Elimination of Human Papillomavirus 16-Positive Tumors by a Mucosal rAd5 Therapeutic Vaccination in a Pre-Clinical Murine Study.

Therapeutic vaccination can harness the body's cellular immune system to target and destroy cancerous cells. Several treatment options are available to eliminate pre-cancerous and cancerous lesions caused by human papillomaviruses (HPV), but may not result in a long-term cure. Therapeutic vaccination may offer an effective, durable, and minimally intrusive alternative. We developed mucosally delivered, recombinant, non-replicating human adenovirus type 5 (rAd5)-vectored vaccines that encode HPV16's oncogenic proteins E6 and E7 alongside a molecular dsRNA adjuvant. The induction of antigen-specific T cells and the therapeutic efficacy of rAd5 were evaluated in a mouse model of HPV tumorigenesis where E6E7-transformed cells, TC-1, were implanted subcutaneously in C57BL/6 mice. After tumor growth, mice were treated intranasally with rAd5 vaccines expressing the wildtype form of E6E7 (rAd5-16/E6E7Wt) in combination with an anti-PD-1 antibody or isotype control. Animals treated with rAd5-16/E6E7Wt with and without anti-PD-1 had significant reductions in tumor volume and increased survival compared to controls. Further, animals treated with rAd5-16/E6E7Wt had increased CD4+ and CD8+ tumor-infiltrating lymphocytes (TILs) and produced a cytotoxic tumor microenvironment. In a second study, the immunogenicity of a non-transformative form of E6E7 (rAd5-16/E6E7Mu) and a vaccine encoding predicted T cell epitopes of E6E7 (rAd5-16/E6E7epi) were evaluated. These vaccines elicited significant reductions in TC-1 tumor volume and increased survival of animals. Antigen-specific CD8+ T effector memory cells were observed in the animals treated with E6E7-encoding rAd5, but not in the rAd5-empty group. The work described here demonstrates that this mucosal vaccination can be used therapeutically to elicit specific cellular immunity and further identifies a clinical candidate with great potential for the treatment and prevention of human cervical cancer.

Identifying intervention strategies to improve HPV vaccine decision-making using behaviour change theory.

Although the HPV vaccine is highly safe and effective, its uptake is sub-optimal in many countries, including Ireland. There is therefore a need to identify appropriate interventions that will increase HPV vaccine acceptance by parents. In this study, we took a systematic approach to understand the factors that influence HPV vaccine uptake by parents of adolescent girls in Ireland to define suitable behaviour change interventions that would support positive vaccine decision-making in the future. Specifically, we conducted semi-structured interviews, used a Theoretical Domains Framework (TDF)-based topic guide, to gain insight into the knowledge, beliefs, attitudes and current behaviours of parents with respect to their HPV vaccine decision. Transcripts were analysed using the TDF. The Behaviour Change Wheel (BCW) was used to identify relevant intervention functions and the Behaviour Change Technique Taxonomy version 1 (BCTTv1), to identify relevant intervention techniques. All parents discussed the essential role of healthcare providers in vaccine decision-making. Complacency and confidence were important factors in decision-making by vaccine hesitant parents. Five BCW intervention functions were identified as appropriate, namely; education; persuasion; environmental restructuring; modelling and enablement. To our knowledge, this is the first study to systematically evaluate HPV vaccine decision-making using behaviour change theory and identify suitable intervention strategies to promote positive vaccine decision-making using this approach.

A Video-Based Behavioral Intervention Associated with Improved HPV Knowledge and Intention to Vaccinate.

The aim of this study was to design, develop, and evaluate the feasibility of a theory- and evidence-based intervention to improve human papillomavirus (HPV) and HPV vaccine knowledge and intention to vaccinate, among parent-daughter dyads. A theory- and evidence-based online behavioral intervention, "Is the HPV vaccine for me?", was developed to improve HPV and HPV vaccine knowledge and intention to vaccinate. Knowledge, intention to vaccinate, and feasibility of the intervention were evaluated in a prospective, randomized, controlled feasibility trial. A total of 49 parent-daughter dyads completed the baseline knowledge assessment (n = 24 control, n = 25 intervention), and 35 dyads completed the knowledge assessment at week 2 (n = 17 control, n = 18 intervention). The intervention resulted in a statistically significant increase in HPV and HPV vaccine knowledge and intention to vaccinate. All intervention participants found the video interesting, while 96% found it useful. This intervention was found to be useful, effective, safe, and acceptable in this feasibility study.

