells. Upon coinfection of Ad.LCR-rep78 with an HD-Ad vector containing a 27-kb globin-LCR-green fluorescent protein (GFP) transgene cassette flanked by AAV inverted terminal repeats (ITRs) (Ad.AAV-LCR-GFP), transduced cells were cloned and expanded (without selection pressure), and vector integration was analyzed in clones with more than 30% GFP-positive cells. Vector integration into the AAVS1 region was seen in 30% of analyzed integration sites, and GFP expression from these integrants was stable over time. Of the remaining integration sites, 25% were within the genomic globin LCR. In almost 90% of sites, transgene integration occurred via the Ad ITR. This indicates that rescue of the AAV ITR-flanked transgene cassette from Ad.AAV-LCR-GFP is not required for Rep78-mediated integration into AAVS1 and that free ends within the vector genome can be created by breaks within the Ad ITRs, whose structure is apparently recognized by cellular "nicking" enzymes. The finding that 55% of all analyzed integration sites were either within the AAVS1 or globin LCR region demonstrates that a high frequency of targeted integration of a large transgene cassette can be achieved in human hematopoietic stem cell lines.     

Pyocin sensitivity of pseudomonas aeruginosa pretreated with antibiotics.

Strains of Pseudomonas aeruginosa exposed to subinhibitory concentrations of either polymyxin B, gentamicin, or carbenicillin were compared with untreated organisms for changes in sensitivity to pyocin. Pretreatment with polymyxin B or gentamicin resulted in a decreased sensitivity to pyocin, while carbenicllin pretreatment did not alter pyocin sensitivity.     

Enhanced therapeutic anti-tumor immunity induced by co-administration of 5-fluorouracil and adenovirus expressing CD40 ligand

Bystander immune activation by chemotherapy has recently gained extensive interest and provided support for the clinical use of chemotherapeutic agents in combination with immune enhancers. The CD40 ligand (CD40L; CD154) is a potent regulator of the anti-tumor immune response and recombinant adenovirus (RAd)-mediated CD40L gene therapy has been effective in various cancer models and in man. In this study we have assessed the combined effect of local RAd-CD40L and 5-fluorouracil (5-FU) administration on a syngeneic MB49 mouse bladder tumor model. Whereas MB49 cells implanted into immunocompetent mice responded poorly to RAd-CD40L or 5-FU alone, administration of both agents dramatically decreased tumor growth, increased survival of the mice and induced systemic MB49-specific immunity. This combination treatment was ineffective in athymic nude mice, highlighting an important role for T cell mediated anti-tumor immunity for full efficacy. 5-FU up-regulated the expression of Fas and immunogenic cell death markers in MB49 cells and cytotoxic T lymphocytes from mice receiving RAd-CD40L immunotherapy efficiently lysed 5-FU treated MB49 cells in a Fas ligand-dependent manner. Furthermore, local RAd-CD40L and 5-FU administration induced a shift of myeloid-derived suppressor cell phenotype into a less suppressive population. Collectively, these data suggest that RAd-CD40L gene therapy is a promising adjuvant treatment to 5-FU for the management of bladder cancer.     

Protective immunity induced by systemic and mucosal delivery of DNA vaccine expressing glycoprotein B of pseudorabies virus

A murine model immunized by systemic and mucosal delivery of plasmid DNA vaccine expressing glycoprotein B (pCIgB) of pseudorabies virus (PrV) was used to evaluate both the nature of the induced immunity and protection against a virulent virus. With regard to systemic delivery, the intramuscular (i.m.) immunization with pCIgB induced strong PrV-specific IgG responses in serum but was inefficient in generating a mucosal IgA response. Mucosal delivery through intranasal (i.n.) immunization of pCIgB induced both systemic and mucosal immunity at the distal mucosal site. However, the levels of systemic immunity induced by i.n. immunization were less than those induced by i.m. immunization. Moreover, i.n. genetic transfer of pCIgB appeared to induce Th2-biased immunity compared with systemic delivery, as judged by the ratio of PrV-specific IgG isotypes and Th1- and Th2-type cytokines produced by stimulated T cells. Moreover, the immunity induced by i.n. immunization did not provide effective protection against i.n. challenge of a virulent PrV strain, whereas i.m. immunization produced resistance to viral infection. Therefore, although i.n. immunization was a useful route for inducing mucosal immunity at the virus entry site, i.n. immunization did not provide effective protection against the lethal infection of PrV.     

