Induction of antigen-specific CD8+ T cells offers the prospect of immunization against many infectious diseases, but no subunit vaccine has induced CD8+ T cells that correlate with efficacy in humans. Here we demonstrate that a replication-deficient chimpanzee adenovirus vector followed by a modified vaccinia virus Ankara booster induces exceptionally high frequency T-cell responses (median >2400 SFC/106 peripheral blood mononuclear cells) to the liver-stage Plasmodium falciparum malaria antigen ME-TRAP. It induces sterile protective efficacy against heterologous strain sporozoites in three vaccinees (3/14, 21%), and delays time to patency through substantial reduction of liver-stage parasite burden in five more (5/14, 36%), P=0.008 compared with controls. The frequency of monofunctional interferon-γ-producing CD8+ T cells, but not antibodies, correlates with sterile protection and delay in time to patency (Pcorrected=0.005). Vaccine-induced CD8+ T cells provide protection against human malaria, suggesting that a major limitation of previous vaccination approaches has been the insufficient magnitude of induced T cells.

Infection of mice with sporozoites of Plasmodium berghei or Plasmodium yoelii has been used extensively to evaluate liver-stage protection by candidate preerythrocytic malaria vaccines. Unfortunately, repeated success of such vaccines in mice has not translated readily to effective malaria vaccines in humans. Thus, mice may be used better as models to dissect basic parameters required for immunity to Plasmodium-infection than as preclinical vaccine models. In turn, this basic information may aid in the rational design of malaria vaccines. Here, we describe a model of circumsporozoite-specific memory CD8 T cell generation that protects mice against multiple P. berghei sporozoite challenges for at least 19 months. Using this model we defined a threshold frequency of memory CD8 T cells in the blood that predicts long-term sterilizing immunity against liver-stage infection. Importantly, the number of Plasmodium-specific memory CD8 T cells required for immunity greatly exceeds the number required for resistance to other pathogens. In addition, this model allowed us to identify readily individual immunized mice that exceed or fall below the protective threshold before infection, information that should greatly facilitate studies to dissect basic mechanisms of protective CD8 T cell memory against liver-stage Plasmodium infection. Furthermore, the extremely large threshold in memory CD8 T cell frequencies required for long-term protection in mice may have important implications for development of effective malaria vaccines.

Background. Vaccine development in human Plasmodium falciparum malaria has been hampered by the exceptionally high levels of CD8+ T cells required for efficacy. Use of potently immunogenic human adenoviruses as vaccine vectors could overcome this problem, but these are limited by preexisting immunity to human adenoviruses.Methods. From 2007 to 2010, we undertook a phase I dose and route finding study of a new malaria vaccine, a replication-incompetent chimpanzee adenovirus 63 (ChAd63) encoding the preerythrocytic insert multiple epitope thrombospondin-related adhesion protein (ME-TRAP; n = 54 vaccinees) administered alone (n = 28) or with a modified vaccinia virus Ankara (MVA) ME-TRAP booster immunization 8 weeks later (n = 26). We observed an excellent safety profile. High levels of TRAP antigen–specific CD8+ and CD4+ T cells, as detected by interferon γ enzyme-linked immunospot assay and flow cytometry, were induced by intramuscular ChAd63 ME-TRAP immunization at doses of 5 × 1010 viral particles and above. Subsequent administration of MVA ME-TRAP boosted responses to exceptionally high levels, and responses were maintained for up to 30 months postvaccination.Conclusions. The ChAd63 chimpanzee adenovirus vector appears safe and highly immunogenic, providing a viable alternative to human adenoviruses as vaccine vectors for human use.Clinical Trials Registration. NCT00890019.