Low Adenovirus Vaccine Doses Administered to Skin Using Microneedle Patches Induce Better Functional Antibody Immunogenicity as Compared to Systemic Injection.

Adenovirus-based vaccines are demonstrating promising clinical potential for multiple infectious diseases, including COVID-19. However, the immunogenicity of the vector itself decreases its effectiveness as a boosting vaccine due to the induction of strong anti-vector neutralizing immunity. Here we determined how dissolvable microneedle patches (DMN) for skin immunization can overcome this issue, using a clinically-relevant adenovirus-based Plasmodium falciparum malaria vaccine, AdHu5-PfRH5, in mice. Incorporation of vaccine into patches significantly enhanced its thermostability compared to the liquid form. Conventional high dose repeated immunization by the intramuscular (IM) route induced low antigen-specific IgG titres and high anti-vector immunity. A low priming dose of vaccine, by the IM route, but more so using DMN patches, induced the most efficacious immune responses, assessed by parasite growth inhibitory activity (GIA) assays. Administration of low dose AdHu5-PfRH5 using patches to the skin, boosted by high dose IM, induced the highest antigen-specific serum IgG response after boosting, the greatest skewing of the antibody response towards the antigen and away from the vector, and the highest efficacy. This study therefore demonstrates that repeated use of the same adenovirus vaccine can be highly immunogenic towards the transgene if a low dose is used to prime the response. It also provides a method of stabilizing adenovirus vaccine, in easy-to-administer dissolvable microneedle patches, permitting storage and distribution out of cold chain.

Melanoma-conditioned medium promotes cytotoxic immune responses by murine bone marrow-derived monocytes despite their expression of 'M2' markers.

Macrophages have been shown to infiltrate a wide range of malignancies and are often considered to promote tumour survival, growth and spread. However, the source and behaviour of discrete tumour-associated macrophage populations are still poorly understood. Here we show a novel method for the rational development of bone marrow-derived monocytes appropriate for the study of processes which involve the contribution of circulating inflammatory monocytes. We have shown that in response to tumour-conditioned medium, these cells upregulate CD206 and CD115, markers traditionally associated with M2-type macrophages. Treated cells show reduced capacity for cytokine secretion but significantly impact CD4+ and CD8+ T-cell proliferation and polarization. Coculture with conditioned bone marrow-derived monocytes significantly reduced CD4+ T-cell proliferation but increased CD8+ T-cell proliferation and granzyme B expression with significant induction of IFNγ secretion by both CD4+ and CD8+ T cells, indicating that these cells may have a role in promoting anti-cancer immunity.

Immunomodulatory Therapeutic Strategies in Stroke.

The role of immunity in all stages of stroke is increasingly being recognized, from the pathogenesis of risk factors to tissue repair, leading to the investigation of a range of immunomodulatory therapies. In the acute phase of stroke, proposed therapies include drugs targeting pro-inflammatory cytokines, matrix metalloproteinases, and leukocyte infiltration, with a key objective to reduce initial brain cell toxicity. Systemically, the early stages of stroke are also characterized by stroke-induced immunosuppression, where downregulation of host defences predisposes patients to infection. Therefore, strategies to modulate innate immunity post-stroke have garnered greater attention. A complementary objective is to reduce longer-term sequelae by focusing on adaptive immunity. Following stroke onset, the integrity of the blood-brain barrier is compromised, exposing central nervous system (CNS) antigens to systemic adaptive immune recognition, potentially inducing autoimmunity. Some pre-clinical efforts have been made to tolerize the immune system to CNS antigens pre-stroke. Separately, immune cell populations that exhibit a regulatory phenotype (T- and B- regulatory cells) have been shown to ameliorate post-stroke inflammation and contribute to tissue repair. Cell-based therapies, established in oncology and transplantation, could become a strategy to treat the acute and chronic stages of stroke. Furthermore, a role for the gut microbiota in ischaemic injury has received attention. Finally, the immune system may play a role in remote ischaemic preconditioning-mediated neuroprotection against stroke. The development of stroke therapies involving organs distant to the infarct site, therefore, should not be overlooked. This review will discuss the immune mechanisms of various therapeutic strategies, surveying published data and discussing more theoretical mechanisms of action that have yet to be exploited.

The immune system and stroke: from current targets to future therapy.