Protective immunity induced by a LLO‐deficient Listeria monocytogenes

Listeria monocytogenes is a food‐borne pathogen able to cause serious disease in human and animals. Listeriolysin O (LLO), a major virulence factor secreted by this bacterium, is a vacuole‐specific lysin that facilitates bacterial entrance into the host cytosol. Thus, LLO plays a key role in the translocation and intracellular spread of L. monocytogenes. To study the effect of LLO on virulence and immunopotency, a LLO‐deficient L. monocytogenes mutant was constructed using a shuttle vector followed by homologous recombination. The mutant strain had lost hemolytic activity, which resulted in an extremely reduced virulence, 5 logs lower than that of the parent strain, yzuLM4, in BALB/c mice. The number of bacteria detected in the spleens and livers of mice infected with the mutant was greatly reduced, and the bacteria were rapidly eliminated by the host. Kinetics studies in this murine model of infection showed that the invasion ability of the mutant strain was much lower than that of the parent strain. Moreover, immunization with the mutant strain conferred protective immunity against listerial infection. In particular, stimulation with Ag85B_240‐259, strong specific Th1 type cellular immunity was elicited by vaccination C57BL/6 mice with hly deficient strain delivering Mycobacterium tuberculosis fusion antigen Ag85B‐ESAT‐6 via intravenous inoculation. These results clearly show that highly attenuated LLO‐deficient L. monocytogenes is an attractive vaccine carrier for delivering heterologous antigens.     

Protective cellular immunity against P. falciparum malaria merozoites is associated with a different P7 and P8 residue orientation in the MHC-peptide-TCR complex

Developing a logical and rational methodology for obtaining vaccines, especially against the main parasite causing human malaria (P. falciparum), consists of blocking receptor-ligand interactions. Conserved peptides derived from proteins involved in invasion and having high red blood cell binding ability have thus been identified. Immunization studies using Aotus monkeys have revealed that these peptides were neither immunogenic nor protection inducing. When modified in their critical binding residues, previously identified by Glycine scanning, some of these peptides were immunogenic and non-protection inducers; others induced short-lived antibodies whilst a few were both immunogenic and protection inducing. However, very few of these modified high activity binding peptides (HABPs) reproducibly induced protection without inducing antibody production, but with high cytokine liberation, suggesting that cellular mechanisms had been activated in the protection process. The three-dimensional structure of these peptides inducing protection without producing antibodies was determined by 1H-NMR. Their HLA-DRbeta1* molecule binding ability was also determined to ascertain association between their 3D structure and ability to bind to Major Histocompatibility Complex Class-II molecules (MHC-II). 1H Nuclear Magnetic Resonance analysis and structure calculations clearly showed that these modified HABPs inducing protective cellular immune responses (but not producing antibodies against malaria) adopted special structural configuration to fit into the MHC II-peptide-TCR complex. A different orientation for P7 and P8 TCR contacting residues was clearly recognized when comparing their structure with modified peptides, which induced high antibody titers and protection, suggesting that these residues are involved in activating the immune system associated with antibody production and protection.     

Cell-mediated immunity to mouse adenovirus infection. Macrophage migration inhibition test.

Cell-mediated immunity (CMI) to mouse adenovirus (M-Ad) infection was studied by macrophage migration inhibition test (MMI) as one of in vitro correlates of CMI. Both direct and indirect tests showed clearly that migration of packed peritoneal exudate cells (PEC) (immune mouse or nonimmune guinea pig) was remarkably inhibited; MIF was produced by interactions between immune PEC and infected cell extracts and between immune spleen cells and infected cells or their extracts. The antigen(s) responsible for the above MMI was demonstrated in 6- to 12-hour infected ME cells, and FUdR-treated infected ME cells. Since under these conditions there is S antigen(s) synthesis but not capsid antigen synthesis, the antigen(s) concerned must be an S antigen(s). T cells sensitized to infected cells were shown to be required to induce MMI. The MMI is specific for M-Ad, since no cross MMI was observed between M-Ad and SV40 systems. Time course study of the development of CMI to M-Ad by MMI tests showed that CMI became detectable 4 days post-infection (pi), reached its peak level about 10 days pi, and faded away rapidly in about 10 days thereafter.     