IntroductionPreventative viral vaccines provide protection through induction of immunologic memory, most notably circulating neutralizing antibodies. 1 For some viruses, such as HIV-1, vaccines have failed to induce protective levels of antibodies and the focus of many of the ongoing HIV-1 vaccine efforts has shifted to T-cell responses. 2 Correlates of T-cell-mediated protection to viral infections remain ill-defined because of the not yet fully understood complexity of memory T-cell responses.Replication-defective adenovirus (Ad) vectors are at the forefront of HIV-1 vaccine research and have entered phase 2 clinical trials. [3][4][5] One of the most remarkable features of Ad-based vaccines is their ability to induce exceptionally high and sustained frequencies of transgene product-specific CD8 ϩ T cells that, unlike those induced by other subunit vaccine carriers such as DNA vaccines or poxvirus vectors, do not contract after the initial activation. 6,7 Here we show that replication-defective E1-deleted Ad vector genomes similar to those of Ads acquired by natural infections 8,9 persist. Persistent vector was found in muscle at the site of inoculation, in liver, and in lymphatic tissues of experimental animals. Within lymphatic tissues the vector genomes are enriched in T-cells directed to the antigen encoded by the viral vector. The vector's genome remains transcriptionally active, and the continued presence of transgene products appears to maintain high frequencies of activated antigen-specific CD8 ϩ T cells in addition to a pool of resting memory T cells. Although the concept of persisting vaccines may provide challenges for their eventual use for mass vaccination, concomitantly maintaining high frequencies of effector-like T cells and resting memory T cells may provide a solution to the dilemma of vaccines that rely on T-cell-mediated protection. Materials and methods MiceC57Bl/6 and BALB/c mice were purchased at 6 to 8 weeks of age from Charles River Laboratories (Boston, MA). OT1 and P14 mice were bred at the Animal Facility of the Wistar Institute (Philadelphia, PA) and typed by polymerase chain reaction (PCR) for homozygosity. Animals were treated according to guidelines of the Wistar Institute. Cell linesHEK 293 and HeLa cells were grown in Dulbecco Modified Eagle medium, supplemented with 10% fetal bovine serum. Viruses and viral vectorsAd vectors expressing Gag of HIV-1, the rabies virus glycoprotein or SIINFEKL as a fusion protein with influenza virus nucleoprotein and green fluorescent protein, the glycoprotein of lymphocytic choriomeningitis virus (LCMV), or green fluorescent protein were propagated on HEK 293 cells, purified, and quality-controlled as described previously. 10 Vaccinia virus vectors expressing Gag were grown on HeLa cells and titrated as described. 11 LCMV strain Armstrong was produced as described. 12 Immunization or infection of miceMice were immunized intramuscularly at 6 to 10 weeks of age with vectors diluted in 100 L PBS. Mice were infected with vaccinia virus vectors or L...

In animal models, E1-deleted human adenoviral recombinants of the serotype 5 (AdHu5) have shown high efficacy as vaccine carriers for different Ags including those of HIV-1. Humans are infected by common serotypes of human adenovirus such as AdHu5 early in life and a significant percentage has high levels of neutralizing Abs to these serotypes, which will very likely impair the efficacy of recombinant vaccines based on the homologous virus. To circumvent this problem, a novel replication-defective adenoviral vaccine carrier based on an E1-deleted recombinant of the chimpanzee adenovirus 68 (AdC68) was developed. An AdC68 construct expressing a codon-optimized, truncated form of gag of HIV-1 induces CD8+ T cells to gag in mice which at the height of the immune response encompass nearly 20% of the entire splenic CD8+ T cell population. The vaccine-induced immune response provides protection to challenge with a vaccinia gag recombinant virus. Induction of transgene-specific CD8+ T cells and protection against viral challenge elicited by the AdC68 vaccines is not strongly inhibited in animals preimmune to AdHu5 virus. However, the response elicited by the AdHu5 vaccine is greatly attenuated in AdHu5 preimmune animals.

An E1-deletion-containing adenoviral recombinant based on the chimpanzee serotype 68 (AdC68) was developed to express the rabies virus glycoprotein. Mice immunized with this construct (AdC68rab.gp) developed antibodies to rabies virus and remained resistant to challenge with an otherwise lethal dose of rabies virus. In naïve mice immunized intranasally, the rabies virus-specific antibody responses elicited by AdC68rab.gp were comparable with regard to both titers and isotype profiles to those induced by an adenoviral recombinant based on human serotype 5 (Adhu5) expressing the same transgene product. In contrast, subcutaneous immunization with the AdC68rab.gp vaccine resulted in markedly lower antibody responses to the rabies virus glycoprotein than the corresponding Adhu5 vaccine. Antibodies from AdC68rab.gp-immunized mice were strongly biased towards the immunoglobulin G2a isotype. The antibody response to the rabies virus glycoprotein presented by Adhu5rab.gp was severely compromised in animals preexposed to the homologous adenovirus. In contrast, the rabies virus-specific antibody response to the AdC68rab.gp vaccine was at most marginally affected by preexisting immunity to common human adenovirus serotypes, such as 2, 4, 5, 7, and 12. This novel vaccine carrier thus offers a distinct advantage over adenoviral vaccines based on common human serotypes.E1-deletion-containing replication-defective adenoviral recombinants based on human serotype 5 (Adhu5) have been tested widely as carriers for gene therapy (2, 21). Gene therapy trials demonstrated high-level expression of the transgene product in a variety of different cell types. Nevertheless, expression was transient in vivo due to clearance of adenovirusinfected cells by CD8 ϩ T cells directed against antigens of the adenovirus as well as against the transgene product (4, 26). Vaccine studies based on the rabies virus glycoprotein (22), the circumsporozoite protein of Plasmodium falciparum (17), the E6 and E7 oncoproteins of human papillomavirus type 16 (HPV-16) (9), and others (9;

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