Stroke is a major cause of morbidity and mortality worldwide. Despite the intensive search for new therapies, hundreds of agents targeting various pathophysiological mechanisms have failed clinical trials, and the thrombolytic agent tissue plasminogen activator is currently the only FDA-approved medication for the treatment of acute ischemic stroke. The immune system is involved in all stages of stroke, from the pathogenesis of risk factors to neurotoxicity, to tissue remodeling and repair. There is a bidirectional interaction between the brain and the immune system, with stroke-induced immunosuppression and subsequent infection a principal source of patient mortality. Newer work also points to a role for the gut microbiota in the immune response to stroke, while clinical sequelae such as dementia might now also be explained in immune terms. However, the exact roles of innate and adaptive components have not been fully elucidated, with studies reporting both detrimental and beneficial functions. Time is a key determinant in defining whether immunity and inflammation are neuroprotective or neurotoxic. The local inflammatory milieu also has a clear influence on many proposed treatments. This review examines the individual components of the immune response to stroke, highlighting the most promising future stroke immunotherapies.

Orally administered adenoviral-based vaccine induces respiratory mucosal memory and protection against RSV infection in cotton rats.

A vaccine against Respiratory Syncytial Virus (RSV) is a major unmet need to prevent the significant morbidity and mortality that it causes in society. In addition to efficacy, such a vaccine must not induce adverse events, as previously occurred with a formalin-inactivated vaccine (FI-RSV). In this study, the safety, immunogenicity and efficacy of a molecularly adjuvanted adenovirus serotype 5 based RSV vaccine encoding the fusion (F) protein (Ad-RSVF) is demonstrated in cotton rats. Protective immunity to RSV was induced by Ad-RSVF when administered by an oral route as well as by intranasal and intramuscular routes. Compared to FI-RSV, the Ad-RSVF vaccine induced significantly greater neutralizing antibody responses and protection against RSV infection. Significantly, oral or intranasal immunization each induced protective multi-functional effector and memory B cell responses in the respiratory tract. This study uniquely demonstrates the capacity of an orally administered adenovirus vaccine to induce protective immunity in the respiratory tract against RSV in a pre-clinical model and supports further clinical development of this oral Ad-RSVF vaccine strategy.

Induction of broad immunity by thermostabilised vaccines incorporated in dissolvable microneedles using novel fabrication methods.

Dissolvable microneedle (DMN) patches for immunization have multiple benefits, including vaccine stability and ease-of-use. However, conventional DMN fabrication methods have several drawbacks. Here we describe a novel, microfluidic, drop dispensing-based dissolvable microneedle production method that overcomes these issues. Uniquely, heterogeneous arrays, consisting of microneedles of diverse composition, can be easily produced on the same patch. Robustness of the process was demonstrated by incorporating and stabilizing adenovirus and MVA vaccines. Clinically-available trivalent inactivated influenza vaccine (TIV) in DMN patches is fully stable for greater than 6months at 40°C. Immunization using low dose TIV-loaded DMN patches induced significantly higher antibody responses compared to intramuscular-based immunization in mice. TIV-loaded patches also induced a broader, heterosubtypic neutralizing antibody response. By addressing issues that will be faced in large-scale fill-finish DMN fabrication processes and demonstrating superior thermostable characteristics and immunogenicity, this study progresses the translation of this microneedle platform to eventual clinical deployment.

Induction of CD8(+) T cell responses and protective efficacy following microneedle-mediated delivery of a live adenovirus-vectored malaria vaccine.

There is an urgent need for improvements in vaccine delivery technologies. This is particularly pertinent for vaccination programmes within regions of limited resources, such as those required for adequate provision for disposal of used needles. Microneedles are micron-sized structures that penetrate the stratum corneum of the skin, creating temporary conduits for the needle-free delivery of drugs or vaccines. Here, we aimed to investigate immunity induced by the recombinant simian adenovirus-vectored vaccine ChAd63.ME-TRAP; currently undergoing clinical assessment as a candidate malaria vaccine, when delivered percutaneously by silicon microneedle arrays. In mice, we demonstrate that microneedle-mediated delivery of ChAd63.ME-TRAP induced similar numbers of transgene-specific CD8(+) T cells compared to intradermal (ID) administration with needle-and-syringe, following a single immunisation and after a ChAd63/MVA heterologous prime-boost schedule. When mice immunised with ChAd63/MVA were challenged with live Plasmodium berghei sporozoites, microneedle-mediated ChAd63.ME-TRAP priming demonstrated equivalent protective efficacy as did ID immunisation. Furthermore, responses following ChAd63/MVA immunisation correlated with a specific design parameter of the array used ('total array volume'). The level of transgene expression at the immunisation site and skin-draining lymph node (dLN) was also linked to total array volume. These findings have implications for defining silicon microneedle array design for use with live, vectored vaccines.