Humoral immunity induced in the lower respiratory tract by local immunization with a temperature-sensitive mutant ofPseudomonas aeruginosa

The nature of the humoral immune mechanisms involved in the protection induced after local immunization with a temperature-sensitive (ts) mutant ofPseudomonas aeruginosa was investigated. We had previously shown that intranasal (i.n.) immunization of granulocytopenic mice protected the animals from lethal pulmonary challenge withP. aeruginosa, whereas mice immunized intraperitoneally were unprotected. Intranasal immunization induced high levels of anti-P. aeruginosa IgG and IgA in the lower respiratory tract, whereas only modest levels of IgG (and no IgA) could be detected in lung lavage fluids from mice immunized by the intraperitoneal (i.p.) route with ts mutant E/9/9. Plasma anti-P. aeruginosa IgG levels after i.n. immunization were lower than those observed after i.p. immunization with similar doses of the ts mutant. The main contribution to the protection induced when mice are immunized intranasally appears to be from IgA in the pulmonary secretions, although other immune mechanisms cannot be discounted.     

Protective action of a vaccine made from Salmonella minnesota R 595 in the intranasal infection of mice with P. aeruginosa

The comparative study of heated corpuscular vaccines prepared from S. minnesota mutant R 595, chemotype Re, from S. minnesota strain SF 1111 with defective lipopolysaccharide and from P. aeruginosa strain PA 103 has been carried out. The vaccine prepared from the chemotype Re mutant, in contrast to the vaccine prepared from S. minnesota strain SF 1111, has been found to induce the development of active immunity (and the corresponding antiserum, passive immunity) to P. aeruginosa introduced intranasally into mice, as well as to stimulate the elimination of the cells of P. aeruginosa infective strain from the lungs of the mice. The potency of the vaccine prepared from the chemotype Re mutant has been found to be significantly no different from that of the vaccine prepared from P. aeruginosa strain PA 103.     

Anti-tumor immune responses following neoadjuvant immunotherapy with a recombinant adenovirus expressing HSP72 to rodent tumors

Gene modification of tumor cells is commonly utilized in various strategies of immunotherapy preventive both as treatment and a means to modify tumor growth. Gene transfer prior to surgery as neoadjuvant therapy has not been studied systematically. We addressed, whether direct intra-tumoral injection of a recombinant adenovirus expressing the immunomodulatory molecule, heat shock protein 72 (ADHSP72), administered prior to surgery could result in sustainable anti-tumor immune responses capable of affecting tumor progression and survival in a number of different murine and rat tumor models. Using intra-dermal murine models of melanoma (B16), colorectal carcinoma (CT26), prostate cancer (TrampC2) and a rat model of glioblastoma (9L), tumors were treated with vehicle or GFP expressing adenovirus (ADGFP) or ADHSP72. Tumors were surgically excised after 72 h. Approximately 25–50% of animals in the ADHSP72 treatment group but not in control groups showed sustained resistance to subsequent tumor challenge. Tumor resistance was associated with development of anti-tumor cellular immune responses. Efficacy of ADHSP72 as neoadjuvant therapy was dependent on the size of the initial tumor with greater likelihood of immune response generation and tumor resistance associated with smaller tumor size at initial treatment. ADHSP72 neoadjuvant therapy resulted in prolonged survival of animals upon re-challenge with autologous tumor cells compared to ADGFP or vehicle control groups. To study the effects on tumor progression of distant metastases, a single tumor focus of animals with multifocal intra-dermal tumors was treated. ADHSP72 diminished progression of the secondary tumor focus and prolonged survival, but only when the secondary tumor focus was <50 mm³ . Our results indicate that gene modification of tumors prior to surgical intervention may be beneficial to prevent recurrence in specific circumstances.     

Construction and characterization of a replication-deficient adenovirus expressing rat-soluble interleukin-6 receptor.

BACKGROUND: The pleiotropic cytokine interleukin-6 mediates its multiple effects at the cell level through a multimeric receptor consisting of a binding protein (gp80) and a signal transducer (gp130). A soluble form of gp80 (sIL-6R or gp55) is found released from the surface of cells and appears to possess interleukin-6 (IL-6) agonist activity. Increases in circulating levels of sIL-6R have been reported in different pathological conditions but the precise role of this protein in vivo remains unknown. MATERIALS AND METHODS: The cDNA encoding the extracellular domain of the rat IL-6R (sIL-6R) with an appropriate leader sequence has been cloned into the E1 region of an adenovirus vector under the control of the hCMV promoter (Ad5.sIL-6R). RESULTS: Infection of different human or rodent cell lines with Ad5.sIL-6R leads to extended production of recombinant sIL-6R protein into the culture media. The kinetics of transgene expression depends both on the cell type and the species. sIL-6R produced in this manner is biologically active as it confers responsiveness of human hepatoma cells (HepG2) to rat IL-6 stimulation. Adenovirus vectors have been shown to be highly effective for transient delivery of cytokines in vivo. Antibodies against recombinant rat soluble IL-6R were generated and an ELISA developed that allowed us to quantify sIL-6R concentrations. The sIL-6R expressing adenovirus vector has been instilled intratracheally into rats and induced an increase in lung sIL-6R concentration from Day 1 up to Day 10. We demonstrate the potency of our system to deliver in vivo or in vitro soluble cytokine receptors in a prolonged but transient manner.     

Protective immunity against Taenia crassiceps murine cysticercosis induced by DNA vaccination with a Taenia saginata tegument antigen

This study investigated the protective capacity of the recombinant Taenia saginata Tso18 antigen administered as a DNA vaccine in the Taenia crassiceps murine model of cysticercosis. This Tso18 DNA sequence, isolated from a T. saginata oncosphere cDNA library, has homologies with Taenia solium and Echinococcus sp. It was cloned in the pcDNA3.1 plasmid and injected once intramuscularly into mice. Compared to saline-vaccinated control mice, immunization reduced the parasite burden by 57.3-81.4%, while lower levels of non-specific protection were induced in control mice injected with the plasmid pcDNA3.1 (18.8-33.1%) or a plasmid with irrelevant construct, pcDNA3.1/3D15 (33.4-38.8%). Importantly, significant levels of protection were observed between the pcDNA3.1/Tso18 plasmid and pcDNA3.1/3D15 plasmid immunized mice. Mice immunized with pTso18 synthesized low levels of, primarily IgG1 sub-class, antibodies. These antibodies were shown to recognize a 66 kDa antigen fraction of T. crassiceps and T. solium. Splenocytes enriched in both CD4+CD8- and CD4-CD8+ T cells from these vaccinated mice proliferated in vitro when exposed to antigens from both T. solium and T. crassiceps cestodes. Immunolocalization studies revealed the Tso18 antigen in oncospheres of T. saginata and T. solium, in the adult tapeworm and in the tegument of T. solium cysticerci. The protective capacity of this antigen and its extensive distribution in different stages, species and genera of cestodes points to the potential of Tso18 antigen for the possible design of a vaccine against cestodes.     

Protective immunity to progressive tumors can be induced by antigen presented on regressor tumors

Immunization of animals with 1591-RE tumor cells, a highly immunogenic UV-induced epithelia cell tumor from C3H/HeN mice, that were haptenated with trinitrophenol (TNP) leads to protective immunity against a challenge of TNP-haptenated 3152-PRO tumor cells, a progressive highly malignant. MCA-induced fibrosarcoma from syngeneic mice. Animals that rejected TNP-1591-RE and subsequently TNP-3152-PRO tumor cells showed increased tumor-specific resistance to another challenge of 3152-PRO tumor cells, even when these fibrosarcoma cells had not been haptenated with TNP. Induction of protection required the presence of TNP-hapten groups on both 1591-RE and 3152-PRO during the initial immunization, and could be induced by immunization with other haptenated syngeneic highly immunogenic regressor tumor lines. In addition, TNP-haptenated progressor variants of the 1591-RE were ineffective in generating protection, suggesting that the immunogenicity of the haptenated tumor used for the initial immunization was a determining factor in whether or not protective immunity against the highly malignant tumor was later generated. Protection required at least two T cell types: a Lyt-1-2+ T cells, and a Lyt-1+2- T cell that also expressed I-J determinants and was Vicia villosa lectin adherent, suggesting it was not a classical helper T cell. These results suggest that presentation of a hapten by highly immunogenic tumor cells can lead to enhanced protective immunity to poorly immunogenic noncross-reactive tumors that co-express the same hapten, and rejection of these haptenated poorly immunogenic tumors leads to enhanced protection against a subsequent challenge of the same, but not noncross-reactive progressor tumors.     