Microneedle-mediated immunization of an adenovirus-based malaria vaccine enhances antigen-specific antibody immunity and reduces anti-vector responses compared to the intradermal route.

Substantial effort has been placed in developing efficacious recombinant attenuated adenovirus-based vaccines. However induction of immunity to the vector is a significant obstacle to its repeated use. Here we demonstrate that skin-based delivery of an adenovirus-based malaria vaccine, HAdV5-PyMSP142, to mice using silicon microneedles induces equivalent or enhanced antibody responses to the encoded antigen, however it results in decreased anti-vector responses, compared to intradermal delivery. Microneedle-mediated vaccine priming and resultant induction of low anti-vector antibody titres permitted repeated use of the same adenovirus vaccine vector. This resulted in significantly increased antigen-specific antibody responses in these mice compared to ID-treated mice. Boosting with a heterologous vaccine; MVA-PyMSP142 also resulted in significantly greater antibody responses in mice primed with HAdV5-PyMSP142 using MN compared to the ID route. The highest protection against blood-stage malaria challenge was observed when a heterologous route of immunization (MN/ID) was used. Therefore, microneedle-mediated immunization has potential to both overcome some of the logistic obstacles surrounding needle-and-syringe-based immunization as well as to facilitate the repeated use of the same adenovirus vaccine thereby potentially reducing manufacturing costs of multiple vaccines. This could have important benefits in the clinical ease of use of adenovirus-based immunization strategies.

Immunity against heterosubtypic influenza virus induced by adenovirus and MVA expressing nucleoprotein and matrix protein-1.

Alternate prime/boost vaccination regimens employing recombinant replication-deficient adenovirus or MVA, expressing Influenza A virus nucleoprotein and matrix protein 1, induced antigen-specific T cell responses in intradermally (ID) vaccinated mice; with the strongest responses resulting from Ad/MVA immunization. In BALB/C mice the immunodominant response was shifted from the previously identified immunodominant epitope to a novel epitope when the antigen was derived from A/Panama/2007/1999 rather than A/PR/8. Alternate immunization routes did not affect the magnitude of antigen-specific systemic IFN-γ response, but higher CD8(+) T-cell IFN-γ immune responses were seen in the bronchoalveolar lavage following intransal (IN) boosting after intramuscular (IM) priming, whilst higher splenic antigen-specific CD8(+) T cell IFN-γ was seen following IM boosting. Partial protection against heterologous influenza virus challenge was achieved following either IM/IM or IM/IN but not ID/ID immunization. These data may be of relevance for the design of optimal immunization regimens for human influenza vaccines, especially for influenza-naïve infants.

Coated microneedle arrays for transcutaneous delivery of live virus vaccines.

Vaccines are sensitive biologics that require continuous refrigerated storage to maintain their viability. The vast majority of vaccines are also administered using needles and syringes. The need for cold chain storage and the significant logistics surrounding needle-and-syringe vaccination is constraining the success of immunization programs. Recombinant live viral vectors are a promising platform for the development of vaccines against a number of infectious diseases, however these viruses must retain infectivity to be effective. Microneedles offer an effective and painless method for delivery of vaccines directly into skin that in the future could provide solutions to current vaccination issues. Here we investigated methods of coating live recombinant adenovirus and modified vaccinia virus Ankara (MVA) vectors onto solid microneedle arrays. An effective spray-coating method, using conventional pharmaceutical processes, was developed, in tandem with suitable sugar-based formulations, which produces arrays with a unique coating of viable virus in a dry form around the shaft of each microneedle on the array. Administration of live virus-coated microneedle arrays successfully resulted in virus delivery, transcutaneous infection and induced an antibody or CD8(+) T cell response in mice that was comparable to that obtained by needle-and-syringe intradermal immunization. To our knowledge, this is the first report of successful vaccination with recombinant live viral vectored vaccines coated on microneedle delivery devices.

Nanoparticle-based drug delivery: case studies for cancer and cardiovascular applications.