Protective immunity to UV radiation-induced skin tumours induced by skin grafts and epidermal cells

There is little evidence that cutaneous dendritic cells (DC), including epidermal Langerhans cells (LC), can induce immunity to UV radiation (UVR)-induced skin tumours. Here, it is shown that cells within skin can induce protective antitumour immunity against a UVR-induced fibrosarcoma. Transplantation of the skin overlying subcutaneous tumours onto na?ve recipients could induce protective antitumour immunity, probably because the grafting stimulated the tumour Ag-loaded DC to migrate to local lymph nodes. This suggests that cutaneous APC can present tumour Ag to induce protective antitumour immunity. Previously, it has been shown that immunization of mice with MHC class II+ epidermal cells (EC) pulsed with tumour extracts could induce delayed-type hypersensitivity against tumour cells. Here, this same immunization protocol could induce protective immunity against a minimum tumorigenic dose of UVR-induced fibrosarcoma cells, but not higher doses. Epidermal cells obtained from semiallogeneic donors and pulsed with tumour extract could also induce protective immunity. However, presentation of BSA Ag from the culture medium was found to contribute to this result using semiallogeneic EC. The results suggest that LC overlying skin tumours may be able to induce protective immunity to UVR-induced tumours if stimulated to migrate from the skin.     

Protective effect of a prime-boost strategy with plasmid DNA followed by recombinant adenovirus expressing TgAMA1 as vaccines against Toxoplasma gondii infection in mice

A heterologous prime-boost strategy with priming plasmid DNA followed by recombinant virus expressing relevant antigens is known to stimulate protective immunity against intracellular parasites. In this study, we have evaluated a heterologous prime-boost strategy for immunizing mice against Toxoplasma gondii infection. Our results revealed that the prime-boost strategy using both plasmid DNA and adenoviral vector encoding TgAMA1 may stimulate both humoral and Th1/Th2 cellular immune responses specific for TgAMA1. Moreover, C57BL/6 mice immunized with the pAMA1/Ad5Null, pNull/Ad5AMA1, and pAMA1/Ad5AMA1 constructs showed survival rates of 12.5%, 37.5%, and 50%, respectively. In contrast, all the pNull/Ad5Null immunized mice died after infection with the PLK-GFP strain of T. gondii. Brain cyst burden was reduced by 23% in mice immunized with pAMA1/Ad5AMA1 compared with the pNull/Ad5AMA1 immunized mice. These results demonstrate that the heterologous DNA priming and recombinant adenovirus boost strategy may provide protective immunity against T. gondii infection.     

Protective antitumor immunity induced by a costimulatory thalidomide analog in conjunction with whole tumor cell vaccination is mediated by increased Th1-type immunity

Thalidomide and its novel T cell costimulatory analogs (immunomodulatory drugs) are currently being assessed in the treatment of patients with advanced cancer. However, neither tumor-specific T cell costimulation nor effective antitumor activity has been demonstrated in vivo. In this study, we assessed the ability of an immunomodulatory drug (CC-4047/ACTIMID) to prime a tumor-specific immune response following tumor cell vaccination. We found that the presence of CC-4047 during the priming phase strongly enhanced antitumor immunity in the vaccinated group, and this correlated with protection from subsequent live tumor challenge. Protection was associated with tumor-specific production of IFN-gamma and was still observed following a second challenge with live tumor cells 60 days later. Furthermore, CD8(+) and CD4(+) splenocyte fractions from treated groups secreted increased IFN-gamma and IL-2 in response to tumor cells in vitro. Coculture of naive splenocytes with anti-CD3 mAb in the presence of CC-4047 directly costimulated T cells and increased Th1-type cytokines. Our results are the first to demonstrate that a costimulatory thalidomide analog can prime protective, long-lasting, tumor-specific, Th1-type responses in vivo and further support their ongoing clinical development as novel anti-cancer agents.     