Nanoparticles (NPs) comprised of nanoengineered complexes are providing new opportunities for enabling targeted delivery of a range of therapeutics and combinations. A range of functionalities can be included within a nanoparticle complex, including surface chemistry that allows attachment of cell-specific ligands for targeted delivery, surface coatings to increase circulation times for enhanced bioavailability, specific materials on the surface or in the nanoparticle core that enable storage of a therapeutic cargo until the target site is reached, and materials sensitive to local or remote actuation cues that allow controlled delivery of therapeutics to the target cells. However, despite the potential benefits of NPs as smart drug delivery and diagnostic systems, much research is still required to evaluate potential toxicity issues related to the chemical properties of NP materials, as well as their size and shape. The need to validate each NP for safety and efficacy with each therapeutic compound or combination of therapeutics is an enormous challenge, which forces industry to focus mainly on those nanoparticle materials where data on safety and efficacy already exists, i.e., predominantly polymer NPs. However, the enhanced functionality affordable by inclusion of metallic materials as part of nanoengineered particles provides a wealth of new opportunity for innovation and new, more effective, and safer therapeutics for applications such as cancer and cardiovascular diseases, which require selective targeting of the therapeutic to maximize effectiveness while avoiding adverse effects on non-target tissues.

Effective induction of high-titer antibodies by viral vector vaccines.

Protein-in-adjuvant vaccines have shown limited success against difficult diseases such as blood-stage malaria. Here we show that a recombinant adenovirus-poxvirus prime-boost immunization regime (known to induce strong T cell immunogenicity) can also induce very strong antigen-specific antibody responses, and we identify a simple complement-based adjuvant to further enhance immunogenicity. Antibodies induced against a blood-stage malaria antigen by this viral vector platform are highly effective against Plasmodium yoelii parasites in mice and against Plasmodium falciparum in vitro.

Single-dose immunogenicity and protective efficacy of simian adenoviral vectors against Plasmodium berghei.

Simian adenoviral vectors (SAd) offer an attractive alternative to standard human adenovirus serotype 5 (AdH5) subunit vaccination, due to pre-existing immunity affecting vaccine performance. We have used a mouse model of liver-stage malaria to test the efficiency of three chimpanzee-origin adenoviral vectors, AdC6, AdC7 and AdC9 containing ME.TRAP as an insert. AdC7 and AdC9 elicited strong immunogenicity ( approximately 20% of CD8(+) T cells in spleen), equivalent to or outperforming AdH5 and inducing sterile protection in 92% (C9), 83% (H5 and C7) and 67% (C6) of the mice, providing the first evidence of single-dose protection to Plasmodium berghei. Protection was afforded by the SAd despite high levels of pre-existing immunity to AdH5. Phenotypic analysis showed that all adenoviral vectors (Ad) elicited CD8(+) T cell responses with an effector memory T cell (T(EM)) phenotype. By contrast, vaccination with poxviral vectors did not confer protection to P. berghei and induced a predominantly CD8(+) central memory T cell (T(CM)) response. Multifunctional CD8(+) T cell responses (co-expressing IFN-gamma, TNF-alpha and IL-2) were also induced by the Ad in higher percentages than the poxviral vectors. Our data suggest that T(EM) cells are important as a first line of defense against fast-replicating pathogens such as murine Plasmodium and demonstrate the potential of replication-defective SAd as future malaria vaccines for humans.

Anti-CD25 antibody enhancement of vaccine-induced immunogenicity: increased durable cellular immunity with reduced immunodominance.

An efficacious vaccine strategy must be capable of inducing strong responses of an appropriate phenotype that are long lasting and sufficiently broad to prevent pathogen escape mechanisms. In the present study, we use anti-CD25 mAb to augment vaccine-induced immunity in mice. We demonstrate that coformulation of Ab and poxviral- or adenoviral-vectored vaccines induces significantly increased T cell responses to a malaria Ag; prior anti-CD25 Ab administration was not required for this effect. Furthermore, this vaccination approach subverts immunodominant epitope hierarchies by enhancing responses to subdominant epitopes induced by recombinant modified vaccinia virus Ankara immunization. Administration of anti-CD25 with a vaccine also induces more durable immunity compared with vaccine alone; significantly higher T cell responses were observed 100 days after the primary immunization. Enhanced immunogenicity is observed for multiple vaccine types with enhanced CD4+ and CD8+ T cell responses induced by bacillus Calmette-Guérin and a recombinant subunit protein vaccine to hepatitis B virus and with multiple Ags of tumor, viral, bacterial, and parasitic origin. Vaccine strategies incorporating anti-CD25 lead to improved protection against pre-erythrocytic malaria challenge. These data underpin new strategies for the design and development of more efficacious vaccines in clinical settings.

Novel protein and poxvirus-based vaccine combinations for simultaneous induction of humoral and cell-mediated immunity.

The presence of both cell-mediated and humoral immunity is important in protection from and clearance of a number of infectious pathogens. We describe novel vaccine regimens using combinations of plasmid DNA, poxvirus and protein to induce strong Ag-specific T cell and Ab responses simultaneously in a murine model. Intramuscular (i.m.) immunization with plasmid DNA encoding the middle Ag of hepatitis B (DNA) concurrently with a commercial hepatitis B virus (HBV) vaccine (Engerix-B) followed by boosting immunizations with both modified vaccinia virus Ankara (MVA) encoding the middle Ag of HBV and Engerix-B induced high levels of CD4(+) and CD8(+) T cells and high titer Ab responses to hepatitis B surface Ag (HbsAg). Substitution of Engerix-B with adjuvant-free rHBsAg induced similar T cell responses and greatly enhanced Ab levels. Repeated immunizations with recombinant or nonrecombinant MVA mixed with Ag induced higher titers of Abs compared with immunization with either Ag or Engerix-B further demonstrating this novel adjuvant effect of MVA. The poxviruses NYVAC, fowlpox (FP9) and ALVAC, and to a lesser extent, adenovirus, also displayed similar adjuvant properties when used in combination with rHBsAg. The use of poxviruses as an adjuvant for protein to concurrently induce Ag-specific T cells and Abs could be applied to the development of vaccines for many diseases, including HIV and malaria, where both cell mediated and humoral immunity may be important for protection.

Progress in DNA-based heterologous prime-boost immunization strategies for malaria.

An effective vaccine against malaria is urgently required to relieve the immense human suffering and mortality caused by this parasite. A successful subunit vaccine against the liver stage of malaria will require the induction of high levels of protective T cells. Despite success in small animal models, DNA vaccines fail to induce strong cellular immune responses in humans. However, DNA vaccines can induce a T-cell response that can be strongly boosted by recombinant viral vectors. We have evaluated this heterologous prime-boost approach using the Plasmodium berghei mouse model for immunogenicity and protective efficacy against malaria challenge using combinations of plasmid DNA, recombinant modified vaccinia virus Ankara, fowlpox virus, and non-replicating adenovirus. We have proceeded to test immunogenicity and efficacy of successful heterologous prime-boost vaccines in phase I/IIa trials in malaria naïve subjects in the UK and in semi-immune individuals in The Gambia. In these clinical trials, remarkably high levels of effector T-cell responses have been induced and significant protection documented in a human sporozoite challenge model. We summarize the preclinical design and development of these heterologous prime-boost vaccines and discuss the encouraging results that have been observed in vaccinated humans.

A Plasmodium falciparum candidate vaccine based on a six-antigen polyprotein encoded by recombinant poxviruses.

To generate broadly protective T cell responses more similar to those acquired after vaccination with radiation-attenuated Plasmodium falciparum sporozoites, we have constructed candidate subunit malaria vaccines expressing six preerythrocytic antigens linked together to produce a 3240-aa-long polyprotein (L3SEPTL). This polyprotein was expressed by a plasmid DNA vaccine vector (DNA) and by two attenuated poxvirus vectors, modified vaccinia virus Ankara (MVA) and fowlpox virus of the FP9 strain. MVAL3SEPTL boosted anti-thrombospondin-related adhesive protein (anti-TRAP) and anti-liver stage antigen 1 (anti-LSA1) CD8(+) T cell responses when primed by single antigen TRAP- or LSA1-expressing DNAs, respectively, but not by DNA-L3SEPTL. However, prime boost regimes involving two heterologous viral vectors expressing L3SEPTL induced a strong cellular response directed against an LSA1 peptide located in the C-terminal region of the polyprotein. Peptide-specific T cells secreted IFN-gamma and were cytotoxic. IFN-gamma-secreting T cells specific for each of the six antigens were induced after vaccination with L3SEPTL, supporting the use of polyprotein inserts to induce multispecific T cells against P. falciparum. The use of polyprotein constructs in nonreplicating poxviruses should broaden the target antigen range of vaccine-induced immunity and increase the number of potential epitopes available for immunogenetically diverse human populations.