A booster vaccine expressing a latency-associated antigen augments BCG induced immunity and confers enhanced protection against tuberculosis

Background

In spite of a consistent protection against tuberculosis (TB) in children, Mycobacterium bovis Bacille Calmette-Guerin (BCG) fails to provide adequate protection against the disease in adults as well as against reactivation of latent infections or exogenous reinfections. It has been speculated that failure to generate adequate memory T cell response, elicitation of inadequate immune response against latency-associated antigens and inability to impart long-term immunity against M. tuberculosis infections are some of the key factors responsible for the limited efficiency of BCG in controlling TB.

Methods/Principal Findings

In this study, we evaluated the ability of a DNA vaccine expressing α-crystallin- a key latency antigen of M. tuberculosis to boost the BCG induced immunity. ‘BCG prime – DNA boost’ regimen (B/D) confers robust protection in guinea pigs along with a reduced pathology in comparison to BCG vaccination (1.37 log₁₀ and 1.96 log₁₀ fewer bacilli in lungs and spleen, respectively; p<0.01). In addition, B/D regimen also confers enhanced protection in mice. Further, we show that B/D immunization in mice results in a heightened frequency of PPD and antigen specific multi-functional CD4 T cells (3⁺) simultaneously producing interferon (IFN)γ, tumor necrosis factor (TNF)α and interleukin (IL)2.

Conclusions/Significance

These results clearly indicate the superiority of α-crystallin based B/D regimen over BCG. Our study, also demonstrates that protection against TB is predictable by an increased frequency of 3⁺ Th1 cells with superior effector functions. We anticipate that this study would significantly contribute towards the development of superior booster vaccines for BCG vaccinated individuals. In addition, this regimen can also be expected to reduce the risk of developing active TB due to reactivation of latent infection.     

Protective immunity induced by vaccination with SAG1 gene-transfected cells against Toxoplasma gondii-infection in mice

To develop a vaccine by augmenting the protective cellular immunity against Toxoplasma gondii (T. gondii)-infection, T gondii SAG1 gene-transfectants were established by using RMA.S (H-2b), a murine transporter associated with the antigen processing (TAP) molecule-deficient lymphoma line, as a host antigen-presenting cell (APC). Immunization of C57BL/6 mice with the SAG1-transfected RMA.S induced CD8+ cytotoxic T lymphocytes (CTL) specific for not only SAG1-transfected RMA.S but also T gondii-infected RMA.S, and elicited protective responses to infection with a virulent T. gondii strain, RH.     

Protective immunity to vaccinia virus induced by vaccination with multiple recombinant outer membrane proteins of intracellular and extracellular virions

Infectious intracellular and extracellular forms of vaccinia virus have different outer membrane proteins, presenting multiple targets to the immune system. We investigated the immunogenicity of soluble forms of L1, an outer membrane protein of the intracellular mature virus, and of A33 and B5, outer membrane proteins of the extracellular enveloped virus. The recombinant proteins, in 10-microg amounts mixed with a Ribi- or saponin-type adjuvant, were administered subcutaneously to mice. Antibody titers to each protein rose sharply after the first and second boosts, reaching levels that surpassed those induced by percutaneous immunization with live vaccinia virus. Immunoglobulin G1 (IgG1) antibody predominated after the protein immunizations, indicative of a T-helper cell type 2 response, whereas live vaccinia virus induced mainly IgG2a, indicative of a T-helper cell type 1 response. Mice immunized with any one of the recombinant proteins survived an intranasal challenge with 5 times the 50% lethal dose of the pathogenic WR strain of vaccinia virus. Measurements of weight loss indicated that the A33 immunization most effectively prevented disease. The superiority of protein combinations was demonstrated when the challenge virus dose was increased 20-fold. The best protection was obtained with a vaccine made by combining recombinant proteins of the outer membranes of intracellular and extracellular virus. Indeed, mice immunized with A33 plus B5 plus L1 or with A33 plus L1 were better protected than mice immunized with live vaccinia virus. Three immunizations with the three-protein combination were necessary and sufficient for complete protection. These studies suggest the feasibility of a multiprotein smallpox vaccine.